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Sample records for zinc-finger transcription factor

  1. Mechanisms of inhibition of zinc-finger transcription factors by selenium compounds ebselen and selenite.

    Science.gov (United States)

    Larabee, Jason L; Hocker, James R; Hanas, Jay S

    2009-03-01

    The anti-inflammatory selenium compounds, ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one) and selenite, were found to alter the DNA binding mechanisms and structures of cysteine-rich zinc-finger transcription factors. As assayed by DNase I protection, DNA binding by TFIIIA (transcription factor IIIA, prototypical Cys(2)His(2) zinc finger protein), was inhibited by micromolar amounts of ebselen. In a gel shift assay, ebselen inhibited the Cys(2)His(2) zinc finger-containing DNA binding domain (DBD) of the NF-kappaB mediated transcription factor Sp1. Ebselen also inhibited DNA binding by the p50 subunit of the pro-inflammatory Cys-containing NF-kappaB transcription factor. Electrospray ionization mass spectrometry (ESI-MS) was utilized to elucidate mechanisms of chemical interaction between ebselen and a zinc-bound Cys(2)His(2) zinc finger polypeptide modeled after the third finger of Sp1 (Sp1-3). Exposing Sp1-3 to micromolar amounts of ebselen resulted in Zn(2+) release from this peptide and the formation of a disulfide bond by oxidation of zinc finger SH groups, the likely mechanism for DNA binding inhibition. Selenite was shown by ESI-MS to also eject zinc from Sp1-3 as well as induce disulfide bond formation through SH oxidation. The selenite-dependent inhibition/oxidation mechanism differed from that of ebselen by inducing the formation of a stable selenotrisulfide bond. Selenite-induced selenotrisulfide formation was dependent upon the structure of the Cys(2)His(2) zinc finger as alteration in the finger structure enhanced this reaction as well as selenite-dependent zinc release. Ebselen and selenite-dependent inhibition/oxidation of Cys-rich zinc finger proteins, with concomitant release of zinc and finger structural changes, points to mechanisms at the atomic and protein level for selenium-induced alterations in Cys-rich proteins, and possible amelioration of certain inflammatory, neurodegenerative, and oncogenic responses.

  2. Role of zinc finger structure in nuclear localization of transcription factor Sp1

    International Nuclear Information System (INIS)

    Ito, Tatsuo; Azumano, Makiko; Uwatoko, Chisana; Itoh, Kohji; Kuwahara, Jun

    2009-01-01

    Transcription factor Sp1 is localized in the nucleus and regulates gene expression. Our previous study demonstrated that the carboxyl terminal region of Sp1 containing 3-zinc finger region as DNA binding domain can also serve as nuclear localization signal (NLS). However, the nuclear transport mechanism of Sp1 has not been well understood. In this study, we performed a gene expression study on mutant Sp1 genes causing a set of amino acid substitutions in zinc finger domains to elucidate nuclear import activity. Nuclear localization of the GFP-fused mutant Sp1 proteins bearing concomitant substitutions in the first and third zinc fingers was highly inhibited. These mutant Sp1 proteins had also lost the binding ability as to the GC box sequence. The results suggest that the overall tertiary structure formed by the three zinc fingers is essential for nuclear localization of Sp1 as well as dispersed basic amino acids within the zinc fingers region.

  3. Interaction of Sp1 zinc finger with transport factor in the nuclear localization of transcription factor Sp1

    International Nuclear Information System (INIS)

    Ito, Tatsuo; Kitamura, Haruka; Uwatoko, Chisana; Azumano, Makiko; Itoh, Kohji; Kuwahara, Jun

    2010-01-01

    Research highlights: → Sp1 zinc fingers themselves interact with importin α. → Sp1 zinc finger domains play an essential role as a nuclear localization signal. → Sp1 can be transported into the nucleus in an importin-dependent manner. -- Abstract: Transcription factor Sp1 is localized in the nucleus and regulates the expression of many cellular genes, but the nuclear transport mechanism of Sp1 is not well understood. In this study, we revealed that GST-fused Sp1 protein bound to endogenous importin α in HeLa cells via the Sp1 zinc finger domains, which comprise the DNA binding domain of Sp1. It was found that the Sp1 zinc finger domains directly interacted with a wide range of importin α including the armadillo (arm) repeat domain and the C-terminal acidic domain. Furthermore, it turned out that all three zinc fingers of Sp1 are essential for binding to importin α. Taken together, these results suggest that the Sp1 zinc finger domains play an essential role as a NLS and Sp1 can be transported into the nucleus in an importin-dependent manner even though it possesses no classical NLSs.

  4. Design, construction, and analysis of specific zinc finger nucleases for microphthalmia - associate transcription factor

    Directory of Open Access Journals (Sweden)

    Wenwen Wang

    2012-08-01

    Full Text Available This work studied the design, construction, and cleavage analysis of zinc finger nucleases (ZFNs that could cut the specific sequences within microphthalmia - associate transcription factor (mitfa of zebra fish. The target site and ZFPs were selected and designed with zinc finger tools, while the ZFPs were synthesized using DNAWorks and two-step PCR. The ZFNs were constructed, expressed, purified, and analyzed in vitro. As expected, the designed ZFNs could create a double-stand break (DSB at the target site in vitro. The DNAWorks, two-step PCR, and an optimized process of protein expression were firstly induced in the construction of ZFNs successfully, which was an effective and simplified protocol. These results could be useful for further application of ZFNs - mediated gene targeting.

  5. Overexpression of a New Zinc Finger Protein Transcription Factor OsCTZFP8 Improves Cold Tolerance in Rice

    Directory of Open Access Journals (Sweden)

    Yong-Mei Jin

    2018-01-01

    Full Text Available Cold stress is one of the most important abiotic stresses in rice. C2H2 zinc finger proteins play important roles in response to abiotic stresses in plants. In the present study, we isolated and functionally characterized a new C2H2 zinc finger protein transcription factor OsCTZFP8 in rice. OsCTZFP8 encodes a C2H2 zinc finger protein, which contains a typical zinc finger motif, as well as a potential nuclear localization signal (NLS and a leucine-rich region (L-box. Expression of OsCTZFP8 was differentially induced by several abiotic stresses and was strongly induced by cold stress. Subcellular localization assay and yeast one-hybrid analysis revealed that OsCTZFP8 was a nuclear protein and has transactivation activity. To characterize the function of OsCTZFP8 in rice, the full-length cDNA of OsCTZFP8 was isolated and transgenic rice with overexpression of OsCTZFP8 driven by the maize ubiquitin promoter was generated using Agrobacterium-mediated transformation. Among 46 independent transgenic lines, 6 single-copy homozygous overexpressing lines were selected by Southern blot analysis and Basta resistance segregation assay in both T1 and T2 generations. Transgenic rice overexpressing OsCTZFP8 exhibited cold tolerant phenotypes with significantly higher pollen fertilities and seed setting rates than nontransgenic control plants. In addition, yield per plant of OsCTZFP8-expressing lines was significantly (p<0.01 higher than that of nontransgenic control plants under cold treatments. These results demonstrate that OsCTZFP8 was a C2H2 zinc finger transcription factor that plays an important role in cold tolerance in rice.

  6. Regulation of Nitrogen Metabolism by GATA Zinc Finger Transcription Factors in Yarrowia lipolytica

    OpenAIRE

    Pomraning, Kyle R.; Bredeweg, Erin L.; Baker, Scott E.

    2017-01-01

    ABSTRACT Fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeast Yarrowia lipolytica, growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors...

  7. The artificial zinc finger coding gene 'Jazz' binds the utrophin promoter and activates transcription.

    Science.gov (United States)

    Corbi, N; Libri, V; Fanciulli, M; Tinsley, J M; Davies, K E; Passananti, C

    2000-06-01

    Up-regulation of utrophin gene expression is recognized as a plausible therapeutic approach in the treatment of Duchenne muscular dystrophy (DMD). We have designed and engineered new zinc finger-based transcription factors capable of binding and activating transcription from the promoter of the dystrophin-related gene, utrophin. Using the recognition 'code' that proposes specific rules between zinc finger primary structure and potential DNA binding sites, we engineered a new gene named 'Jazz' that encodes for a three-zinc finger peptide. Jazz belongs to the Cys2-His2 zinc finger type and was engineered to target the nine base pair DNA sequence: 5'-GCT-GCT-GCG-3', present in the promoter region of both the human and mouse utrophin gene. The entire zinc finger alpha-helix region, containing the amino acid positions that are crucial for DNA binding, was specifically chosen on the basis of the contacts more frequently represented in the available list of the 'code'. Here we demonstrate that Jazz protein binds specifically to the double-stranded DNA target, with a dissociation constant of about 32 nM. Band shift and super-shift experiments confirmed the high affinity and specificity of Jazz protein for its DNA target. Moreover, we show that chimeric proteins, named Gal4-Jazz and Sp1-Jazz, are able to drive the transcription of a test gene from the human utrophin promoter.

  8. Fear-of-intimacy-mediated zinc transport controls the function of zinc-finger transcription factors involved in myogenesis.

    Science.gov (United States)

    Carrasco-Rando, Marta; Atienza-Manuel, Alexandra; Martín, Paloma; Burke, Richard; Ruiz-Gómez, Mar

    2016-06-01

    Zinc is a component of one-tenth of all human proteins. Its cellular concentration is tightly regulated because its dyshomeostasis has catastrophic health consequences. Two families of zinc transporters control zinc homeostasis in organisms, but there is little information about their specific developmental roles. We show that the ZIP transporter Fear-of-intimacy (Foi) is necessary for the formation of Drosophila muscles. In foi mutants, myoblasts segregate normally, but their specification is affected, leading to the formation of a misshapen muscle pattern and distorted midgut. The observed phenotypes could be ascribed to the inactivation of specific zinc-finger transcription factors (ZFTFs), supporting the hypothesis that they are a consequence of intracellular depletion of zinc. Accordingly, foi phenotypes can be rescued by mesodermal expression of other ZIP members with similar subcellular localization. We propose that Foi acts mostly as a transporter to regulate zinc intracellular homeostasis, thereby impacting on the activity of ZFTFs that control specific developmental processes. Our results additionally suggest a possible explanation for the presence of large numbers of zinc transporters in organisms based on differences in ion transport specificity and/or degrees of activity among transporters. © 2016. Published by The Company of Biologists Ltd.

  9. C2H2 type of zinc finger transcription factors in foxtail millet define response to abiotic stresses.

    Science.gov (United States)

    Muthamilarasan, Mehanathan; Bonthala, Venkata Suresh; Mishra, Awdhesh Kumar; Khandelwal, Rohit; Khan, Yusuf; Roy, Riti; Prasad, Manoj

    2014-09-01

    C2H2 type of zinc finger transcription factors (TFs) play crucial roles in plant stress response and hormone signal transduction. Hence considering its importance, genome-wide investigation and characterization of C2H2 zinc finger proteins were performed in Arabidopsis, rice and poplar but no such study was conducted in foxtail millet which is a C4 Panicoid model crop well known for its abiotic stress tolerance. The present study identified 124 C2H2-type zinc finger TFs in foxtail millet (SiC2H2) and physically mapped them onto the genome. The gene duplication analysis revealed that SiC2H2s primarily expanded in the genome through tandem duplication. The phylogenetic tree classified these TFs into five groups (I-V). Further, miRNAs targeting SiC2H2 transcripts in foxtail millet were identified. Heat map demonstrated differential and tissue-specific expression patterns of these SiC2H2 genes. Comparative physical mapping between foxtail millet SiC2H2 genes and its orthologs of sorghum, maize and rice revealed the evolutionary relationships of C2H2 type of zinc finger TFs. The duplication and divergence data provided novel insight into the evolutionary aspects of these TFs in foxtail millet and related grass species. Expression profiling of candidate SiC2H2 genes in response to salinity, dehydration and cold stress showed differential expression pattern of these genes at different time points of stresses.

  10. Engineered zinc-finger transcription factors inhibit the replication and transcription of HBV in vitro and in vivo.

    Science.gov (United States)

    Luo, Wei; Wang, Junxia; Xu, Dengfeng; Bai, Huili; Zhang, Yangli; Zhang, Yuhong; Li, Xiaosong

    2018-04-01

    In the present study, an artificial zinc-finger transcription factor eukaryotic expression vector specifically recognizing and binding to the hepatitis B virus (HBV) enhancer (Enh) was constructed, which inhibited the replication and expression of HBV DNA. The HBV EnhI‑specific pcDNA3.1‑artificial transcription factor (ATF) vector was successfully constructed, and then transformed or injected into HepG2.2.15 cells and HBV transgenic mice, respectively. The results demonstrated that the HBV EnhI (1,070‑1,234 bp)‑specific ATF significantly inhibited the replication and transcription of HBV DNA in vivo and in vitro. The HBV EnhI‑specific ATF may be a meritorious component of progressive combination therapies for eliminating HBV DNA in infected patients. A radical cure for chronic HBV infection may become feasible by using this bioengineering technology.

  11. Molecular Characterization of the Schistosoma mansoni Zinc Finger Protein SmZF1 as a Transcription Factor

    Science.gov (United States)

    D'Astolfo, Diego S.; Cardoso, Fernanda C.; Rajão, Matheus A.; Mourão, Marina M.; Gava, Elisandra; Oliveira, Sérgio C.; Macedo, Andréa M.; Machado, Carlos R.; Pena, Sérgio D. J.; Kitten, Gregory T.; Franco, Glória R.

    2009-01-01

    Background During its development, the parasite Schistosoma mansoni is exposed to different environments and undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Characterization of proteins involved in the regulation of these processes is of importance for the understanding of schistosome biology. Proteins containing zinc finger motifs usually participate in regulatory processes and are considered the major class of transcription factors in eukaryotes. It has already been shown, by EMSA (Eletrophoretic Mobility Shift Assay), that SmZF1, a S. mansoni zinc finger (ZF) protein, specifically binds both DNA and RNA oligonucleotides. This suggests that this protein might act as a transcription factor in the parasite. Methodology/Principal Findings In this study we extended the characterization of SmZF1 by determining its subcellular localization and by verifying its ability to regulate gene transcription. We performed immunohistochemistry assays using adult male and female worms, cercariae and schistosomula to analyze the distribution pattern of SmZF1 and verified that the protein is mainly detected in the cells nuclei of all tested life cycle stages except for adult female worms. Also, SmZF1 was heterologously expressed in mammalian COS-7 cells to produce the recombinant protein YFP-SmZF1, which was mainly detected in the nucleus of the cells by confocal microscopy and Western blot assays. To evaluate the ability of this protein to regulate gene transcription, cells expressing YFP-SmZF1 were tested in a luciferase reporter system. In this system, the luciferase gene is downstream of a minimal promoter, upstream of which a DNA region containing four copies of the SmZF1 putative best binding site (D1-3DNA) was inserted. SmZF1 increased the reporter gene transcription by two fold (p≤0.003) only when its specific binding site was present. Conclusion Taken together, these results strongly support the hypothesis

  12. ZNF328, a novel human zinc-finger protein, suppresses transcriptional activities of SRE and AP-1

    International Nuclear Information System (INIS)

    Ou Ying; Wang Shenqiu; Cai Zhenyu; Wang Yuequn; Wang Canding; Li Yongqing; Li Fang; Yuan Wuzhou; Liu Bisheng; Wu Xiushan; Liu Mingyao

    2005-01-01

    The zinc finger proteins containing the Kruppel-associated box domain (KRAB-ZFPs) are the single largest class of transcription factors in human genome. Many of the KRAB-ZFPs are involved in cardiac development or cardiovascular diseases. Here, we have identified a novel human KRAB zinc finger gene, named ZNF328, from the human fetal heart cDNA library. The complete sequence of ZNF328 cDNA contains a 2376-bp open reading frame (ORF) and encodes a 792 amino acid protein with an N-terminal KRAB domain and classical zinc finger C 2 H 2 motifs in the C-terminus. Northern blot analysis indicates that the protein is expressed in most of the examined human adult and embryonic tissues. ZNF328 is a transcription suppressor when fused to Gal-4 DNA-binding domain and cotransfected with VP-16. Overexpression of ZNF328 in COS-7 cells inhibits the transcriptional activities of SRE and AP-1. Deletion analysis with a series of truncated fusion proteins indicates that the KRAB motif is a basal repression domain when cotransfected with VP-16. Similar results were obtained when the truncated fusion proteins were assayed for the transcriptional activities of SRE and AP-1. These results suggest that ZNF328 protein may act as a transcriptional repressor in mitogen-activated protein kinase (MAPK) signaling pathway to mediate cellular functions

  13. Nucleolin modulates the subcellular localization of GDNF-inducible zinc finger protein 1 and its roles in transcription and cell proliferation

    International Nuclear Information System (INIS)

    Dambara, Atsushi; Morinaga, Takatoshi; Fukuda, Naoyuki; Yamakawa, Yoshinori; Kato, Takuya; Enomoto, Atsushi; Asai, Naoya; Murakumo, Yoshiki; Matsuo, Seiichi; Takahashi, Masahide

    2007-01-01

    GZF1 is a zinc finger protein induced by glial cell-line-derived neurotrophic factor (GDNF). It is a sequence-specific transcriptional repressor with a BTB/POZ (Broad complex, Tramtrack, Bric a brac/Poxvirus and zinc finger) domain and ten zinc finger motifs. In the present study, we used immunoprecipitation and mass spectrometry to identify nucleolin as a GZF1-binding protein. Deletion analysis revealed that zinc finger motifs 1-4 of GZF1 mediate its association with nucleolin. When zinc fingers 1-4 were deleted from GZF1 or nucleolin expression was knocked down by short interference RNA (siRNA), nuclear localization of GZF1 was impaired. These results suggest that nucleolin is involved in the proper subcellular distribution of GZF1. In addition, overexpression of nucleolin moderately inhibited the transcriptional repressive activity of GZF1 whereas knockdown of nucleolin expression by siRNA enhanced its activity. Thus, the repressive activity of GZF1 is modulated by the level at which nucleolin is expressed. Finally, we found that knockdown of GZF1 and nucleolin expression markedly impaired cell proliferation. These findings suggest that the physiological functions of GZF1 may be regulated by the protein's association with nucleolin

  14. ZNF322, a novel human C2H2 Krueppel-like zinc-finger protein, regulates transcriptional activation in MAPK signaling pathways

    International Nuclear Information System (INIS)

    Li Yongqing; Wang Yuequn; Zhang Caibo; Yuan Wuzhou; Wang Jun; Zhu Chuanbing; Chen Lei; Huang Wen; Zeng Weiqi; Wu Xiushan; Liu Mingyao

    2004-01-01

    Cardiac differentiation involves a cascade of coordinated gene expression that regulates cell proliferation and matrix protein formation in a defined temporal-spatial manner. The C 2 H 2 zinc finger-containing transcription factors have been implicated as critical regulators of multiple cardiac-expressed genes and are important for human heart development and diseases. Here we have identified and characterized a novel zinc-finger gene named ZNF322 using degenerated primers from a human embryo heart cDNA library. The gene contains four exons and spans 23.2 kb in chromosome 6p22.1 region, and transcribes a 2.7 kb mRNA that encodes a protein with 402 amino acid residues. The predicted protein contains 9 tandem C 2 H 2 -type zinc-finger motifs. Northern blot analysis shows that ZNF322 is expressed in every human tissue examined at adult stage and during embryonic developmental stages from 80 days to 24 weeks. When overexpressed in COS-7 cells, ZNF322-EGFP fusion protein is detected in the nucleus and cytoplasm. Reporter gene assays show that ZNF322 is a transcriptional activator. Furthermore, overexpression of ZNF322 in COS-7 cells activates the transcriptional activity of SRE and AP-1. Together, these results suggest that ZNF322 is a member of the zinc-finger transcription factor family and may act as a positive regulator in gene transcription mediated by the MAPK signaling pathways

  15. Zinc finger protein 521 antagonizes early B-cell factor 1 and modulates the B-lymphoid differentiation of primary hematopoietic progenitors

    NARCIS (Netherlands)

    Mega, Tiziana; Lupia, Michela; Amodio, Nicola; Horton, Sarah J.; Mesuraca, Maria; Pelaggi, Daniela; Agosti, Valter; Grieco, Michele; Chiarella, Emanuela; Spina, Raffaella; Moore, Malcolm A. S.; Schuringa, Jan Jacob; Bond, Heather M.; Morrone, Giovanni

    2011-01-01

    Zinc finger protein 521 (EHZF/ZNF521) is a multi-functional transcription co-factor containing 30 zinc fingers and an N-terminal motif that binds to the nucleosome remodelling and histone deacetylase (NuRD) complex. ZNF521 is believed to be a relevant player in the regulation of the homeostasis of

  16. SUMOylation of the KRAB zinc-finger transcription factor PARIS/ZNF746 regulates its transcriptional activity

    International Nuclear Information System (INIS)

    Nishida, Tamotsu; Yamada, Yoshiji

    2016-01-01

    Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS in a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC-1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms. -- Highlights: •PARIS can be SUMOylated in vivo and in vitro. •SUMOylation of PARIS functions in the repression of PGC-1a promoter activity. •PIASy interacts with PARIS and enhances its SUMOylation. •PIASy influences PARIS-mediated repression of PGC-1a promoter activity.

  17. SUMOylation of the KRAB zinc-finger transcription factor PARIS/ZNF746 regulates its transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Nishida, Tamotsu, E-mail: nishida@gene.mie-u.ac.jp; Yamada, Yoshiji

    2016-05-13

    Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS in a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC-1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms. -- Highlights: •PARIS can be SUMOylated in vivo and in vitro. •SUMOylation of PARIS functions in the repression of PGC-1a promoter activity. •PIASy interacts with PARIS and enhances its SUMOylation. •PIASy influences PARIS-mediated repression of PGC-1a promoter activity.

  18. Solution structure of an archaeal DNA binding protein with an eukaryotic zinc finger fold.

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    Florence Guillière

    Full Text Available While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers.

  19. Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains

    International Nuclear Information System (INIS)

    Vallee, B.L.; Auld, D.S.; Coleman, J.E.

    1991-01-01

    The authors recognize three distinct motifs of DNA-binding zinc proteins: (i) zinc fingers, (ii) zinc clusters, and (iii) zinc twists. Until very recently, x-ray crystallographic or NMR three-dimensional structure analyses of DNA-binding zinc proteins have not been available to serve as standards of reference for the zinc binding sites of these families of proteins. Those of the DNA-binding domains of the fungal transcription factor GAL4 and the rat glucocorticoid receptor are the first to have been determined. Both proteins contain two zinc binding sites, and in both, cysteine residues are the sole zinc ligands. In GAL4, two zinc atoms are bound to six cysteine residues which form a zinc cluster akin to that of metallothionein; the distance between the two zinc atoms of GAL4 is ∼3.5 angstrom. In the glucocorticoid receptor, each zinc atom is bound to four cysteine residues; the interatomic zinc-zinc distance is ∼13 angstrom, and in this instance, a zinc twist is represented by a helical DNA recognition site located between the two zinc atoms. Zinc clusters and zinc twists are here recognized as two distinctive motifs in DNA-binding proteins containing multiple zinc atoms. For native zinc fingers, structural data do not exist as yet; consequently, the interatomic distances between zinc atoms are not known. As further structural data become available, the structural and functional significance of these different motifs in their binding to DNA and other proteins participating in the transmission of the genetic message will become apparent

  20. The creation of the artificial RING finger from the cross-brace zinc finger by α-helical region substitution

    International Nuclear Information System (INIS)

    Miyamoto, Kazuhide; Togiya, Kayo

    2010-01-01

    The creation of the artificial RING finger as ubiquitin-ligating enzyme (E3) has been demonstrated. In this study, by the α-helical region substitution between the EL5 RING finger and the Williams-Beuren syndrome transcription factor (WSTF) PHD finger, the artificial E3 (WSTF PHD R ING finger) was newly created. The experiments of the chemical modification of residues Cys and the circular dichroism spectra revealed that the WSTF PHD R ING finger binds two zinc atoms and adopts the zinc-dependent ordered-structure. In the substrate-independent ubiquitination assay, the WSTF PHD R ING finger functions as E3 and was poly- or mono-ubiquitinated. The present strategy is very simple and convenient, and consequently it might be widely applicable to the creation of various artificial E3 RING fingers with the specific ubiquitin-conjugating enzyme (E2)-binding capability.

  1. Conversion of Human Fibroblasts to Stably Self-Renewing Neural Stem Cells with a Single Zinc-Finger Transcription Factor

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    Ebrahim Shahbazi

    2016-04-01

    Full Text Available Direct conversion of somatic cells into neural stem cells (NSCs by defined factors holds great promise for mechanistic studies, drug screening, and potential cell therapies for different neurodegenerative diseases. Here, we report that a single zinc-finger transcription factor, Zfp521, is sufficient for direct conversion of human fibroblasts into long-term self-renewable and multipotent NSCs. In vitro, Zfp521-induced NSCs maintained their characteristics in the absence of exogenous factor expression and exhibited morphological, molecular, developmental, and functional properties that were similar to control NSCs. In addition, the single-seeded induced NSCs were able to form NSC colonies with efficiency comparable with control NSCs and expressed NSC markers. The converted cells were capable of surviving, migrating, and attaining neural phenotypes after transplantation into neonatal mouse and adult rat brains, without forming tumors. Moreover, the Zfp521-induced NSCs predominantly expressed rostral genes. Our results suggest a facilitated approach for establishing human NSCs through Zfp521-driven conversion of fibroblasts.

  2. The pro1(+) gene from Sordaria macrospora encodes a C6 zinc finger transcription factor required for fruiting body development.

    Science.gov (United States)

    Masloff, S; Pöggeler, S; Kück, U

    1999-05-01

    During sexual morphogenesis, the filamentous ascomycete Sordaria macrospora differentiates into multicellular fruiting bodies called perithecia. Previously it has been shown that this developmental process is under polygenic control. To further understand the molecular mechanisms involved in fruiting body formation, we generated the protoperithecia forming mutant pro1, in which the normal development of protoperithecia into perithecia has been disrupted. We succeeded in isolating a cosmid clone from an indexed cosmid library, which was able to complement the pro1(-) mutation. Deletion analysis, followed by DNA sequencing, subsequently demonstrated that fertility was restored to the pro1 mutant by an open reading frame encoding a 689-amino-acid polypeptide, which we named PRO1. A region from this polypeptide shares significant homology with the DNA-binding domains found in fungal C6 zinc finger transcription factors, such as the GAL4 protein from yeast. However, other typical regions of C6 zinc finger proteins, such as dimerization elements, are absent in PRO1. The involvement of the pro1(+) gene in fruiting body development was further confirmed by trying to complement the mutant phenotype with in vitro mutagenized and truncated versions of the pro1 open reading frame. Southern hybridization experiments also indicated that pro1(+) homologues are present in other sexually propagating filamentous ascomycetes.

  3. Regulation of Nitrogen Metabolism by GATA Zinc Finger Transcription Factors in Yarrowia lipolytica

    Energy Technology Data Exchange (ETDEWEB)

    Pomraning, Kyle R.; Bredeweg, Erin L.; Baker, Scott E.

    2017-02-15

    ABSTRACT

    Fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeastYarrowia lipolytica, growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism inY. lipolytica. Deletion of the GATA transcription factor genesgzf3andgzf2resulted in nitrogen source-specific growth defects and greater accumulation of lipids when the cells were growing on a simple nitrogen source. Deletion ofgzf1, which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion ofgzf3results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, whilegzf2is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressormig1, including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism.

    IMPORTANCENitrogen source is

  4. Zinc fingers 1, 2, 5 and 6 of transcriptional regulator, PRDM4, are required for its nuclear localisation

    Energy Technology Data Exchange (ETDEWEB)

    Tunbak, Hale, E-mail: h.tunbak@ucl.ac.uk [The Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT (United Kingdom); Georgiou, Christiana, E-mail: christiana.georgiou.10@ucl.ac.uk [The Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT (United Kingdom); Guan, Cui, E-mail: c.guan@qmul.ac.uk [School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS (United Kingdom); Richardson, William David, E-mail: w.richardson@ucl.ac.uk [The Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT (United Kingdom); Chittka, Alexandra, E-mail: a.chittka@ucl.ac.uk [The Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT (United Kingdom)

    2016-05-27

    PRDM4 is a member of the PRDM family of transcriptional regulators which control various aspects of cellular differentiation and proliferation. PRDM proteins exert their biological functions both in the cytosol and the nucleus of cells. All PRDM proteins are characterised by the presence of two distinct structural motifs, the PR/SET domain and the zinc finger (ZF) motifs. We previously observed that deletion of all six zinc fingers found in PRDM4 leads to its accumulation in the cytosol, whereas overexpressed full length PRDM4 is found predominantly in the nucleus. Here, we investigated the requirements for single zinc fingers in the nuclear localisation of PRDM4. We demonstrate that ZF's 1, 2, 5 and 6 contribute to the accumulation of PRDM4 in the nucleus. Their effect is additive as deleting either ZF1-2 or ZF 5–6 redistributes PRDM4 protein from being almost exclusively nuclear to cytosolic and nuclear. We investigated the potential mechanism of nuclear shuttling of PRDM4 via the importin α/β-mediated pathway and find that PRDM4 nuclear targeting is independent of α/β-mediated nuclear import. -- Highlights: •Zinc fingers 1, 2, 5, and 6 are necessary for efficient nuclear localisation of PRDM4. •Zinc fingers 3 and 4 are dispensable for nuclear localisation of PRDM4. •Zinc knuckle is dispensable for nuclear localisation of PRDM4. •PRDM4 nuclear transport is independent of importin α/β-mediated pathway of nuclear import.

  5. Lead inhibition of DNA-binding mechanism of Cys(2)His(2) zinc finger proteins.

    Science.gov (United States)

    Hanas, J S; Rodgers, J S; Bantle, J A; Cheng, Y G

    1999-11-01

    The association of lead with chromatin in cells suggests that deleterious metal effects may in part be mediated through alterations in gene function. To elucidate if and how lead may alter DNA binding of cysteine-rich zinc finger proteins, lead ions were analyzed for their ability to alter the DNA binding mechanism of the Cys(2)His(2) zinc finger protein transcription factor IIIA (TFIIIA). As assayed by DNase I protection, the interaction of TFIIIA with the 50-bp internal control region of the 5S ribosomal gene was partially inhibited by 5 microM lead ions and completely inhibited by 10 to 20 microM lead ions. Preincubation of free TFIIIA with lead resulted in DNA-binding inhibition, whereas preincubation of a TFIIIA/5S RNA complex with lead did not result in DNA-binding inhibition. Because 5S RNA binds TFIIIA zinc fingers, this result is consistent with an inhibition mechanism via lead binding to zinc fingers. The complete loss of DNase I protection on the 5S gene indicates the mechanism of inhibition minimally involves the N-terminal fingers of TFIIIA. Inhibition was not readily reversible and occurred in the presence of an excess of beta-mercaptoethanol. Inhibition kinetics were fast, progressing to completion in approximately 5 min. Millimolar concentrations of sulfhydryl-specific arsenic ions were not inhibitory for TFIIIA binding. Micromolar concentrations of lead inhibited DNA binding by Sp1, another Cys(2)His(2) finger protein, but not by the nonfinger protein AP2. Inhibition of Cys(2)His(2) zinc finger transcription factors by lead ions at concentrations near those known to have deleterious physiological effects points to new molecular mechanisms for lead toxicity in promoting disease.

  6. Localized frustration and binding-induced conformational change in recognition of 5S RNA by TFIIIA zinc finger.

    Science.gov (United States)

    Tan, Cheng; Li, Wenfei; Wang, Wei

    2013-12-19

    Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

  7. Activation of transcriptional activities of AP-1 and SRE by a new zinc-finger protein ZNF641

    International Nuclear Information System (INIS)

    Qi Xingzhu; Li Yongqing; Xiao Jing; Yuan Wuzhou; Yan Yan; Wang Yuequn; Liang Shuyuan; Zhu Chuanbing; Chen Yingduan; Liu Mingyao; Wu Xiushan

    2006-01-01

    Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes in cell signal transduction connecting cell-surface receptors to critical regulatory targets within cells and control cell survival, adaptation, and proliferation. Previous studies revealed that zinc-finger proteins are involved in the regulation of the MAPK signaling pathways. Here, we report the identification and characterization of a novel human zinc-finger protein, ZNF641. The cDNA of ZNF641 is 4.9 kb, encoding 438 amino acids in the nucleus. The protein is highly conserved in evolution across different vertebrate species from mouse to human. Northern blot analysis indicates that ZNF641 is expressed in most of the examined human tissues, with a high level in skeletal muscle. Overexpression of pCMV-Tag2B-ZNF641 in the COS-7 cells activates the transcriptional activities of AP-1 and SRE. Deletion analysis indicates that the linker between KRAB box and C 2 H 2 -type zinc-fingers represents the basal activation domain. These results suggest that ZNF641 may be a positive regulator in MAPK-mediated signaling pathways that lead to the activation of AP-1 and SRE

  8. Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine.

    Science.gov (United States)

    Gersbach, Charles A; Perez-Pinera, Pablo

    2014-08-01

    New technologies have recently been developed to control the expression of human genes in their native genomic context by engineering synthetic transcription factors that can be targeted to any DNA sequence. The ability to precisely regulate any gene as it occurs naturally in the genome provides a means to address a variety of diseases and disorders. This approach also circumvents some of the traditional challenges of gene therapy. In this editorial, we review the technologies that have enabled targeted human gene activation, including the engineering of transcription factors based on zinc finger proteins, transcription activator-like effectors and the CRISPR/Cas9 system. Additionally, we highlight examples in which these methods have been developed for therapeutic applications and discuss challenges and opportunities.

  9. Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome

    Directory of Open Access Journals (Sweden)

    Böhm Siegfried

    2004-07-01

    Full Text Available Background The classical C2H2 zinc finger domain is involved in a wide range of functions and can bind to DNA, RNA and proteins. The comparison of zinc finger proteins in several eukaryotes has shown that there is a lot of lineage specific diversification and expansion. Although the number of characterized plant proteins that carry the classical C2H2 zinc finger motifs is growing, a systematic classification and analysis of a plant genome zinc finger gene set is lacking. Results We found through in silico analysis 176 zinc finger proteins in Arabidopsis thaliana that hence constitute the most abundant family of putative transcriptional regulators in this plant. Only a minority of 33 A. thaliana zinc finger proteins are conserved in other eukaryotes. In contrast, the majority of these proteins (81% are plant specific. They are derived from extensive duplication events and form expanded families. We assigned the proteins to different subgroups and families and focused specifically on the two largest and evolutionarily youngest families (A1 and C1 that are suggested to be primarily involved in transcriptional regulation. The newly defined family A1 (24 members comprises proteins with tandemly arranged zinc finger domains. Family C1 (64 members, earlier described as the EPF-family in Petunia, comprises proteins with one isolated or two to five dispersed fingers and a mostly invariant QALGGH motif in the zinc finger helices. Based on the amino acid pattern in these helices we could describe five different signature sequences prevalent in C1 zinc finger domains. We also found a number of non-finger domains that are conserved in these families. Conclusions Our analysis of the few evolutionarily conserved zinc finger proteins of A. thaliana suggests that most of them could be involved in ancient biological processes like RNA metabolism and chromatin-remodeling. In contrast, the majority of the unique A. thaliana zinc finger proteins are known or

  10. Zinc finger protein 521 overexpression increased transcript levels of ...

    Indian Academy of Sciences (India)

    2016-02-12

    Feb 12, 2016 ... Zinc finger protein 521 is highly expressed in brain, neural stem cells and early progenitors of the human .... Membranes were blocked for 1 h with 10% skim milk and ..... fat-like development of white fat and thermogenesis.

  11. The Promyelocytic Leukemia Zinc Finger Protein: Two Decades of Molecular Oncology

    International Nuclear Information System (INIS)

    Suliman, Bandar Ali; Xu, Dakang; Williams, Bryan Raymond George

    2012-01-01

    The promyelocytic leukemia zinc finger (PLZF) protein, also known as Zbtb16 or Zfp145, was first identified in a patient with acute promyelocytic leukemia, where a reciprocal chromosomal translocation t(11;17)(q23;q21) resulted in a fusion with the RARA gene encoding retinoic acid receptor alpha. The wild-type Zbtb16 gene encodes a transcription factor that belongs to the POK (POZ and Krüppel) family of transcriptional repressors. In addition to nine Krüppel-type sequence-specific zinc fingers, which make it a member of the Krüppel-like zinc finger protein family, the PLZF protein contains an N-terminal BTB/POZ domain and RD2 domain. PLZF has been shown to be involved in major developmental and biological processes, such as spermatogenesis, hind limb formation, hematopoiesis, and immune regulation. PLZF is localized mainly in the nucleus where it exerts its transcriptional repression function, and many post-translational modifications affect this ability and also have an impact on its cytoplasmic/nuclear dissociation. PLZF achieves its transcriptional regulation by binding to many secondary molecules to form large multi-protein complexes that bind to the regulatory elements in the promoter region of the target genes. These complexes are also capable of physically interacting with its target proteins. Recently, PLZF has become implicated in carcinogenesis as a tumor suppressor gene, since it regulates the cell cycle and apoptosis in many cell types. This review will examine the major advances in our knowledge of PLZF biological activities that augment its value as a therapeutic target, particularly in cancer and immunological diseases.

  12. Zinc-fingers and homeoboxes 1 (ZHX1) binds DNA methyltransferase (DNMT) 3B to enhance DNMT3B-mediated transcriptional repression

    International Nuclear Information System (INIS)

    Kim, Sung-Hak; Park, Jinah; Choi, Moon-Chang; Kim, Hwang-Phill; Park, Jung-Hyun; Jung, Yeonjoo; Lee, Ju-Hee; Oh, Do-Youn; Im, Seock-Ah; Bang, Yung-Jue; Kim, Tae-You

    2007-01-01

    DNA methyltransferases (DNMT) 3B is a de novo DNMT that represses transcription independent of DNMT activity. In order to gain a better insight into DNMT3B-mediated transcriptional repression, we performed a yeast two-hybrid analysis using DNMT3B as a bait. Of the various binding candidates, ZHX1, a member of zinc-finger and homeobox protein, was found to interact with DNMT3B in vivo and in vitro. N-terminal PWWP domain of DNMT3B was required for its interaction with homeobox motifs of ZHX1. ZHX1 contains nuclear localization signal at C-terminal homeobox motif, and both ZHX1 and DNMT3B were co-localized in nucleus. Furthermore, we found that ZHX1 enhanced the transcriptional repression mediated by DNMT3B when DNMT3B is directly targeted to DNA. These results showed for First the direct linkage between DNMT and zinc-fingers homeoboxes protein, leading to enhanced gene silencing by DNMT3B

  13. The solution structure of the N-terminal zinc finger of GATA-1 reveals a specific binding face for the transcriptional co-factor FOG

    International Nuclear Information System (INIS)

    Kowalski, K.; Czolij, R.; King, G.F.; Crossley, M.; Mackay, J.P.

    1999-01-01

    Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional 1H/15N NMR spectroscopy, and we use 1H/15N spin relaxation measurements to investigate its backbone dynamics. The structure consists of two distorted β-hairpins and a single α-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NF. Notably, this face is well removed from the putative DNA- binding face of the NF, an observation which is suggestive of simultaneous roles for the NF; that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions

  14. Expression and function of the zinc finger transcription factor Sp6-9 in the spider Parasteatoda tepidariorum.

    Science.gov (United States)

    Königsmann, Tatiana; Turetzek, Natascha; Pechmann, Matthias; Prpic, Nikola-Michael

    2017-11-01

    Zinc finger transcription factors of the Sp6-9 group are evolutionarily conserved in all metazoans and have important functions in, e.g., limb formation and heart development. The function of Sp6-9-related genes has been studied in a number of vertebrates and invertebrates, but data from chelicerates (spiders and allies) was lacking so far. We have isolated the ortholog of Sp6-9 from the common house spider Parasteatoda tepidariorum and the cellar spider Pholcus phalangioides. We show that the Sp6-9 gene in these spider species is expressed in the developing appendages thus suggesting a conserved role in limb formation. Indeed, RNAi with Sp6-9 in P. tepidariorum leads not only to strong limb defects, but also to the loss of body segments and head defects in more strongly affected animals. Together with a new expression domain in the early embryo, these data suggest that Sp6-9 has a dual role P. tepidariorum. The early role in head and body segment formation is not known from other arthropods, but the role in limb formation is evolutionarily highly conserved.

  15. Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression.

    Science.gov (United States)

    Lomniczi, Alejandro; Wright, Hollis; Castellano, Juan Manuel; Matagne, Valerie; Toro, Carlos A; Ramaswamy, Suresh; Plant, Tony M; Ojeda, Sergio R

    2015-12-16

    In primates, puberty is unleashed by increased GnRH release from the hypothalamus following an interval of juvenile quiescence. GWAS implicates Zinc finger (ZNF) genes in timing human puberty. Here we show that hypothalamic expression of several ZNFs decreased in agonadal male monkeys in association with the pubertal reactivation of gonadotropin secretion. Expression of two of these ZNFs, GATAD1 and ZNF573, also decreases in peripubertal female monkeys. However, only GATAD1 abundance increases when gonadotropin secretion is suppressed during late infancy. Targeted delivery of GATAD1 or ZNF573 to the rat hypothalamus delays puberty by impairing the transition of a transcriptional network from an immature repressive epigenetic configuration to one of activation. GATAD1 represses transcription of two key puberty-related genes, KISS1 and TAC3, directly, and reduces the activating histone mark H3K4me2 at each promoter via recruitment of histone demethylase KDM1A. We conclude that GATAD1 epitomizes a subset of ZNFs involved in epigenetic repression of primate puberty.

  16. Zinc finger transcription factors displaced SREBP proteins as the major Sterol regulators during Saccharomycotina evolution.

    Directory of Open Access Journals (Sweden)

    Sarah L Maguire

    2014-01-01

    Full Text Available In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs, which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1 and C. albicans (Cph2 have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1 and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina.

  17. Zinc Finger Transcription Factors Displaced SREBP Proteins as the Major Sterol Regulators during Saccharomycotina Evolution

    Science.gov (United States)

    Maguire, Sarah L.; Wang, Can; Holland, Linda M.; Brunel, François; Neuvéglise, Cécile; Nicaud, Jean-Marc; Zavrel, Martin; White, Theodore C.; Wolfe, Kenneth H.; Butler, Geraldine

    2014-01-01

    In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs), which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1) and C. albicans (Cph2) have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1) and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina. PMID:24453983

  18. Zinc-finger proteins in health and disease.

    Science.gov (United States)

    Cassandri, Matteo; Smirnov, Artem; Novelli, Flavia; Pitolli, Consuelo; Agostini, Massimiliano; Malewicz, Michal; Melino, Gerry; Raschellà, Giuseppe

    2017-01-01

    Zinc-finger proteins (ZNFs) are one of the most abundant groups of proteins and have a wide range of molecular functions. Given the wide variety of zinc-finger domains, ZNFs are able to interact with DNA, RNA, PAR (poly-ADP-ribose) and other proteins. Thus, ZNFs are involved in the regulation of several cellular processes. In fact, ZNFs are implicated in transcriptional regulation, ubiquitin-mediated protein degradation, signal transduction, actin targeting, DNA repair, cell migration, and numerous other processes. The aim of this review is to provide a comprehensive summary of the current state of knowledge of this class of proteins. Firstly, we describe the actual classification of ZNFs, their structure and functions. Secondly, we focus on the biological role of ZNFs in the development of organisms under normal physiological and pathological conditions.

  19. PML-associated repressor of transcription (PAROT), a novel KRAB-zinc finger repressor, is regulated through association with PML nuclear bodies

    International Nuclear Information System (INIS)

    Fleischer, Sandra; Wiemann, Stefan; Will, Hans; Hofmann, Thomas G.

    2006-01-01

    Promyelocytic leukemia nuclear bodies (PML-NBs) are implicated in transcriptional regulation. Here we identify a novel transcriptional repressor, PML-associated repressor of transcription (PAROT), which is regulated in its repressor activity through recruitment to PML-NBs. PAROT is a Krueppel-associated box ( KRAB) zinc-finger (ZNF) protein, which comprises an amino terminal KRAB-A and KRAB-B box, a linker domain and 8 tandemly repeated C 2 H 2 -ZNF motifs at its carboxy terminus. Consistent with its domain structure, when tethered to DNA, PAROT represses transcription, and this is partially released by the HDAC inhibitor trichostatin A. PAROT colocalizes with members of the heterochromatin protein 1 (HP1) family and with transcriptional intermediary factor-1β/KRAB-associated protein 1 (TIF-1β/KAP1), a transcriptional corepressor for the KRAB-ZNF family. Interestingly, PML isoform IV, in contrast to PML-III, efficiently recruits PAROT and TIF-1β from heterochromatin to PML-NBs. PML-NB recruitment of PAROT partially releases its transcriptional repressor activity, indicating that PAROT can be regulated through subnuclear compartmentalization. Taken together, our data identify a novel transcriptional repressor and provide evidence for its regulation through association with PML-NBs

  20. A Zinc-Finger-Family Transcription Factor, AbVf19, Is Required for the Induction of a Gene Subset Important for Virulence in Alternaria brassicicola

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, Akhil [Univ. of Hawaii, Manoa, HI (United States); Ohm, Robin A. [USDOE Joint Genome Inst., Walnut Creek, CA (United States); Oxiles, Lindsay [Univ. of Hawaii, Manoa, HI (United States); Brooks, Fred [Univ. of Hawaii, Manoa, HI (United States); Lawrence, Christopher B. [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Grigoriev, Igor V. [USDOE Joint Genome Inst., Walnut Creek, CA (United States); Cho, Yangrae [Univ. of Hawaii, Manoa, HI (United States)

    2011-10-26

    Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen with a broad host range within the family Brassicaceae. It produces secondary metabolites that marginally affect virulence. Cell wall degrading enzymes (CDWE) have been considered important for pathogenesis but none of them individually have been identified as significant virulence factors in A. brassicicola. In this study, knockout mutants of a gene, AbVf19, were created and produced considerably smaller lesions than the wild type on inoculated host plants. The presence of tandem zinc-finger domains in the predicted amino acid sequence and nuclear localization of AbVf19- reporter protein suggested that it was a transcription factor. Gene expression comparisons using RNA-seq identified 74 genes being downregulated in the mutant during a late stage of infection. Among the 74 downregulated genes, 28 were putative CWDE genes. These were hydrolytic enzyme genes that composed a small fraction of genes within each family of cellulases, pectinases, cutinases, and proteinases. The mutants grew slower than the wild type on an axenic medium with pectin as a major carbon source. This study demonstrated the existence and the importance of a transcription factor that regulates a suite of genes that are important for decomposing and utilizing plant material during the late stage of plant infection.

  1. ZNF649, a novel Kruppel type zinc-finger protein, functions as a transcriptional suppressor

    International Nuclear Information System (INIS)

    Yang Hong; Yuan Wuzhou; Wang Ying; Zhu Chuanbing; Liu Bisheng; Wang Yuequn; Yang, Dan; Li Yongqing; Wang Canding; Wu Xiushan; Liu Mingyao

    2005-01-01

    Cardiac differentiation involves a cascade of coordinated gene expression that regulates cell proliferation and matrix protein formation in a defined temporo-spatial manner. Many of the KRAB-ZFPs are involved in cardiac development or cardiovascular diseases. Here we report the identification and characterization of a novel human zinc-finger gene named ZNF649. The cDNA of ZNF649 is 3176 bp, encoding a protein of 505 amino acids in the nuclei. Northern blot analysis indicates that ZNF649 is expressed in most of the examined human adult and embryonic tissues. ZNF649 is a transcription suppressor when fused to GAL-4 DNA-binding domain and cotransfected with VP-16. Overexpression of ZNF649 in COS-7 cells inhibits the transcriptional activities of SRE and AP-1. Deletion analysis with a series of truncated fusion proteins indicates that the KRAB motif is a basal repression domain when the truncated fusion proteins were assayed for the transcriptional activities of SRE and AP-1. These results suggest that ZNF649 protein may act as a transcriptional repressor in mitogen-activated protein kinase signaling pathway to mediate cellular functions

  2. WRKY transcription factor superfamily: Structure, origin and functions

    African Journals Online (AJOL)

    terminal ends contain the WRKYGQR amino acid sequence and a zinc-finger motif. WRKY transcription factors can regulate the expression of target genes that contain the W-box elements (C/T)TGAC(C/T) in the promoter regions by specifically ...

  3. Sda1, a Cys2-His2 zinc finger transcription factor, is involved in polyol metabolism and fumonisin B1 production in Fusarium verticillioides.

    Directory of Open Access Journals (Sweden)

    Martha Malapi-Wight

    Full Text Available The ubiquitous ascomycete Fusarium verticillioides causes ear rot and stalk rot of maize, both of which reduce grain quality and yield. Additionally, F. verticillioides produces the mycotoxin fumonisin B1 (FB1 during infection of maize kernels, and thus potentially compromises human and animal health. The current knowledge is fragmentary regarding the regulation of FB1 biosynthesis, particularly when considering interplay with environmental factors such as nutrient availability. In this study, SDA1 of F. verticillioides, predicted to encode a Cys-2 His-2 zinc finger transcription factor, was shown to play a key role in catabolizing select carbon sources. Growth of the SDA1 knock-out mutant (Δsda1 was completely inhibited when sorbitol was the sole carbon source and was severely impaired when exclusively provided mannitol or glycerol. Deletion of SDA1 unexpectedly increased FB1 biosynthesis, but reduced arabitol and mannitol biosynthesis, as compared to the wild-type progenitor. Trichoderma reesei ACE1, a regulator of cellulase and xylanase expression, complemented the F. verticillioides Δsda1 mutant, which indicates that Ace1 and Sda1 are functional orthologs. Taken together, the data indicate that Sda1 is a transcriptional regulator of carbon metabolism and toxin production in F. verticillioides.

  4. The zinc finger transcription factor 191 is required for early embryonic development and cell proliferation

    International Nuclear Information System (INIS)

    Li Jianzhong; Chen Xia; Yang Hua; Wang Shuiliang; Guo Baoyu; Yu Long; Wang Zhugang; Fu Jiliang

    2006-01-01

    Human zinc finger protein 191 (ZNF191/ZNF24) was cloned and characterized as a SCAN family member, which shows 94% identity to its mouse homologue zinc finger protein 191 (Zfp191). ZNF191 can specifically interact with an intronic polymorphic TCAT repeat (HUMTH01) in the tyrosine hydroxylase (TH) gene. Allelic variations of HUMTH01 have been stated to have a quantitative silencing effect on TH gene expression and to correlate with quantitative and qualitative changes in the binding by ZNF191. Zfp191 is widely expressed during embryonic development and in multiple tissues and organs in adult. To investigate the functions of Zfp191 in vivo, we have used homologous recombination to generate mice that are deficient in Zfp191. Heterozygous Zfp191 +/- mice are normal and fertile. Homozygous Zfp191 -/- embryos are severely retarded in development and die at approximately 7.5 days post-fertilization. Unexpectedly, in Zfp191 -/- and Zfp191 +/- embryos, TH gene expression is not affected. Blastocyst outgrowth experiments and the RNA interference-mediated knockdown of ZNF191 in cultured cells revealed an essential role for Zfp191 in cell proliferation. In further agreement with this function, no viable Zfp191 -/- cell lines were obtained by derivation of embryonic stem (ES) cells from blastocysts of Zfp191 +/- intercrosses or by forced homogenotization of heterozygous ES cells at high concentrations of G418. These data show that Zfp191 is indispensable for early embryonic development and cell proliferation

  5. Utrophin up-regulation by an artificial transcription factor in transgenic mice.

    Directory of Open Access Journals (Sweden)

    Elisabetta Mattei

    2007-08-01

    Full Text Available Duchenne Muscular Dystrophy (DMD is a severe muscle degenerative disease, due to absence of dystrophin. There is currently no effective treatment for DMD. Our aim is to up-regulate the expression level of the dystrophin related gene utrophin in DMD, complementing in this way the lack of dystrophin functions. To this end we designed and engineered several synthetic zinc finger based transcription factors. In particular, we have previously shown that the artificial three zinc finger protein named Jazz, fused with the appropriate effector domain, is able to drive the transcription of a test gene from the utrophin promoter "A". Here we report on the characterization of Vp16-Jazz-transgenic mice that specifically over-express the utrophin gene at the muscular level. A Chromatin Immunoprecipitation assay (ChIP demonstrated the effective access/binding of the Jazz protein to active chromatin in mouse muscle and Vp16-Jazz was shown to be able to up-regulate endogenous utrophin gene expression by immunohistochemistry, western blot analyses and real-time PCR. To our knowledge, this is the first example of a transgenic mouse expressing an artificial gene coding for a zinc finger based transcription factor. The achievement of Vp16-Jazz transgenic mice validates the strategy of transcriptional targeting of endogenous genes and could represent an exclusive animal model for use in drug discovery and therapeutics.

  6. A conserved function of the zinc finger transcription factor Sp8/9 in allometric appendage growth in the milkweed bug Oncopeltus fasciatus.

    Science.gov (United States)

    Schaeper, Nina D; Prpic, Nikola-Michael; Wimmer, Ernst A

    2009-08-01

    The genes encoding the closely related zinc finger transcription factors Buttonhead (Btd) and D-Sp1 are expressed in the developing limb primordia of Drosophila melanogaster and are required for normal growth of the legs. The D-Sp1 homolog of the red flour beetle Tribolium castaneum, Sp8 (appropriately termed Sp8/9), is also required for the proper growth of the leg segments. Here we report on the isolation and functional study of the Sp8/9 gene from the milkweed bug Oncopeltus fasciatus. We show that Sp8/9 is expressed in the developing appendages throughout development and that the downregulation of Sp8/9 via RNAi leads to antennae, rostrum, and legs with shortened and fused segments. This supports a conserved role of Sp8/9 in allometric leg segment growth. However, all leg segments including the claws are present and the expression of the leg genes Distal-less, dachshund, and homothorax are proportionally normal, thus providing no evidence for a role of Sp8/9 in appendage specification.

  7. Effect of the linkers between the zinc fingers in zinc finger protein 809 on gene silencing and nuclear localization

    Energy Technology Data Exchange (ETDEWEB)

    Ichida, Yu, E-mail: ichida-y@ncchd.go.jp; Utsunomiya, Yuko; Onodera, Masafumi

    2016-03-18

    Zinc finger protein 809 (ZFP809) belongs to the Kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family and functions in repressing the expression of Moloney murine leukemia virus (MoMLV). ZFP809 binds to the primer-binding site (PBS)located downstream of the MoMLV-long terminal repeat (LTR) and induces epigenetic modifications at integration sites, such as repressive histone modifications and de novo DNA methylation. KRAB-ZFPs contain consensus TGEKP linkers between C2H2 zinc fingers. The phosphorylation of threonine residues within linkers leads to the inactivation of zinc finger binding to target sequences. ZFP809 also contains consensus linkers between zinc fingers. However, the function of ZFP809 linkers remains unknown. In the present study, we constructed ZFP809 proteins containing mutated linkers and examined their ability to silence transgene expression driven by MLV, binding ability to MLV PBS, and cellular localization. The results of the present study revealed that the linkers affected the ability of ZFP809 to silence transgene expression. Furthermore, this effect could be partly attributed to changes in the localization of ZFP809 proteins containing mutated linkers. Further characterization of ZFP809 linkers is required for understanding the functions and features of KRAB-ZFP-containing linkers. - Highlights: • ZFP809 has three consensus linkers between the zinc fingers. • Linkers are required for ZFP809 to silence transgene expression driven by MLV-LTR. • Linkers affect the precise nuclear localization of ZFP809.

  8. Genetic analysis of Kruppel-like zinc finger 11 variants in 5864 Danish individuals: potential effect on insulin resistance and modified signal transducer and activator of transcription-3 binding by promoter variant -1659G>C

    DEFF Research Database (Denmark)

    Gutiérrez-Aguilar, Ruth; Froguel, Philippe; Hamid, Yasmin H

    2008-01-01

    CONTEXT: The transcription factor Krüppel-like zinc finger 11 (KLF11) has been suggested to contribute to genetic risk of type 2 diabetes (T2D). Our previous results showed that four KLF11 variants, in strong linkage disequilibrium (LD block including +185 A>G/Gln62Arg and -1659 G>C) were...

  9. The Zinc Finger of Prolyl Hydroxylase Domain Protein 2 Is Essential for Efficient Hydroxylation of Hypoxia-Inducible Factor α.

    Science.gov (United States)

    Arsenault, Patrick R; Song, Daisheng; Chung, Yu Jin; Khurana, Tejvir S; Lee, Frank S

    2016-09-15

    Prolyl hydroxylase domain protein 2 (PHD2) (also known as EGLN1) is a key oxygen sensor in mammals that posttranslationally modifies hypoxia-inducible factor α (HIF-α) and targets it for degradation. In addition to its catalytic domain, PHD2 contains an evolutionarily conserved zinc finger domain, which we have previously proposed recruits PHD2 to the HSP90 pathway to promote HIF-α hydroxylation. Here, we provide evidence that this recruitment is critical both in vitro and in vivo We show that in vitro, the zinc finger can function as an autonomous recruitment domain to facilitate interaction with HIF-α. In vivo, ablation of zinc finger function by a C36S/C42S Egln1 knock-in mutation results in upregulation of the erythropoietin gene, erythrocytosis, and augmented hypoxic ventilatory response, all hallmarks of Egln1 loss of function and HIF stabilization. Hence, the zinc finger ordinarily performs a critical positive regulatory function. Intriguingly, the function of this zinc finger is impaired in high-altitude-adapted Tibetans, suggesting that their adaptation to high altitude may, in part, be due to a loss-of-function EGLN1 allele. Thus, these findings have important implications for understanding both the molecular mechanism of the hypoxic response and human adaptation to high altitude. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  10. A multiscale approach to simulating the conformational properties of unbound multi-C₂H₂ zinc finger proteins.

    Science.gov (United States)

    Liu, Lei; Wade, Rebecca C; Heermann, Dieter W

    2015-09-01

    The conformational properties of unbound multi-Cys2 His2 (mC2H2) zinc finger proteins, in which zinc finger domains are connected by flexible linkers, are studied by a multiscale approach. Three methods on different length scales are utilized. First, atomic detail molecular dynamics simulations of one zinc finger and its adjacent flexible linker confirmed that the zinc finger is more rigid than the flexible linker. Second, the end-to-end distance distributions of mC2H2 zinc finger proteins are computed using an efficient atomistic pivoting algorithm, which only takes excluded volume interactions into consideration. The end-to-end distance distribution gradually changes its profile, from left-tailed to right-tailed, as the number of zinc fingers increases. This is explained by using a worm-like chain model. For proteins of a few zinc fingers, an effective bending constraint favors an extended conformation. Only for proteins containing more than nine zinc fingers, is a somewhat compacted conformation preferred. Third, a mesoscale model is modified to study both the local and the global conformational properties of multi-C2H2 zinc finger proteins. Simulations of the CCCTC-binding factor (CTCF), an important mC2H2 zinc finger protein for genome spatial organization, are presented. © 2015 Wiley Periodicals, Inc.

  11. Krüppel-like factors: Three fingers in control

    Directory of Open Access Journals (Sweden)

    Swamynathan Shivalingappa K

    2010-04-01

    Full Text Available Abstract Krüppel-like factors (KLFs, members of the zinc-finger family of transcription factors capable of binding GC-rich sequences, have emerged as critical regulators of important functions all over the body. They are characterised by a highly conserved C-terminal DNA-binding motif containing three C2H2 zinc-finger domains, with variable N-terminal regulatory domains. Currently, there are 17 KLFs annotated in the human genome. In spite of their structural similarity to one another, the genes encoding different KLFs are scattered all over the genome. By virtue of their ability to activate and/or repress the expression of a large number of genes, KLFs regulate a diverse array of developmental events and cellular processes, such as erythropoiesis, cardiac remodelling, adipogenesis, maintenance of stem cells, epithelial barrier formation, control of cell proliferation and neoplasia, flow-mediated endothelial gene expression, skeletal and smooth muscle development, gluconeogenesis, monocyte activation, intestinal and conjunctival goblet cell development, retinal neuronal regeneration and neonatal lung development. Characteristic features, nomenclature, evolution and functional diversities of the human KLFs are reviewed here.

  12. Krüppel-like factors: three fingers in control.

    Science.gov (United States)

    Swamynathan, Shivalingappa K

    2010-04-01

    Krüppel-like factors (KLFs), members of the zinc-finger family of transcription factors capable of binding GC-rich sequences, have emerged as critical regulators of important functions all over the body. They are characterised by a highly conserved C-terminal DNA-binding motif containing three C2H2 zinc-finger domains, with variable N-terminal regulatory domains. Currently, there are 17 KLFs annotated in the human genome. In spite of their structural similarity to one another, the genes encoding different KLFs are scattered all over the genome. By virtue of their ability to activate and/or repress the expression of a large number of genes, KLFs regulate a diverse array of developmental events and cellular processes, such as erythropoiesis, cardiac remodelling, adipogenesis, maintenance of stem cells, epithelial barrier formation, control of cell proliferation and neoplasia, flow-mediated endothelial gene expression, skeletal and smooth muscle development, gluconeogenesis, monocyte activation, intestinal and conjunctival goblet cell development, retinal neuronal regeneration and neonatal lung development. Characteristic features, nomenclature, evolution and functional diversities of the human KLFs are reviewed here.

  13. Zinc finger protein 521 antagonizes early B-cell factor 1 and modulates the B-lymphoid differentiation of primary hematopoietic progenitors.

    Science.gov (United States)

    Mega, Tiziana; Lupia, Michela; Amodio, Nicola; Horton, Sarah J; Mesuraca, Maria; Pelaggi, Daniela; Agosti, Valter; Grieco, Michele; Chiarella, Emanuela; Spina, Raffaella; Moore, Malcolm A S; Schuringa, Jan Jacob; Bond, Heather M; Morrone, Giovanni

    2011-07-01

    Zinc finger protein 521 (EHZF/ZNF521) is a multi-functional transcription co-factor containing 30 zinc fingers and an amino-terminal motif that binds to the nucleosome remodelling and histone deacetylase (NuRD) complex. ZNF521 is believed to be a relevant player in the regulation of the homeostasis of the hematopoietic stem/progenitor cell compartment, however the underlying molecular mechanisms are still largely unknown. Here, we show that this protein plays an important role in the control of B-cell development by inhibiting the activity of early B-cell factor-1 (EBF1), a master factor in B-lineage specification. In particular, our data demonstrate that: (1) ZNF521 binds to EBF1 via its carboxyl-terminal portion and this interaction is required for EBF1 inhibition; (2) NuRD complex recruitment by ZNF521 is not essential for the inhibition of transactivation of EBF1-dependent promoters; (3) ZNF521 represses EBF1 target genes in a human B-lymphoid molecular context; and (4) RNAi-mediated silencing of ZNF521/Zfp521 in primary human and murine hematopoietic progenitors strongly enhances the generation of B-lymphocytes in vitro. Taken together, our data indicate that ZNF521 can antagonize B-cell development and lend support to the notion that it may contribute to conserve the multipotency of primitive lympho-myeloid progenitors by preventing or delaying their EBF1-driven commitment toward the B-cell lineage.

  14. ZifBASE: a database of zinc finger proteins and associated resources

    Directory of Open Access Journals (Sweden)

    Punetha Ankita

    2009-09-01

    Full Text Available Abstract Background Information on the occurrence of zinc finger protein motifs in genomes is crucial to the developing field of molecular genome engineering. The knowledge of their target DNA-binding sequences is vital to develop chimeric proteins for targeted genome engineering and site-specific gene correction. There is a need to develop a computational resource of zinc finger proteins (ZFP to identify the potential binding sites and its location, which reduce the time of in vivo task, and overcome the difficulties in selecting the specific type of zinc finger protein and the target site in the DNA sequence. Description ZifBASE provides an extensive collection of various natural and engineered ZFP. It uses standard names and a genetic and structural classification scheme to present data retrieved from UniProtKB, GenBank, Protein Data Bank, ModBase, Protein Model Portal and the literature. It also incorporates specialized features of ZFP including finger sequences and positions, number of fingers, physiochemical properties, classes, framework, PubMed citations with links to experimental structures (PDB, if available and modeled structures of natural zinc finger proteins. ZifBASE provides information on zinc finger proteins (both natural and engineered ones, the number of finger units in each of the zinc finger proteins (with multiple fingers, the synergy between the adjacent fingers and their positions. Additionally, it gives the individual finger sequence and their target DNA site to which it binds for better and clear understanding on the interactions of adjacent fingers. The current version of ZifBASE contains 139 entries of which 89 are engineered ZFPs, containing 3-7F totaling to 296 fingers. There are 50 natural zinc finger protein entries ranging from 2-13F, totaling to 307 fingers. It has sequences and structures from literature, Protein Data Bank, ModBase and Protein Model Portal. The interface is cross linked to other public

  15. Characterization and chondrocyte differentiation stage-specific expression of KRAB zinc-finger protein gene ZNF470

    International Nuclear Information System (INIS)

    Hering, Thomas M.; Kazmi, Najam H.; Huynh, Tru D.; Kollar, John; Xu, Laura; Hunyady, Aaron B.; Johnstone, Brian

    2004-01-01

    As part of a study to identify novel transcriptional regulators of chondrogenesis-related gene expression, we have cloned and characterized cDNA for zinc-finger protein 470 (ZNF470), the human ortholog of which encodes a 717 amino acid residue protein containing 17 Cys 2 His 2 zinc-finger domains, as well as KRAB-A and KRAB-B motifs. The cDNA library used to isolate the initial ZNF470 clone was prepared from human bone marrow-derived mesenchymal progenitor cells at an intermediate stage of chondrogenic differentiation. We have determined the intron-exon structure of the human ZNF470 gene, which has been mapped to a zinc-finger cluster in a known imprinted region of human chromosome 19q13.4. ZNF470 is expressed at high levels in human testis and is expressed at low or undetectible levels in other adult tissues. Human ZNF470 expressed in mammalian cells as an EGFP fusion protein localizes predominantly to the nucleus, consistent with a role in transcriptional regulation. ZNF470, analyzed by quantitative real time PCR, was transiently expressed before the maximal expression of COL2A1 during chondrogenic differentiation in vitro. We have also characterized the bovine ortholog of human ZNF470, which encodes a 508 amino acid residue protein having 10 zinc-finger domains. A bovine ZNF470 cDNA clone was used to examine expression of ZNF470 in bovine articular chondrocytes treated with retinoic acid to stimulate dedifferentiation. Bovine ZNF470 expression was undetectable in freshly isolated bovine articular chondrocytes, but was dramatically upregulated in dedifferentiated retinoic acid-treated chondrocytes. These results, in two model systems, suggest a possible role for ZNF470 in the regulation of chondrogenesis-specific gene expression

  16. Characterization of two novel nuclear BTB/POZ domain zinc finger isoforms. Association with differentiation of hippocampal neurons, cerebellar granule cells, and macroglia

    DEFF Research Database (Denmark)

    Mitchelmore, Cathy; Kjaerulff, Karen M; Pedersen, Hans C

    2002-01-01

    BTB/POZ (broad complex tramtrack bric-a-brac/poxvirus and zinc finger) zinc finger factors are a class of nuclear DNA-binding proteins involved in development, chromatin remodeling, and cancer. However, BTB/POZ domain zinc finger factors linked to development of the mammalian cerebral cortex......, cerebellum, and macroglia have not been described previously. We report here the isolation and characterization of two novel nuclear BTB/POZ domain zinc finger isoforms, designated HOF(L) and HOF(S), that are specifically expressed in early hippocampal neurons, cerebellar granule cells, and gliogenic...

  17. Luciferase-Zinc-Finger System for the Rapid Detection of Pathogenic Bacteria.

    Science.gov (United States)

    Shi, Chu; Xu, Qing; Ge, Yue; Jiang, Ling; Huang, He

    2017-08-09

    Rapid and reliable detection of pathogenic bacteria is crucial for food safety control. Here, we present a novel luciferase-zinc finger system for the detection of pathogens that offers rapid and specific profiling. The system, which uses a zinc-finger protein domain to probe zinc finger recognition sites, was designed to bind the amplified conserved regions of 16S rDNA, and the obtained products were detected using a modified luciferase. The luciferase-zinc finger system not only maintained luciferase activity but also allowed the specific detection of different bacterial species, with a sensitivity as low as 10 copies and a linear range from 10 to 10 4 copies per microliter of the specific PCR product. Moreover, the system is robust and rapid, enabling the simultaneous detection of 6 species of bacteria in artificially contaminated samples with excellent accuracy. Thus, we envision that our luciferase-zinc finger system will have far-reaching applications.

  18. The Transcriptional Repressive Activity of KRAB Zinc Finger Proteins Does Not Correlate with Their Ability to Recruit TRIM28.

    Directory of Open Access Journals (Sweden)

    Kristin E Murphy

    Full Text Available KRAB domain Zinc finger proteins are one of the most abundant families of transcriptional regulators in higher vertebrates. The prevailing view is that KRAB domain proteins function as potent transcriptional repressors by recruiting TRIM28 and promoting heterochromatin spreading. However, the extent to which all KRAB domain proteins are TRIM28-dependent transcriptional repressors is currently unclear. Our studies on mouse ZFP568 revealed that TRIM28 recruitment by KRAB domain proteins is not sufficient to warrant transcriptional repressive activity. By using luciferase reporter assays and yeast two-hybrid experiments, we tested the ability of ZFP568 and other mouse KRAB domain proteins to repress transcription and bind TRIM28. We found that some mouse KRAB domain proteins are poor transcriptional repressors despite their ability to recruit TRIM28, while others showed strong KRAB-dependent transcriptional repression, but no TRIM28 binding. Together, our results show that the transcriptional repressive activity of KRAB-ZNF proteins does not correlate with their ability to recruit TRIM28, and provide evidence that KRAB domains can regulate transcription in a TRIM28-independent fashion. Our findings challenge the current understanding of the molecular mechanisms used by KRAB domain proteins to control gene expression and highlight that a high percentage of KRAB domain proteins in the mouse genome differ from the consensus KRAB sequence at amino acid residues that are critical for TRIM28 binding and/or repressive activity.

  19. Promyelocytic leukaemia zinc finger maintains self-renewal of male germline stem cells (mGSCs) and its expression pattern in dairy goat testis.

    Science.gov (United States)

    Song, W; Zhu, H; Li, M; Li, N; Wu, J; Mu, H; Yao, X; Han, W; Liu, W; Hua, J

    2013-08-01

    Previous studies have shown that promyelocytic leukaemia zinc finger (PLZF) is a spermatogonia-specific transcription factor in the testis, required to regulate self-renewal and maintenance of the spermatogonia stem cell. Up to now, expression and function of PLZF in the goat testis has not been known. The objectives of this study were to investigate PLZF expression pattern in the dairy goat and its effect on male goat germline stem cell (mGSC) self-renewal and differentiation. Testis development and expression patterns of PLZF in the dairy goat were analysed by haematoxylin and eosin staining, immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, effects of PLZF overexpression on mGSC self-renewal and differentiation were evaluated by quantitative RT-PCR (QRT-PCR), immunofluorescence and BrdU incorporation assay. Promyelocytic leukaemia zinc finger was essential for dairy goat testis development and expression of several proliferation and pluripotency-associated proteins including OCT4, C-MYC were upregulated by PLZF overexpression. The study demonstrated that PLZF played a key role in maintaining self-renewal of mGSCs and its overexpression enhanced expression of proliferation-associated genes. Promyelocytic leukaemia zinc finger could function in the dairy goat as well as in other species in maintaining self-renewal of germline stem cells and this study provides a model to study the mechanism on self-renewal and differentiation of mGSCs in livestock. © 2013 John Wiley & Sons Ltd.

  20. The maize INDETERMINATE1 flowering time regulator defines a highly conserved zinc finger protein family in higher plants

    Directory of Open Access Journals (Sweden)

    Colasanti Joseph

    2006-06-01

    Full Text Available Abstract Background The maize INDETERMINATE1 gene, ID1, is a key regulator of the transition to flowering and the founding member of a transcription factor gene family that encodes a protein with a distinct arrangement of zinc finger motifs. The zinc fingers and surrounding sequence make up the signature ID domain (IDD, which appears to be found in all higher plant genomes. The presence of zinc finger domains and previous biochemical studies showing that ID1 binds to DNA suggests that members of this gene family are involved in transcriptional regulation. Results Comparison of IDD genes identified in Arabidopsis and rice genomes, and all IDD genes discovered in maize EST and genomic databases, suggest that ID1 is a unique member of this gene family. High levels of sequence similarity amongst all IDD genes from maize, rice and Arabidopsis suggest that they are derived from a common ancestor. Several unique features of ID1 suggest that it is a divergent member of the maize IDD family. Although no clear ID1 ortholog was identified in the Arabidopsis genome, highly similar genes that encode proteins with identity extending beyond the ID domain were isolated from rice and sorghum. Phylogenetic comparisons show that these putative orthologs, along with maize ID1, form a group separate from other IDD genes. In contrast to ID1 mRNA, which is detected exclusively in immature leaves, several maize IDD genes showed a broad range of expression in various tissues. Further, Western analysis with an antibody that cross-reacts with ID1 protein and potential orthologs from rice and sorghum shows that all three proteins are detected in immature leaves only. Conclusion Comparative genomic analysis shows that the IDD zinc finger family is highly conserved among both monocots and dicots. The leaf-specific ID1 expression pattern distinguishes it from other maize IDD genes examined. A similar leaf-specific localization pattern was observed for the putative ID1 protein

  1. ZFNGenome: A comprehensive resource for locating zinc finger nuclease target sites in model organisms

    Directory of Open Access Journals (Sweden)

    Voytas Daniel F

    2011-01-01

    Full Text Available Abstract Background Zinc Finger Nucleases (ZFNs have tremendous potential as tools to facilitate genomic modifications, such as precise gene knockouts or gene replacements by homologous recombination. ZFNs can be used to advance both basic research and clinical applications, including gene therapy. Recently, the ability to engineer ZFNs that target any desired genomic DNA sequence with high fidelity has improved significantly with the introduction of rapid, robust, and publicly available techniques for ZFN design such as the Oligomerized Pool ENgineering (OPEN method. The motivation for this study is to make resources for genome modifications using OPEN-generated ZFNs more accessible to researchers by creating a user-friendly interface that identifies and provides quality scores for all potential ZFN target sites in the complete genomes of several model organisms. Description ZFNGenome is a GBrowse-based tool for identifying and visualizing potential target sites for OPEN-generated ZFNs. ZFNGenome currently includes a total of more than 11.6 million potential ZFN target sites, mapped within the fully sequenced genomes of seven model organisms; S. cerevisiae, C. reinhardtii, A. thaliana, D. melanogaster, D. rerio, C. elegans, and H. sapiens and can be visualized within the flexible GBrowse environment. Additional model organisms will be included in future updates. ZFNGenome provides information about each potential ZFN target site, including its chromosomal location and position relative to transcription initiation site(s. Users can query ZFNGenome using several different criteria (e.g., gene ID, transcript ID, target site sequence. Tracks in ZFNGenome also provide "uniqueness" and ZiFOpT (Zinc Finger OPEN Targeter "confidence" scores that estimate the likelihood that a chosen ZFN target site will function in vivo. ZFNGenome is dynamically linked to ZiFDB, allowing users access to all available information about zinc finger reagents, such as the

  2. Engineering drought tolerant tomato plants over-expressing BcZAT12 gene encoding a C₂H₂ zinc finger transcription factor.

    Science.gov (United States)

    Rai, Avinash Chandra; Singh, Major; Shah, Kavita

    2013-01-01

    Efficient genetic transformation of cotyledonary explants of tomato (Solanum lycopersicum, cv. H-86, Kashi vishesh) was obtained. Disarmed Agrobacterium tumifaciens strain GV 3101 was used in conjugation with binary vector pBinAR containing a construct consisting of the coding sequence of the BcZAT12 gene under the regulatory control of the stress inducible Bclea1a promoter. ZAT12 encodes a C₂H₂ zinc finger protein which confers multiple abiotic stress tolerance to plants. Integration of ZAT12 gene into nuclear genome of individual kanamycin resistant transformed T₀ tomato lines was confirmed by Southern blot hybridization with segregation analysis of T(1) plants showing Mendelian inheritance of the transgene. Expression of ZAT12 in drought-stressed transformed tomato lines was verified in T₂ generation plants using RT-PCR. Of the six transformed tomato lines (ZT1-ZT6) the transformants ZT1 and ZT5 showed maximum expression of BcZAT12 gene transcripts when exposed to 7 days drought stress. Analysis of relative water content (RWC), electrolyte leakage (EL), chlorophyll colour index (CCI), H₂O₂ level and catalase activity suggested that tomato BcZAT12 transformants ZT1 and ZT5 have significantly increased levels of drought tolerance. These results suggest that BcZAT12 transformed tomato cv. H-86 has real potential for molecular breeding programs aimed at augmenting yield of tomato in regions affected with drought stress. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. ZNF383, a novel KRAB-containing zinc finger protein, suppresses MAPK signaling pathway

    International Nuclear Information System (INIS)

    Cao Lei; Wang Zhi; Zhu Chuanbing; Zhao Yulian; Yuan Wuzhou; Li Jing; Wang Yuequn; Ying Zhaochu; Li Yongqing; Yu Weishi; Wu Xiushan; Liu Mingyao

    2005-01-01

    Mitogen-activated protein kinases (MAPKs) are major components of pathways controlling embryogenesis, cell differentiation, cell proliferation, and cell death. One of the most explored functions of MAPK signaling is the regulation of gene expression by direct or indirect phosphorylation and subsequent activation of transcription factors. In this article, we isolated a novel KRAB-related zinc finger gene named ZNF383 from an early embryo heart cDNA library. The cDNA of ZNF383 is 2220 bp, encoding a protein of 475 amino acids. The protein is conserved in evolution across different species. Northern blot analysis indicates that a 2.2 kb transcript specific for ZNF383 is detected in most of the examined human adult and embryonic tissues with a higher level in skeletal muscle. In COS-7 cells, ZNF383 protein is localized to nucleus and cytoplasm. ZNF383 is a transcription repressor when fused to Gal-4 DNA-binding domain and cotransfected with VP-16. Deletion analysis indicates that the KRAB box of ZNF383 is responsible for the transcriptional repressor activity. Overexpression of ZNF383 in cells inhibits the transcriptional activities of AP-1 and SRE, suggesting that ZNF383 may act as a negative regulator in MAPK-mediated signaling pathways

  4. Structural and dynamical characterization of the Miz-1 zinc fingers 5-8 by solution-state NMR

    Energy Technology Data Exchange (ETDEWEB)

    Bernard, David; Bedard, Mikaeel; Bilodeau, Josee; Lavigne, Pierre, E-mail: pierre.lavigne@usherbrooke.ca [Universite de Sherbrooke, Departement de Biochimie, Faculte de Medecine et des Sciences de la Sante, Institut de Pharmacologie de Sherbrooke (Canada)

    2013-10-15

    Myc-interacting zinc finger protein-1 (Miz-1) is a BTB/POZ transcription factor that activates the transcription of cytostatic genes, such as p15{sup INK4B} or p21{sup CIP1}. The C-terminus of Miz-1 contains 13 consensus C{sub 2}H{sub 2} zinc finger domains (ZF). ZFs 1-4 have been shown to interact with SMAD3/4, while the remaining ZFs are expected to bind the promoters of target genes. We have noted unusual features in ZF 5 and the linker between ZFs 5 and 6. Indeed, a glutamate is found instead of the conserved basic residue two positions before the second zinc-coordinating histidine on the ZF 5 helix, and the linker sequence is DTDKE in place of the classical TGEKP sequence. In a canonical {beta}{beta}{alpha} fold, such unusual primary structure elements should cause severe electrostatic repulsions. In this context, we have characterized the structure and the dynamics of a Miz-1 construct comprising ZFs 5-8 (Miz 5-8) by solution-state NMR. Whilst ZFs 5, 7 and 8 were shown to adopt the classical {beta}{beta}{alpha} fold for C{sub 2}H{sub 2} ZFs, the number of long-range NOEs was insufficient to define a classical fold for ZF 6. We show by using {sup 15}N-relaxation dispersion experiments that this lack of NOEs is due to the presence of extensive motions on the {mu}s-ms timescale. Since this negatively charged region would have to be located near the phosphodiester backbone in a DNA complex, we propose that in addition to promoting conformational searches, it could serve as a hinge region to keep ZFs 1-4 away from DNA.

  5. Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers

    Directory of Open Access Journals (Sweden)

    Muriaux Delphine

    2007-08-01

    Full Text Available Abstract Background The HIV-1 nucleocapsid protein (NC is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT, gRNA dimerization and packaging, and virion assembly. Results We previously reported a role for the first NC zinc finger in virion structure and replication 1. To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired. Conclusion These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses.

  6. A novel zinc finger protein Zfp277 mediates transcriptional repression of the Ink4a/arf locus through polycomb repressive complex 1

    DEFF Research Database (Denmark)

    Negishi, Masamitsu; Saraya, Atsunori; Mochizuki, Shinobu

    2010-01-01

    . METHODOLOGY/PRINCIPAL FINDINGS: We examined the function of Zinc finger domain-containing protein 277 (Zfp277), a novel zinc finger protein that interacts with the PcG protein Bmi1. Zfp277 binds to the Ink4a/Arf locus in a Bmi1-independent manner and interacts with polycomb repressor complex (PRC) 1 through...... is essential for the recruitment of PRC1 to the Ink4a/Arf locus. Our findings also highlight dynamic regulation of both Zfp277 and PcG proteins by the oxidative stress pathways....

  7. Functional analysis of a novel KRAB/C2H2 zinc finger protein Mipu1

    International Nuclear Information System (INIS)

    Jiang, Lei; Tang, Daolin; Wang, Kangkai; Zhang, Huali; Yuan, Can; Duan, Dayue; Xiao, Xianzhong

    2007-01-01

    A novel rat gene, Mipu1, encodes a 608 amino acid protein with an amino-terminal KRAB domain and 14 carboxyl-terminal C 2 H 2 zinc finger motifs. Mipu1 is localized to the nucleus through its KRAB domain or the linker adjacent to its zinc finger region. Using the GST-Mipu1 bound to glutathione-Sepharose beads, a consensus putative DNA binding site (5'-TGTCTTATCGAA-3') was extracted from a random oligonucleotide library. EMSA and target detection assay showed that the probe containing the putative site can bind to purified GST-Mipu1 fusion protein. The oligonucleotide containing the putative site was inserted into the pGL3-promotor vector to produce a reporter construct. The expression of reporter gene was repressed by overexpression of Mipu1 in a dose-dependent manner. Mutation analysis of the consensus sequence indicated that the repression mediated by Mipu1 is sequence-dependent. These results suggest that Mipu1 is a nuclear protein, which functions as a transcriptional repressor

  8. ZNF307, a novel zinc finger gene suppresses p53 and p21 pathway

    International Nuclear Information System (INIS)

    Li Jing; Wang Yuequn; Fan Xiongwei; Mo Xiaoyang; Wang Zequn; Li Yongqing; Yin Zhaochu; Deng Yun; Luo Na; Zhu Chuanbing; Liu Mingyao; Ma Qian; Ocorr, Karen; Yuan Wuzhou; Wu Xiushan

    2007-01-01

    We have cloned a novel KRAB-related zinc finger gene, ZNF307, encoding a protein of 545 aa. ZNF307 is conserved across species in evolution and is differentially expressed in human adult and fetal tissues. The fusion protein of EGFP-ZNF307 localizes in the nucleus. Transcriptional activity assays show ZNF307 suppresses transcriptional activity of L8G5-luciferase. Overexpressing ZNF307 in different cell lines also inhibits the transcriptional activities of p53 and p21. Moreover, ZNF307 works by reducing the p53 protein level and p53 protein reduction is achieved by increasing transcription of MDM2 and EP300. ZNF307 might suppress p53-p21 pathway through activating MDM2 and EP300 expression and inducing p53 degradation

  9. Herpes simplex virus induces the marked up-regulation of the zinc finger transcriptional factor INSM1, which modulates the expression and localization of the immediate early protein ICP0

    Directory of Open Access Journals (Sweden)

    Kimura Hiroshi

    2011-05-01

    Full Text Available Abstract Background Herpes simplex viruses (HSVs rapidly shut off macromolecular synthesis in host cells. In contrast, global microarray analyses have shown that HSV infection markedly up-regulates a number of host cell genes that may play important roles in HSV-host cell interactions. To understand the regulatory mechanisms involved, we initiated studies focusing on the zinc finger transcription factor insulinoma-associated 1 (INSM1, a host cell protein markedly up-regulated by HSV infection. Results INSM1 gene expression in HSV-1-infected normal human epidermal keratinocytes increased at least 400-fold 9 h after infection; INSM1 promoter activity was also markedly stimulated. Expression and subcellular localization of the immediate early HSV protein ICP0 was affected by INSM1 expression, and chromatin immunoprecipitation (ChIP assays revealed binding of INSM1 to the ICP0 promoter. Moreover, the role of INSM1 in HSV-1 infection was further clarified by inhibition of HSV-1 replication by INSM1-specific siRNA. Conclusions The results suggest that INSM1 up-regulation plays a positive role in HSV-1 replication, probably by binding to the ICP0 promoter.

  10. Two short basic sequences surrounding the zinc finger of nucleocapsid protein NCp10 of Moloney murine leukemia virus are critical for RNA annealing activity.

    Science.gov (United States)

    De Rocquigny, H; Ficheux, D; Gabus, C; Allain, B; Fournie-Zaluski, M C; Darlix, J L; Roques, B P

    1993-02-25

    The 56 amino acid nucleocapsid protein (NCp10) of Moloney Murine Leukemia Virus, contains a CysX2CysX4HisX4Cys zinc finger flanked by basic residues. In vitro NCp10 promotes genomic RNA dimerization, a process most probably linked to genomic RNA packaging, and replication primer tRNA(Pro) annealing to the initiation site of reverse transcription. To characterize the amino-acid sequences involved in the various functions of NCp10, we have synthesized by solid phase method the native protein and a series of derived peptides shortened at the N- or C-terminus with or without the zinc finger domain. In the latter case, the two parts of the protein were linked by a Glycine - Glycine spacer. The in vitro studies of these peptides show that nucleic acid annealing activities of NCp10 do not require a zinc finger but are critically dependent on the presence of specific sequences located on each side of the CCHC domain and containing proline and basic residues. Thus, deletion of 11R or 49PRPQT, of the fully active 29 residue peptide 11RQGGERRRSQLDRDGGKKPRGPRGPRPQT53 leads to a complete loss of NCp10 activity. Therefore it is proposed that in NCp10, the zinc finger directs the spatial recognition of the target RNAs by the basic domains surrounding the zinc finger.

  11. Role of protein structure and the role of individual fingers in zinc finger protein-DNA recognition: a molecular dynamics simulation study and free energy calculations

    Science.gov (United States)

    Hamed, Mazen Y.

    2018-05-01

    Molecular dynamics and MM_GBSA energy calculations on various zinc finger proteins containing three and four fingers bound to their target DNA gave insights into the role of each finger in the DNA binding process as part of the protein structure. The wild type Zif 268 (PDB code: 1AAY) gave a ΔG value of - 76.1 (14) kcal/mol. Zinc fingers ZF1, ZF2 and ZF3 were mutated in one experiment and in another experiment one finger was cut and the rest of the protein was studied for binding. The ΔΔG values for the Zinc Finger protein with both ZF1 and ZF2 mutated was + 80 kcal/mol, while mutating only ZF1 the ΔΔG value was + 52 kcal/mol (relative to the wild type). Cutting ZF3 and studying the protein consisting only of ZF1 linked to ZF2 gave a ΔΔG value of + 68 kcal/mol. Upon cutting ZF1, the resulting ZF2 linked to ZF3 protein gave a ΔΔG value of + 41 kcal/mol. The above results shed light on the importance of each finger in the binding process, especially the role of ZF1 as the anchoring finger followed in importance by ZF2 and ZF3. The energy difference between the binding of the wild type protein Zif268 (1AAY) and that for individual finger binding to DNA according to the formula: ΔΔGlinkers, otherstructuralfactors = ΔGzif268 - (ΔGF1+F2+F3) gave a value = - 44.5 kcal/mol. This stabilization can be attributed to the contribution of linkers and other structural factors in the intact protein in the DNA binding process. DNA binding energies of variant proteins of the wild type Zif268 which differ in their ZF1 amino acid sequence gave evidence of a good relationship between binding energy and recognition and specificity, this finding confirms the reported vital role of ZF1 in the ZF protein scanning and anchoring to the target DNA sequence. The role of hydrogen bonds in both specific and nonspecific amino acid-DNA contacts is discussed in relation to mutations. The binding energies of variant Zinc Finger proteins confirmed the role of ZF1 in the recognition

  12. Finger millet (Eleucine coracana) flour as a vehicle for fortification with zinc.

    Science.gov (United States)

    Tripathi, Bhumika; Platel, Kalpana

    2010-01-01

    Millets, being less expensive compared to cereals and the staple for the poorer sections of population, could be the choice for fortification with micronutrients such as zinc. In view of this, finger millet, widely grown and commonly consumed in southern India, was explored as a vehicle for fortification with zinc in this investigation. Finger millet flour fortified with either zinc oxide or zinc stearate so as to provide 50mg zinc per kg flour, was specifically examined for the bioaccessibility of the fortified mineral, as measured by in vitro simulated gastrointestinal digestion procedure and storage stability. Addition of the zinc salts increased the bioaccessible zinc content by 1.5-3 times that of the unfortified flour. Inclusion of EDTA along with the fortified salt significantly enhanced the bioaccessibility of zinc from the fortified flours, the increase being three-fold. Inclusion of citric acid along with the zinc salt and EDTA during fortification did not have any additional beneficial effect on zinc bioaccessiblity. Moisture and free fatty acid contents of the stored fortified flours indicated the keeping quality of the same, up to 60 days. Both zinc oxide and zinc stearate were equally effective as fortificants, when used in combination with EDTA as a co-fortificant. The preparation of either roti or dumpling from the fortified flours stored up to 60 days did not result in any significant compromise in the bioaccessible zinc content. Thus, the present study has revealed that finger millet flour can effectively be used as a vehicle for zinc fortification to derive additional amounts of bioaccessible zinc, with reasonably good storage stability, to combat zinc deficiency. Copyright 2009 Elsevier GmbH. All rights reserved.

  13. Investigating the DNA-binding ability of GATA-1-N-terminal zinc finger

    International Nuclear Information System (INIS)

    Wong, R.; Newton, A.; Crossley, M.; Mackay, J.

    2001-01-01

    Erythroid transcription factor GATA-1 interacts with both DNA and other proteins through its zinc finger domains (ZnFs). While it has been known for me time that the C-terminal ZnF binds DNA at GATA sites, only recently has it been observed that the N-terminal finger (NF) is capable of interacting with GATC sites. Further, a number of naturally occurring mutations in NF (V205M, G208S, R216Q, D218G) that lead to anaemia and thrombocytopenia have been identified. We are interested in characterising the NF-DNA interaction and determining the effects of mutation upon this interaction. Using nuclear magnetic resonance (NMR) spectroscopy, we have observed an interaction between recombinant NF and a 16-mer DNA duplex containing a core GATC sequence. This result forms the basis from which residues in NF involved in DNA binding can be identified, and work is being carried out to improve the quality of the NMR data with the aim of determining the solution structure of the NF-DNA complex. The DNA-binding affinity of both wild-type and mutant NFs mentioned above is also being investigated using isothermal titration calorimetry. These data suggest that the strength of the interaction between NF and the 16-mer DNA duplex is in the sub-micromolar range, and comparisons between the DNA-binding affinities of the NF mutants are being made. Together, these studies will help us to understand how GATA-1 acts as a transcriptional regulator and how mutations in NF domain of GATA-1 may lead to blood disorders

  14. A plant EPF-type zinc-finger protein, CaPIF1, involved in defence against pathogens.

    Science.gov (United States)

    Oh, Sang-Keun; Park, Jeong Mee; Joung, Young Hee; Lee, Sanghyeob; Chung, Eunsook; Kim, Soo-Yong; Yu, Seung Hun; Choi, Doil

    2005-05-01

    SUMMARY To understand better the defence responses of plants to pathogen attack, we challenged hot pepper plants with bacterial pathogens and identified transcription factor-encoding genes whose expression patterns were altered during the subsequent hypersensitive response. One of these genes, CaPIF1 (Capsicum annuum Pathogen-Induced Factor 1), was characterized further. This gene encodes a plant-specific EPF-type protein that contains two Cys(2)/His(2) zinc fingers. CaPIF1 expression was rapidly and specifically induced when pepper plants were challenged with bacterial pathogens to which they are resistant. In contrast, challenge with a pathogen to which the plants are susceptible only generated weak CaPIF1 expression. CaPIF1 expression was also strongly induced in pepper leaves by the exogenous application of ethephon, an ethylene-releasing compound, and salicylic acid, whereas methyl jasmonate had only moderate effects. CaPIF1 localized to the nuclei of onion epidermis when expressed as a CaPIF1-smGFP fusion protein. Transgenic tobacco plants over-expressing CaPIF1 driven by the CaMV 35S promoter showed increased resistance to challenge with a tobacco-specific pathogen or non-host bacterial pathogens. These plants also showed constitutive up-regulation of multiple defence-related genes. Moreover, virus-induced silencing of the CaPIF1 orthologue in Nicotiana benthamiana enhanced susceptibility to the same host or non-host bacterial pathogens. These observations provide evidence that an EPF-type Cys(2)/His(2) zinc-finger protein plays a crucial role in the activation of the pathogen defence response in plants.

  15. Transcription factor ZBED6 mediates IGF2 gene expression by regulating promoter activity and DNA methylation in myoblasts

    Science.gov (United States)

    Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were up regulated during C2C12 differentiation. The IGF2 expression levels wer...

  16. Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers.

    Science.gov (United States)

    De Rocquigny, H; Gabus, C; Vincent, A; Fournié-Zaluski, M C; Roques, B; Darlix, J L

    1992-07-15

    The nucleocapsid (NC) of human immunodeficiency virus type 1 consists of a large number of NC protein molecules, probably wrapping the dimeric RNA genome within the virion inner core. NC protein is a gag-encoded product that contains two zinc fingers flanked by basic residues. In human immunodeficiency virus type 1 virions, NCp15 is ultimately processed into NCp7 and p6 proteins. During virion assembly the retroviral NC protein is necessary for core formation and genomic RNA encapsidation, which are essential for virus infectivity. In vitro NCp15 activates viral RNA dimerization, a process most probably linked in vivo to genomic RNA packaging, and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. To characterize the domains of human immunodeficiency virus type 1 NC protein necessary for its various functions, the 72-amino acid NCp7 and several derived peptides were synthesized in a pure form. We show here that synthetic NCp7 with or without the two zinc fingers has the RNA annealing activities of NCp15. Further deletions of the N-terminal 12 and C-terminal 8 amino acids, leading to a 27-residue peptide lacking the finger domains, have little or no effect on NC protein activity in vitro. However deletion of short sequences containing basic residues flanking the first finger leads to a complete loss of NC protein activity. It is proposed that the basic residues and the zinc fingers cooperate to select and package the genomic RNA in vivo. Inhibition of the viral RNA binding and annealing activities associated with the basic residues flanking the first zinc finger of NC protein could therefore be used as a model for the design of antiviral agents.

  17. Functional Profiling of Transcription Factor Genes in Neurospora crassa

    Directory of Open Access Journals (Sweden)

    Alexander J. Carrillo

    2017-09-01

    Full Text Available Regulation of gene expression by DNA-binding transcription factors is essential for proper control of growth and development in all organisms. In this study, we annotate and characterize growth and developmental phenotypes for transcription factor genes in the model filamentous fungus Neurospora crassa. We identified 312 transcription factor genes, corresponding to 3.2% of the protein coding genes in the genome. The largest class was the fungal-specific Zn2Cys6 (C6 binuclear cluster, with 135 members, followed by the highly conserved C2H2 zinc finger group, with 61 genes. Viable knockout mutants were produced for 273 genes, and complete growth and developmental phenotypic data are available for 242 strains, with 64% possessing at least one defect. The most prominent defect observed was in growth of basal hyphae (43% of mutants analyzed, followed by asexual sporulation (38%, and the various stages of sexual development (19%. Two growth or developmental defects were observed for 21% of the mutants, while 8% were defective in all three major phenotypes tested. Analysis of available mRNA expression data for a time course of sexual development revealed mutants with sexual phenotypes that correlate with transcription factor transcript abundance in wild type. Inspection of this data also implicated cryptic roles in sexual development for several cotranscribed transcription factor genes that do not produce a phenotype when mutated.

  18. Functional promoter variant in zinc finger protein 202 predicts severe atherosclerosis and ischemic heart disease

    DEFF Research Database (Denmark)

    Frikke-Schmidt, R.; Nordestgaard, Børge; Grande, Peer

    2008-01-01

    Objectives This study was designed to test the hypotheses that single nucleotide polymorphisms ( SNPs), in zinc finger protein 202 ( ZNF202), predict severe atherosclerosis and ischemic heart disease ( IHD). Background ZNF202 is a transcriptional repressor controlling promoter elements in genes...... involved in vascular maintenance and lipid metabolism. Methods We first determined genotype association for 9 ZNF202 SNPs with severe atherosclerosis ( ankle brachial index >0.7 vs. ...,998 controls. Finally, we determined whether g. -660A>G altered transcriptional activity of the ZNF202 promoter in vitro. Results Cross-sectionally, ZNF202 g. -660 GG versus AA homozygosity predicted an odds ratio for severe atherosclerosis of 2.01 ( 95% confidence interval [CI]: 1.34 to 3.01). Prospectively...

  19. Genomic structure and cloning of two transcript isoforms of human Sp8.

    NARCIS (Netherlands)

    M.A. Milona (Maria-athina); J.E. Gough (Julie); A.J. Edgar (Alasdair)

    2004-01-01

    textabstractBACKGROUND: The Specificity proteins (Sp) are a family of transcription factors that have three highly conserved zinc-fingers located towards the carboxy-terminal that bind GC-boxes and assist in the initiation of gene transcription. Human Sp1-7 genes have been

  20. DUF581 is plant specific FCS-like zinc finger involved in protein-protein interaction.

    Directory of Open Access Journals (Sweden)

    Muhammed Jamsheer K

    Full Text Available Zinc fingers are a ubiquitous class of protein domain with considerable variation in structure and function. Zf-FCS is a highly diverged group of C2-C2 zinc finger which is present in animals, prokaryotes and viruses, but not in plants. In this study we identified that a plant specific domain of unknown function, DUF581 is a zf-FCS type zinc finger. Based on HMM-HMM comparison and signature motif similarity we named this domain as FCS-Like Zinc finger (FLZ domain. A genome wide survey identified that FLZ domain containing genes are bryophytic in origin and this gene family is expanded in spermatophytes. Expression analysis of selected FLZ gene family members of A. thaliana identified an overlapping expression pattern suggesting a possible redundancy in their function. Unlike the zf-FCS domain, the FLZ domain found to be highly conserved in sequence and structure. Using a combination of bioinformatic and protein-protein interaction tools, we identified that FLZ domain is involved in protein-protein interaction.

  1. Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

    Science.gov (United States)

    In, K H; Asano, K; Beier, D; Grobholz, J; Finn, P W; Silverman, E K; Silverman, E S; Collins, T; Fischer, A R; Keith, T P; Serino, K; Kim, S W; De Sanctis, G T; Yandava, C; Pillari, A; Rubin, P; Kemp, J; Israel, E; Busse, W; Ledford, D; Murray, J J; Segal, A; Tinkleman, D; Drazen, J M

    1997-03-01

    Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

  2. The unique N-terminal zinc finger of synaptotagmin-like protein 4 reveals FYVE structure.

    Science.gov (United States)

    Miyamoto, Kazuhide; Nakatani, Arisa; Saito, Kazuki

    2017-12-01

    Synaptotagmin-like protein 4 (Slp4), expressed in human platelets, is associated with dense granule release. Slp4 is comprised of the N-terminal zinc finger, Slp homology domain, and C2 domains. We synthesized a compact construct (the Slp4N peptide) corresponding to the Slp4 N-terminal zinc finger. Herein, we have determined the solution structure of the Slp4N peptide by nuclear magnetic resonance (NMR). Furthermore, experimental, chemical modification of Cys residues revealed that the Slp4N peptide binds two zinc atoms to mediate proper folding. NMR data showed that eight Cys residues coordinate zinc atoms in a cross-brace fashion. The Simple Modular Architecture Research Tool database predicted the structure of Slp4N as a RING finger. However, the actual structure of the Slp4N peptide adopts a unique C 4 C 4 -type FYVE fold and is distinct from a RING fold. To create an artificial RING finger (ARF) with specific ubiquitin-conjugating enzyme (E2)-binding capability, cross-brace structures with eight zinc-ligating residues are needed as the scaffold. The cross-brace structure of the Slp4N peptide could be utilized as the scaffold for the design of ARFs. © 2017 The Protein Society.

  3. Characterization of the SUMO-binding activity of the myeloproliferative and mental retardation (MYM-type zinc fingers in ZNF261 and ZNF198.

    Directory of Open Access Journals (Sweden)

    Catherine M Guzzo

    Full Text Available SUMO-binding proteins interact with SUMO modified proteins to mediate a wide range of functional consequences. Here, we report the identification of a new SUMO-binding protein, ZNF261. Four human proteins including ZNF261, ZNF198, ZNF262, and ZNF258 contain a stretch of tandem zinc fingers called myeloproliferative and mental retardation (MYM-type zinc fingers. We demonstrated that MYM-type zinc fingers from ZNF261 and ZNF198 are necessary and sufficient for SUMO-binding and that individual MYM-type zinc fingers function as SUMO-interacting motifs (SIMs. Our binding studies revealed that the MYM-type zinc fingers from ZNF261 and ZNF198 interact with the same surface on SUMO-2 recognized by the archetypal consensus SIM. We also present evidence that MYM-type zinc fingers in ZNF261 contain zinc, but that zinc is not required for SUMO-binding. Immunofluorescence microscopy studies using truncated fragments of ZNF198 revealed that MYM-type zinc fingers of ZNF198 are necessary for localization to PML-nuclear bodies (PML-NBs. In summary, our studies have identified and characterized the SUMO-binding activity of the MYM-type zinc fingers in ZNF261 and ZNF198.

  4. Engineering and Application of Zinc Finger Proteins and TALEs for Biomedical Research.

    Science.gov (United States)

    Kim, Moon-Soo; Kini, Anu Ganesh

    2017-08-01

    Engineered DNA-binding domains provide a powerful technology for numerous biomedical studies due to their ability to recognize specific DNA sequences. Zinc fingers (ZF) are one of the most common DNA-binding domains and have been extensively studied for a variety of applications, such as gene regulation, genome engineering and diagnostics. Another novel DNA-binding domain known as a transcriptional activator-like effector (TALE) has been more recently discovered, which has a previously undescribed DNA-binding mode. Due to their modular architecture and flexibility, TALEs have been rapidly developed into artificial gene targeting reagents. Here, we describe the methods used to design these DNA-binding proteins and their key applications in biomedical research.

  5. Expression of zinc finger E-box-binding homeobox factor 1 in epithelial ovarian cancer: A clinicopathological analysis of 238 patients

    OpenAIRE

    LI, XIUFANG; HUANG, RUIXIA; LI, RUTH HOLM; TROPE, CLAES G.; NESLAND, JAHN M.; SUO, ZHENHE

    2015-01-01

    A growing body of evidence indicates that aberrant activation of epithelial-to-mesenchymal transition (EMT) plays a key role in tumor cell invasion and metastasis. Zinc finger E-box-binding homeobox factor 1 (ZEB1), as a crucial mediator of EMT, contributes to the malignant progression of various epithelial tumors. To determine whether ZEB1 is involved in the progression of ovarian cancer, we immunohistochemically evaluated the expression of ZEB1 in 238 cases of epithelial ovarian cancer (EOC...

  6. Solution NMR characterization of Sgf73(1-104) indicates that Zn ion is required to stabilize zinc finger motif

    International Nuclear Information System (INIS)

    Lai, Chaohua; Wu, Minhao; Li, Pan; Shi, Chaowei; Tian, Changlin; Zang, Jianye

    2010-01-01

    Zinc finger motif contains a zinc ion coordinated by several conserved amino acid residues. Yeast Sgf73 protein was identified as a component of SAGA (Spt/Ada/Gcn5 acetyltransferase) multi-subunit complex and Sgf73 protein was known to contain two zinc finger motifs. Sgf73(1-104), containing the first zinc finger motif, was necessary to modulate the deubiquitinase activity of SAGA complex. Here, Sgf73(1-104) was over-expressed using bacterial expression system and purified for solution NMR (nuclear magnetic resonance) structural studies. Secondary structure and site-specific relaxation analysis of Sgf73(1-104) were achieved after solution NMR backbone assignment. Solution NMR and circular dichroism analysis of Sgf73(1-104) after zinc ion removal using chelation reagent EDTA (ethylene-diamine-tetraacetic acid) demonstrated that zinc ion was required to maintain stable conformation of the zinc finger motif.

  7. The zinc fingers of the Small Optic Lobes (SOL) calpain bind polyubiquitin.

    Science.gov (United States)

    Hastings, Margaret H; Qiu, Alvin; Zha, Congyao; Farah, Carole A; Mahdid, Yacine; Ferguson, Larissa; Sossin, Wayne S

    2018-05-28

    The Small Optic Lobes (SOL) calpain is a highly conserved member of the calpain family expressed in the nervous system. A dominant negative form of the SOL calpain inhibited consolidation of one form of synaptic plasticity, non-associative facilitation, in sensory-motor neuronal cultures in Aplysia, presumably by inhibiting cleavage of protein kinase Cs (PKCs) into constitutively active protein kinase Ms (PKMs) (Hu et al, 2017a). SOL calpains have a conserved set of 5-6 N-terminal zinc fingers. Bioinformatic analysis suggests that these zinc fingers could bind to ubiquitin. In this study, we show that both the Aplysia and mouse SOL calpain (also known as Calpain 15) zinc fingers bind ubiquitinated proteins, and we confirm that Aplysia SOL binds poly- but not mono or di-ubiquitin. No specific zinc finger is required for polyubiquitin binding. Neither polyubiquitin nor calcium was sufficient to induce purified Aplysia SOL calpain to autolyse or to cleave the atypical PKC to PKM in vitro. In Aplysia, overexpression of the atypical PKC in sensory neurons leads to an activity-dependent cleavage event and an increase in nuclear ubiquitin staining. Activity-dependent cleavage is partially blocked by a dominant negative SOL calpain, but not by a dominant negative classical calpain. The cleaved PKM was stabilized by the dominant negative classical calpain and destabilized by a dominant negative form of the PKM stabilizing proteinKIdney/BRAin protein(KIBRA). These studies provide new insight into SOL calpain's function and regulation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  8. Dynamics of Linker Residues Modulate the Nucleic Acid Binding Properties of the HIV-1 Nucleocapsid Protein Zinc Fingers

    Science.gov (United States)

    Zargarian, Loussiné; Tisné, Carine; Barraud, Pierre; Xu, Xiaoqian; Morellet, Nelly; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2014-01-01

    The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity. PMID:25029439

  9. The PR/SET Domain Zinc Finger Protein Prdm4 Regulates Gene Expression in Embryonic Stem Cells but Plays a Nonessential Role in the Developing Mouse Embryo

    Science.gov (United States)

    Bogani, Debora; Morgan, Marc A. J.; Nelson, Andrew C.; Costello, Ita; McGouran, Joanna F.; Kessler, Benedikt M.

    2013-01-01

    Prdm4 is a highly conserved member of the Prdm family of PR/SET domain zinc finger proteins. Many well-studied Prdm family members play critical roles in development and display striking loss-of-function phenotypes. Prdm4 functional contributions have yet to be characterized. Here, we describe its widespread expression in the early embryo and adult tissues. We demonstrate that DNA binding is exclusively mediated by the Prdm4 zinc finger domain, and we characterize its tripartite consensus sequence via SELEX (systematic evolution of ligands by exponential enrichment) and ChIP-seq (chromatin immunoprecipitation-sequencing) experiments. In embryonic stem cells (ESCs), Prdm4 regulates key pluripotency and differentiation pathways. Two independent strategies, namely, targeted deletion of the zinc finger domain and generation of a EUCOMM LacZ reporter allele, resulted in functional null alleles. However, homozygous mutant embryos develop normally and adults are healthy and fertile. Collectively, these results strongly suggest that Prdm4 functions redundantly with other transcriptional partners to cooperatively regulate gene expression in the embryo and adult animal. PMID:23918801

  10. Zinc finger proteins and other transcription regulators as response proteins in benzo[a]pyrene exposed cells

    International Nuclear Information System (INIS)

    Gao Zhihua; Jin Jinghua; Yang Jun; Yu Yingnian

    2004-01-01

    Proteomic analysis, which combines two-dimensional electrophoresis (2-DE) and mass spectrometry (MS), is an important approach to screen proteins responsive to specific stimuli. Benzo[a]pyrene (B[a]P), a prototype of polycyclic hydrocarbons (PAHs), is a potent procarcinogen generated from the combustion of fossil fuel and cigarette smoke. To further probe the molecular mechanism of mutagenesis and carcinogenesis, and to find potential molecular markers involved in cellular responses to B[a]P exposure, we performed proteomic analysis of whole cellular proteins in human amnion epithelial cells after B[a]P-treatment. Image visualization and statistical analysis indicated that more than 40 proteins showed significant changes following B[a]P-treatment (P<0.05). Among them, 20 proteins existed only in the control groups, while six were only present in B[a]P-treated cells. In addition, the expression of 10 proteins increased whereas 11 decreased after B[a]P-treatment. These proteins were subjected to in-gel tryptic digestion followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis. Using peptide mass fingerprinting (PMF) to search the nrNCBI database, we identified 22 proteins. Most of these proteins have unknown functions and have not been previously connected to a response to B[a]P exposure. To further annotate the characteristics of these proteins, GOblet analysis was carried out and results indicated that they were involved in multiple biological processes including regulation of transcription, cell proliferation, cell aging and other processes. However, expression changes were noted in a number of transcription regulators, including eight zinc finger proteins as well as SNF2L1 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 1), which is closely linked to the chromatin remodeling process. These data may provide new clues to further understand the implication of

  11. Identification of a novel zinc finger protein gene (ZNF298) in the GAP2 of human chromosome 21q

    International Nuclear Information System (INIS)

    Shibuya, Kazunori; Kudoh, Jun; Okui, Michiyo; Shimizu, Nobuyoshi

    2005-01-01

    We have isolated a novel zinc finger protein gene, designated ZNF298, as a candidate gene for a particular phenotype of Down syndrome or bipolar affective disorder (BPAD) which maps to human chromosome 21q22.3. ZNF298 gene consists of 25 exons spanning approximately 80 kb in a direction from the telomere to centromere. There are four kinds of transcripts that harbor three types of 3' UTR. These four transcripts (ZNF298a, ZNF298b, ZNF298c, and ZNF298d) contain putative open reading frames encoding 1178, 1198, 555, and 515 amino acids, respectively. ZNF298 gene was ubiquitously expressed in various tissues at very low level. The protein motif analysis revealed that ZNF298 proteins contain a SET [Su(var)3-9, Enhancer-of-zeste, Trithorax] domain, multiple C2H2-type zinc finger (ZnF C 2H2) domains, several nuclear localization signals (NLSs), and PEST sequences. Nuclear localization of ZNF298 protein was confirmed by transfection of expression vector of GFP-tagged protein into two human cell lines. Interestingly, this gene crosses over a clone gap (GAP2) remaining in the band 21q22.3. We obtained the DNA fragments corresponding to GAP2 using ZNF298 cDNA sequence as anchor primers for PCR and determined its genomic DNA sequence

  12. Prediction of DNA-binding specificity in zinc finger proteins

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... Support Vector Machine (SVM) is a state-of-the-art classifica- tion technique. Using canonical binding model, the C2H2 zinc finger protein–DNA interaction interface is modelled by the pairwise amino acid–base interactions. Using a classification framework, known examples of non-binding ZF–DNA pairs.

  13. Cloning and characterization of a novel human zinc finger gene, hKid3, from a C2H2-ZNF enriched human embryonic cDNA library

    International Nuclear Information System (INIS)

    Gao Li; Sun Chong; Qiu Hongling; Liu Hui; Shao Huanjie; Wang Jun; Li Wenxin

    2004-01-01

    To investigate the zinc finger genes involved in human embryonic development, we constructed a C 2 H 2 -ZNF enriched human embryonic cDNA library, from which a novel human gene named hKid3 was identified. The hKid3 cDNA encodes a 554 amino acid protein with an amino-terminal KRAB domain and 11 carboxyl-terminal C 2 H 2 zinc finger motifs. Northern blot analysis indicates that two hKid3 transcripts of 6 and 8.5 kb express in human fetal brain and kidney. The 6 kb transcript can also be detected in human adult brain, heart, and skeletal muscle while the 8.5 kb transcript appears to be embryo-specific. GFP-fused hKid3 protein is localized to nuclei and the ZF domain is necessary and sufficient for nuclear localization. To explore the DNA-binding specificity of hKid3, an oligonucleotide library was selected by GST fusion protein of hKid3 ZF domain, and the consensus core sequence 5'-CCAC-3' was evaluated by competitive electrophoretic mobility shift assay. Moreover, The KRAB domain of hKid3 exhibits transcription repressor activity when tested in GAL4 fusion protein assay. These results indicate that hKid3 may function as a transcription repressor with regulated expression pattern during human development of brain and kidney

  14. Proliferation and osteo/odontogenic differentiation of stem cells from apical papilla regulated by Zinc fingers and homeoboxes 2: An in vitro study

    International Nuclear Information System (INIS)

    Wan, Fang; Gao, Lifen; Lu, Yating; Ma, Hongxin; Wang, Hongxing; Liang, Xiaohong; Wang, Yan; Ma, Chunhong

    2016-01-01

    In the process of tooth root development, stem cells from the apical papilla (SCAPs) can differentiate into odontoblasts and form root dentin, however, molecules regulating SCAPs differentiation have not been elucidated. Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. It is reported to modulate the development of nerve cells, liver cells, B cells, red blood cells, and so on. However, the role of ZHX2 in tooth root development remains unclear. In this study, we explored the potential role of ZHX2 in the process of SCAPs differentiation. The results showed that overexpression of ZHX2 upregulated the expression of osteo/odontogenic related genes and ALP activity, inhibited the proliferation of SCAPs. Consistently, ZHX2 knockdown reduced SCAPs mineralization and promoted SCAPs proliferation. These results indicated that ZHX2 plays a critical role in the proliferation and osteo/odontogenic differentiation of SCAPs. - Highlights: • Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. • we found another new biological function of ZHX2 for the first time. • ZHX2 inhibit SCAPs proliferation. • ZHX2 promote the osteo/odontogenic differentiation of SCAPs.

  15. Proliferation and osteo/odontogenic differentiation of stem cells from apical papilla regulated by Zinc fingers and homeoboxes 2: An in vitro study

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Fang [Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China); VIP Center, Shandong Provincial Key Laboratory of Oral Biomedicine, School and Hospital of Stomatology, Shandong University, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China); Gao, Lifen [Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China); Lu, Yating [VIP Center, Shandong Provincial Key Laboratory of Oral Biomedicine, School and Hospital of Stomatology, Shandong University, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China); Ma, Hongxin; Wang, Hongxing; Liang, Xiaohong [Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China); Wang, Yan, E-mail: wangyan1965@sdu.edu.cn [VIP Center, Shandong Provincial Key Laboratory of Oral Biomedicine, School and Hospital of Stomatology, Shandong University, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China); Ma, Chunhong, E-mail: machunhong@sdu.edu.cn [Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China)

    2016-01-15

    In the process of tooth root development, stem cells from the apical papilla (SCAPs) can differentiate into odontoblasts and form root dentin, however, molecules regulating SCAPs differentiation have not been elucidated. Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. It is reported to modulate the development of nerve cells, liver cells, B cells, red blood cells, and so on. However, the role of ZHX2 in tooth root development remains unclear. In this study, we explored the potential role of ZHX2 in the process of SCAPs differentiation. The results showed that overexpression of ZHX2 upregulated the expression of osteo/odontogenic related genes and ALP activity, inhibited the proliferation of SCAPs. Consistently, ZHX2 knockdown reduced SCAPs mineralization and promoted SCAPs proliferation. These results indicated that ZHX2 plays a critical role in the proliferation and osteo/odontogenic differentiation of SCAPs. - Highlights: • Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. • we found another new biological function of ZHX2 for the first time. • ZHX2 inhibit SCAPs proliferation. • ZHX2 promote the osteo/odontogenic differentiation of SCAPs.

  16. A novel cold-inducible zinc finger protein from soybean, SCOF-1, enhances cold tolerance in transgenic plants.

    Science.gov (United States)

    Kim, J C; Lee, S H; Cheong, Y H; Yoo, C M; Lee, S I; Chun, H J; Yun, D J; Hong, J C; Lee, S Y; Lim, C O; Cho, M J

    2001-02-01

    Cold stress on plants induces changes in the transcription of cold response genes. A cDNA clone encoding C2H2-type zinc finger protein, SCOF-1, was isolated from soybean. The transcription of SCOF-1 is specifically induced by low temperature and abscisic acid (ABA) but not by dehydration or high salinity. Constitutive overexpression of SCOF-1 induced cold-regulated (COR) gene expression and enhanced cold tolerance of non-acclimated transgenic Arabidopsis and tobacco plants. SCOF-1 localized to the nucleus but did not bind directly to either C-repeat/dehydration (CRT/DRE) or ABA responsive element (ABRE), cis-acting DNA regulatory elements present in COR gene promoters. However, SCOF-1 greatly enhanced the DNA binding activity of SGBF-1, a soybean G-box binding bZIP transcription factor, to ABRE in vitro. SCOF-1 also interacted with SGBF-1 in a yeast two-hybrid system. The SGBF-1 transactivated the beta-glucuronidase reporter gene driven by the ABRE element in Arabidopsis leaf protoplasts. Furthermore, the SCOF-1 enhanced ABRE-dependent gene expression mediated by SGBF-1. These results suggest that SCOF-1 may function as a positive regulator of COR gene expression mediated by ABRE via protein-protein interaction, which in turn enhances cold tolerance of plants.

  17. Characterization and localization of metal-responsive-element-binding transcription factors from tilapia

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, Andrew Pok-Lap; Au, Candy Yee-Man; Chan, William Wai-Lun [Department of Biochemistry, Chinese University of Hong Kong, Sha Tin, N.T., Hong Kong (Hong Kong); Chan, King Ming, E-mail: kingchan@cuhk.edu.hk [Department of Biochemistry, Chinese University of Hong Kong, Sha Tin, N.T., Hong Kong (Hong Kong)

    2010-08-01

    Two isoforms of MTF-1, MTF-1L (long form) and MTF-1S (short form), were cloned in tilapia (Ti) and characterized in a tilapia liver cell line, Hepa-T1. The cloned tiMTF-1L has the characteristics of all of the tiMTF-1S identified so far with the zinc finger domain having six fingers, the acidic-rich, proline-rich, and serine/threonine-rich domains; however, the short form encodes for the zinc finger domain with five zinc fingers only and no other domains. The transient transfection of tiMTF-1L into human HepG2 cells showed both constitutive and zinc-induced metal-responsive-element (MRE)-driven reporter gene expression. However, the transfection of tiMTF-1S (which lacks all three transactivation domains) into a human cell line showed reduced transcriptional activities compared with an endogenous control in both basal- and Zn{sup 2+}-induced conditions. The tiMTF-1 isoforms were tagged with GFP and transfected into Hepa-T1 cells (tilapia hepatocytes). The nuclear translocation of tiMTF-1L was observed when the cells were exposed to a sufficient concentration of metals for 6 h. However, tiMTF-1S, was localized in the nucleus with or without metal treatment. Electrophoretic mobility shift assay (EMSA) confirmed that both of the isoforms were able to bind to the MRE specifically in vitro. Tissue distribution studies showed that tiMTF-1L was more abundant than tiMTF-1S in all of the tissues tested.

  18. Characterization and localization of metal-responsive-element-binding transcription factors from tilapia

    International Nuclear Information System (INIS)

    Cheung, Andrew Pok-Lap; Au, Candy Yee-Man; Chan, William Wai-Lun; Chan, King Ming

    2010-01-01

    Two isoforms of MTF-1, MTF-1L (long form) and MTF-1S (short form), were cloned in tilapia (Ti) and characterized in a tilapia liver cell line, Hepa-T1. The cloned tiMTF-1L has the characteristics of all of the tiMTF-1S identified so far with the zinc finger domain having six fingers, the acidic-rich, proline-rich, and serine/threonine-rich domains; however, the short form encodes for the zinc finger domain with five zinc fingers only and no other domains. The transient transfection of tiMTF-1L into human HepG2 cells showed both constitutive and zinc-induced metal-responsive-element (MRE)-driven reporter gene expression. However, the transfection of tiMTF-1S (which lacks all three transactivation domains) into a human cell line showed reduced transcriptional activities compared with an endogenous control in both basal- and Zn 2+ -induced conditions. The tiMTF-1 isoforms were tagged with GFP and transfected into Hepa-T1 cells (tilapia hepatocytes). The nuclear translocation of tiMTF-1L was observed when the cells were exposed to a sufficient concentration of metals for 6 h. However, tiMTF-1S, was localized in the nucleus with or without metal treatment. Electrophoretic mobility shift assay (EMSA) confirmed that both of the isoforms were able to bind to the MRE specifically in vitro. Tissue distribution studies showed that tiMTF-1L was more abundant than tiMTF-1S in all of the tissues tested.

  19. The zinc finger E-box-binding homeobox 1 (Zeb1) promotes the conversion of mouse fibroblasts into functional neurons.

    Science.gov (United States)

    Yan, Long; Li, Yue; Shi, Zixiao; Lu, Xiaoyin; Ma, Jiao; Hu, Baoyang; Jiao, Jianwei; Wang, Hongmei

    2017-08-04

    The zinc finger E-box-binding transcription factor Zeb1 plays a pivotal role in the epithelial-mesenchymal transition. Numerous studies have focused on the molecular mechanisms by which Zeb1 contributes to this process. However, the functions of Zeb1 beyond the epithelial-mesenchymal transition remain largely elusive. Using a transdifferentiation system to convert mouse embryonic fibroblasts (MEFs) into functional neurons via the neuronal transcription factors achaete-scute family bHLH (basic helix-loop-helix) transcription factor1 ( Ascl1 ), POU class 3 homeobox 2 (POU3F2/ Brn2 ), and neurogenin 2 (Neurog2, Ngn2 ) (ABN), we found that Zeb1 was up-regulated during the early stages of transdifferentiation. Knocking down Zeb1 dramatically attenuated the transdifferentiation efficiency, whereas Zeb1 overexpression obviously increased the efficiency of transdifferentiation from MEFs to neurons. Interestingly, Zeb1 improved the transdifferentiation efficiency induced by even a single transcription factor ( e.g. Asc1 or Ngn2 ). Zeb1 also rapidly promoted the maturation of induced neuron cells to functional neurons and improved the formation of neuronal patterns and electrophysiological characteristics. Induced neuron cells could form functional synapse in vivo after transplantation. Genome-wide RNA arrays showed that Zeb1 overexpression up-regulated the expression of neuron-specific genes and down-regulated the expression of epithelial-specific genes during conversion. Taken together, our results reveal a new role for Zeb1 in the transdifferentiation of MEFs into neurons. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Glycoengineering of Human Cell Lines Using Zinc Finger Nuclease Gene Targeting

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Bennett, Eric Paul; Clausen, Henrik

    2013-01-01

    Lectin affinity chromatography is a powerful technique for isolation of glycoproteins carrying a specific glycan structure of interest. However, the enormous diversity of glycans present on the cell surface, as well as on individual proteins, makes it difficult to isolate an entire glycoproteome...... with one or even a series of lectins. Here we present a technique to generate cell lines with homogenous truncated O-glycans using zinc finger nuclease gene targeting. Because of their simplified O-glycoproteome, the cells have been named SimpleCells. Glycoproteins from SimpleCells can be isolated...... in a single purification step by lectin chromatography performed on a long lectin column. This protocol describes Zinc finger nuclease gene targeting of human cells to simplify the glycoproteome, as well as lectin chromatography and isolation of glycopeptides from total cell lysates of SimpleCells....

  1. A DHHC-type zinc finger protein gene regulates shoot branching in ...

    African Journals Online (AJOL)

    hope&shola

    Arabidopsis. Key words: Arabidopsis, DHHC-type zinc finger protein, At5g04270, shoot branching. ..... and human HIP14 (Ducker et al., 2004), were isolated and identified to .... the control of branching in the rms1 mutant of pea. Plant Physiol.

  2. GhZFP1, a novel CCCH-type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5.

    Science.gov (United States)

    Guo, Ying-Hui; Yu, Yue-Ping; Wang, Dong; Wu, Chang-Ai; Yang, Guo-Dong; Huang, Jin-Guang; Zheng, Cheng-Chao

    2009-01-01

    * Zinc finger proteins are a superfamily involved in many aspects of plant growth and development. However, CCCH-type zinc finger proteins involved in plant stress tolerance are poorly understood. * A cDNA clone designated Gossypium hirsutum zinc finger protein 1 (GhZFP1), which encodes a novel CCCH-type zinc finger protein, was isolated from a salt-induced cotton (G. hirsutum) cDNA library using differential hybridization screening and further studied in transgenic tobacco Nicotiana tabacum cv. NC89. Using yeast two-hybrid screening (Y2H), proteins GZIRD21A (GhZFP1 interacting and responsive to dehydration protein 21A) and GZIPR5 (GhZFP1 interacting and pathogenesis-related protein 5), which interacted with GhZFP1, were isolated. * GhZFP1 contains two typical zinc finger motifs (Cx8Cx5Cx3H and Cx5Cx4Cx3H), a putative nuclear export sequence (NES) and a potential nuclear localization signal (NLS). Transient expression analysis using a GhZFP1::GFP fusion gene in onion epidermal cells indicated a nuclear localization for GhZFP1. RNA blot analysis showed that the GhZFP1 transcript was induced by salt (NaCl), drought and salicylic acid (SA). The regions in GhZFP1 that interact with GZIRD21A and GZIPR5 were identified using truncation mutations. * Overexpression of GhZFP1 in transgenic tobacco enhanced tolerance to salt stress and resistance to Rhizoctonia solani. Therefore, it appears that GhZFP1 might be involved as an important regulator in plant responses to abiotic and biotic stresses.

  3. Elevated expression and potential roles of human Sp5, a member of Sp transcription factor family, in human cancers

    International Nuclear Information System (INIS)

    Chen Yongxin; Guo Yingqiu; Ge Xijin; Itoh, Hirotaka; Watanabe, Akira; Fujiwara, Takeshi; Kodama, Tatsuhiko; Aburatani, Hiroyuki

    2006-01-01

    In this report, we describe the expression and function of human Sp5, a member of the Sp family of zinc finger transcription factors. Like other family members, the Sp5 protein contains a Cys2His2 zinc finger DNA binding domain at the C-terminus. Our experiments employing Gal4-Sp5 fusion proteins reveal multiple transcriptional domains, including a N-terminal activity domain, an intrinsic repressive element, and a C-terminal synergistic domain. Elevated expression of Sp5 was noted in several human tumors including hepatocellular carcinoma, gastric cancer, and colon cancer. To study the effects of the Sp5 protein on growth properties of human cancer cells and facilitate the identification of its downstream genes, we combined an inducible gene expression system with microarray analysis to screen for its transcriptional targets. Transfer of Sp5 into MCF-7 cells that expressed no detectable endogenous Sp5 protein elicited significant growth promotion activity. Several of the constitutively deregulated genes have been associated with tumorigenesis (CDC25C, CEACAM6, TMPRSS2, XBP1, MYBL1, ABHD2, and CXCL12) and Wnt/β-Catenin signaling pathways (BAMBI, SIX1, IGFBP5, AES, and p21 WAF1 ). This information could be utilized for further mechanistic research and for devising optimized therapeutic strategies against human cancers

  4. Zinc finger protein 598 inhibits cell survival by promoting UV-induced apoptosis.

    Science.gov (United States)

    Yang, Qiaohong; Gupta, Romi

    2018-01-19

    UV is one of the major causes of DNA damage induced apoptosis. However, cancer cells adopt alternative mechanisms to evade UV-induced apoptosis. To identify factors that protect cancer cells from UV-induced apoptosis, we performed a genome wide short-hairpin RNA (shRNA) screen, which identified Zinc finger protein 598 (ZNF598) as a key regulator of UV-induced apoptosis. Here, we show that UV irradiation transcriptionally upregulates ZNF598 expression. Additionally, ZNF598 knockdown in cancer cells inhibited UV-induced apoptosis. In our study, we observe that ELK1 mRNA level as well as phosphorylated ELK1 levels was up regulated upon UV irradiation, which was necessary for UV irradiation induced upregulation of ZNF598. Cells expressing ELK1 shRNA were also resistant to UV-induced apoptosis, and phenocopy ZNF598 knockdown. Upon further investigation, we found that ZNF598 knockdown inhibits UV-induced apoptotic gene expression, which matches with decrease in percentage of annexin V positive cell. Similarly, ectopic expression of ZNF598 promoted apoptotic gene expression and also increased annexin V positive cells. Collectively, these results demonstrate that ZNF598 is a UV irradiation regulated gene and its loss results in resistance to UV-induced apoptosis.

  5. The strategy of fusion genes construction determines efficient expression of introduced transcription factors.

    Science.gov (United States)

    Adamus, Tomasz; Konieczny, Paweł; Sekuła, Małgorzata; Sułkowski, Maciej; Majka, Marcin

    2014-01-01

    The main goal in gene therapy and biomedical research is an efficient transcription factors (TFs) delivery system. SNAIL, a zinc finger transcription factor, is strongly involved in tumor, what makes its signaling pathways an interesting research subject. The necessity of tracking activation of intracellular pathways has prompted fluorescent proteins usage as localization markers. Advanced molecular cloning techniques allow to generate fusion proteins from fluorescent markers and transcription factors. Depending on fusion strategy, the protein expression levels and nuclear transport ability are significantly different. The P2A self-cleavage motif through its cleavage ability allows two single proteins to be simultaneously expressed. The aim of this study was to compare two strategies for introducing a pair of genes using expression vector system. We have examined GFP and SNAI1 gene fusions by comprising common nucleotide polylinker (multiple cloning site) or P2A motif in between them, resulting in one fusion or two independent protein expressions respectively. In each case transgene expression levels and translation efficiency as well as nuclear localization of expressed protein have been analyzed. Our data showed that usage of P2A motif provides more effective nuclear transport of SNAIL transcription factor than conventional genes linker. At the same time the fluorescent marker spreads evenly in subcellular space.

  6. Zinc(II) and the single-stranded DNA binding protein of bacteriophage T4

    International Nuclear Information System (INIS)

    Gauss, P.; Krassa, K.B.; McPheeters, D.S.; Nelson, M.A.; Gold, L.

    1987-01-01

    The DNA binding domain of the gene 32 protein of the bacteriophage T4 contains a single zinc-finger sequence. The gene 32 protein is an extensively studied member of a class of proteins that bind relatively nonspecifically to single-stranded DNA. The authors have sequenced and characterized mutations in gene 32 whose defective proteins are activated by increasing the Zn(II) concentration in the growth medium. The results identify a role for the gene 32 protein in activation of T4 late transcription. Several eukaryotic proteins with zinc fingers participate in activation of transcription, and the gene 32 protein of T4 should provide a simple, well-characterized system in which genetics can be utilized to study the role of a zinc finger in nucleic acid binding and gene expression

  7. X-ray Absorption Spectroscopy Combined with Time-Dependent Density Functional Theory Elucidates Differential Substitution Pathways of Au(I) and Au(III) with Zinc Fingers.

    Science.gov (United States)

    Abbehausen, Camilla; de Paiva, Raphael Enoque Ferraz; Bjornsson, Ragnar; Gomes, Saulo Quintana; Du, Zhifeng; Corbi, Pedro Paulo; Lima, Frederico Alves; Farrell, Nicholas

    2018-01-02

    A combination of two elements' (Au, Zn) X-ray absorption spectroscopy (XAS) and time-dependent density functional theory (TD-DFT) allowed the elucidation of differential substitution pathways of Au(I) and Au(III) compounds reacting with biologically relevant zinc fingers (ZnFs). Gold L 3 -edge XAS probed the interaction of gold and the C-terminal Cys 2 HisCys finger of the HIV-1 nucleocapsid protein NCp7, and the Cys 2 His 2 human transcription factor Sp1. The use of model compounds helped assign oxidation states and the identity of the gold-bound ligands. The computational studies accurately reproduced the experimental XAS spectra and allowed the proposition of structural models for the interaction products at early time points. The direct electrophilic attack on the ZnF by the highly thiophilic Au(I) resulted in a linear P-Au-Cys coordination sphere after zinc ejection whereas for the Sp1, loss of PEt 3 results in linear Cys-Au-Cys or Cys-Au-His arrangements. Reactions with Au(III) compounds, on the other hand, showed multiple binding modes. Prompt reaction between [AuCl(dien)] 2+ and [Au(dien)(DMAP)] 3+ with Sp1 showed a partially reduced Au center and a final linear His-Au-His coordination. Differently, in the presence of NCp7, [AuCl(dien)] 2+ readily reduces to Au(I) and changes from square-planar to linear geometry with Cys-Au-His coordination, while [Au(dien)(DMAP)] 3+ initially maintains its Au(III) oxidation state and square-planar geometry and the same first coordination sphere. The latter is the first observation of a "noncovalent" interaction of a Au(III) complex with a zinc finger and confirms early hypotheses that stabilization of Au(III) occurs with N-donor ligands. Modification of the zinc coordination sphere, suggesting full or partial zinc ejection, is observed in all cases, and for [Au(dien)(DMAP)] 3+ this represents a novel mechanism for nucleocapsid inactivation. The combination of XAS and TD-DFT presents the first direct experimental

  8. The ascorbate peroxidase APX1 is a direct target of a zinc finger transcription factor ZFP36 and a late embryogenesis abundant protein OsLEA5 interacts with ZFP36 to co-regulate OsAPX1 in seed germination in rice.

    Science.gov (United States)

    Huang, Liping; Jia, Jing; Zhao, Xixi; Zhang, MengYao; Huang, Xingxiu; E Ji; Ni, Lan; Jiang, Mingyi

    2018-01-01

    Seed germination is a vital developmental process. Abscisic acid (ABA) is an essential repressor of seed germination, while ROS (reactive oxygen species) also plays a vital role in regulating seed germination. ABA could inhibit the production of ROS in seed germination, but the mechanism of ABA reduced ROS production in seed germination was hitherto unknown. Here, by ChIP (chromatin immunoprecipitation)-seq, we found that ZFP36, a rice zinc finger transcription factor, could directly bind to the promoter of OsAPX1, coding an ascorbate peroxidase (APX) which has the most affinity for H 2 O 2 (substrate; a type of ROS), and act as a transcriptional activator of OsAPX1 promoter. Moreover, ZFP36 could interact with a late embryogenesis abundant protein OsLEA5 to co-regulate the promoter activity of OsAPX1. The seed germination is highly inhibited in ZFP36 overexpression plants under ABA treatment, while an RNA interference (RNAi) mutant of OsLEA5 rice seeds were less sensitive to ABA, and exogenous ASC (ascorbate acid) could alleviate the inhibition induced by ABA. Thus, our conclusion is that OsAPX1 is a direct target of ZFP36 and OsLEA5 could interact with ZFP36 to co-regulate ABA-inhibited seed germination by controlling the expression of OsAPX1. Copyright © 2017. Published by Elsevier Inc.

  9. Gain, loss and divergence in primate zinc-finger genes: a rich resource for evolution of gene regulatory differences between species.

    Directory of Open Access Journals (Sweden)

    Katja Nowick

    Full Text Available The molecular changes underlying major phenotypic differences between humans and other primates are not well understood, but alterations in gene regulation are likely to play a major role. Here we performed a thorough evolutionary analysis of the largest family of primate transcription factors, the Krüppel-type zinc finger (KZNF gene family. We identified and curated gene and pseudogene models for KZNFs in three primate species, chimpanzee, orangutan and rhesus macaque, to allow for a comparison with the curated set of human KZNFs. We show that the recent evolutionary history of primate KZNFs has been complex, including many lineage-specific duplications and deletions. We found 213 species-specific KZNFs, among them 7 human-specific and 23 chimpanzee-specific genes. Two human-specific genes were validated experimentally. Ten genes have been lost in humans and 13 in chimpanzees, either through deletion or pseudogenization. We also identified 30 KZNF orthologs with human-specific and 42 with chimpanzee-specific sequence changes that are predicted to affect DNA binding properties of the proteins. Eleven of these genes show signatures of accelerated evolution, suggesting positive selection between humans and chimpanzees. During primate evolution the most extensive re-shaping of the KZNF repertoire, including most gene additions, pseudogenizations, and structural changes occurred within the subfamily homininae. Using zinc finger (ZNF binding predictions, we suggest potential impact these changes have had on human gene regulatory networks. The large species differences in this family of TFs stands in stark contrast to the overall high conservation of primate genomes and potentially represents a potent driver of primate evolution.

  10. The transcription factor Rbf1 is the master regulator for b-mating type controlled pathogenic development in Ustilago maydis.

    Directory of Open Access Journals (Sweden)

    Kai Heimel

    Full Text Available In the phytopathogenic basidiomycete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled by the heterodimeric bE/bW transcription factor complex encoded by the b-mating type locus. The formation of the active bE/bW heterodimer leads to the formation of filaments, induces a G2 cell cycle arrest, and triggers pathogenicity. Here, we identify a set of 345 bE/bW responsive genes which show altered expression during these developmental changes; several of these genes are associated with cell cycle coordination, morphogenesis and pathogenicity. 90% of the genes that show altered expression upon bE/bW-activation require the zinc finger transcription factor Rbf1, one of the few factors directly regulated by the bE/bW heterodimer. Rbf1 is a novel master regulator in a multilayered network of transcription factors that facilitates the complex regulatory traits of sexual and pathogenic development.

  11. Genome-wide strategies identify downstream target genes of chick connective tissue-associated transcription factors.

    Science.gov (United States)

    Orgeur, Mickael; Martens, Marvin; Leonte, Georgeta; Nassari, Sonya; Bonnin, Marie-Ange; Börno, Stefan T; Timmermann, Bernd; Hecht, Jochen; Duprez, Delphine; Stricker, Sigmar

    2018-03-29

    Connective tissues support organs and play crucial roles in development, homeostasis and fibrosis, yet our understanding of their formation is still limited. To gain insight into the molecular mechanisms of connective tissue specification, we selected five zinc-finger transcription factors - OSR1, OSR2, EGR1, KLF2 and KLF4 - based on their expression patterns and/or known involvement in connective tissue subtype differentiation. RNA-seq and ChIP-seq profiling of chick limb micromass cultures revealed a set of common genes regulated by all five transcription factors, which we describe as a connective tissue core expression set. This common core was enriched with genes associated with axon guidance and myofibroblast signature, including fibrosis-related genes. In addition, each transcription factor regulated a specific set of signalling molecules and extracellular matrix components. This suggests a concept whereby local molecular niches can be created by the expression of specific transcription factors impinging on the specification of local microenvironments. The regulatory network established here identifies common and distinct molecular signatures of limb connective tissue subtypes, provides novel insight into the signalling pathways governing connective tissue specification, and serves as a resource for connective tissue development. © 2018. Published by The Company of Biologists Ltd.

  12. The transcription factor Nerfin-1 prevents reversion of neurons into neural stem cells.

    Science.gov (United States)

    Froldi, Francesca; Szuperak, Milan; Weng, Chen-Fang; Shi, Wei; Papenfuss, Anthony T; Cheng, Louise Y

    2015-01-15

    Cellular dedifferentiation is the regression of a cell from a specialized state to a more multipotent state and is implicated in cancer. However, the transcriptional network that prevents differentiated cells from reacquiring stem cell fate is so far unclear. Neuroblasts (NBs), the Drosophila neural stem cells, are a model for the regulation of stem cell self-renewal and differentiation. Here we show that the Drosophila zinc finger transcription factor Nervous fingers 1 (Nerfin-1) locks neurons into differentiation, preventing their reversion into NBs. Following Prospero-dependent neuronal specification in the ganglion mother cell (GMC), a Nerfin-1-specific transcriptional program maintains differentiation in the post-mitotic neurons. The loss of Nerfin-1 causes reversion to multipotency and results in tumors in several neural lineages. Both the onset and rate of neuronal dedifferentiation in nerfin-1 mutant lineages are dependent on Myc- and target of rapamycin (Tor)-mediated cellular growth. In addition, Nerfin-1 is required for NB differentiation at the end of neurogenesis. RNA sequencing (RNA-seq) and chromatin immunoprecipitation (ChIP) analysis show that Nerfin-1 administers its function by repression of self-renewing-specific and activation of differentiation-specific genes. Our findings support the model of bidirectional interconvertibility between neural stem cells and their post-mitotic progeny and highlight the importance of the Nerfin-1-regulated transcriptional program in neuronal maintenance. © 2015 Froldi et al.; Published by Cold Spring Harbor Laboratory Press.

  13. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Vakhrushev, Sergey Y; Vester-Christensen, Malene B

    2011-01-01

    Zinc-finger nuclease (ZFN) gene targeting is emerging as a versatile tool for engineering of multiallelic gene deficiencies. A longstanding obstacle for detailed analysis of glycoproteomes has been the extensive heterogeneities in glycan structures and attachment sites. Here we applied ZFN target...

  14. Enhanced cellulase production from Trichoderma reesei Rut-C30 by engineering with an artificial zinc finger protein library.

    Science.gov (United States)

    Zhang, Fei; Bai, Fengwu; Zhao, Xinqing

    2016-10-01

    Trichoderma reesei Rut-C30 is a well-known cellulase producer, and improvement of its cellulase production is of great interest. An artificial zinc finger protein (AZFP) library is constructed for expression in T. reesei Rut-C30, and a mutant strain T. reesei U3 is selected based on its enhanced cellulase production. The U3 mutant shows a 55% rise in filter paper activity and 8.1-fold increased β-glucosidase activity, when compared to the native strain T. reesei Rut-C30. It is demonstrated that enhanced β-glucosidase activity was due to elevated transcription level of β-glucosidase gene in the U3 mutant. Moreover, significant elevation in transcription levels of several putative Azfp-U3 target genes is detected in the U3 mutant, including genes encoding hypothetical transcription factors and a putative glycoside hydrolase. Furthermore, U3 cellulase shows 115% higher glucose yield from pretreated corn stover, when compared to the cellulase of T. reesei Rut-C30. These results demonstrate that AZFP can be used to improve cellulase production in T. reesei Rut-C30. Our current work offers the establishment of an alternative strategy to develop fungal cell factories for improved production of high value industrial products. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Arabidopsis thaliana BTB/ POZ-MATH proteins interact with members of the ERF/AP2 transcription factor family.

    Science.gov (United States)

    Weber, Henriette; Hellmann, Hanjo

    2009-11-01

    In Arabidopsis thaliana, the BTB/POZ-MATH (BPM) proteins comprise a small family of six members. They have been described previously to use their broad complex, tram track, bric-a-brac/POX virus and zinc finger (BTB/POZ) domain to assemble with CUL3a and CUL3b and potentially to serve as substrate adaptors to cullin-based E3-ligases in plants. In this article, we show that BPMs can also assemble with members of the ethylene response factor/Apetala2 transcription factor family, and that this is mediated by their meprin and TRAF (tumor necrosis factor receptor-associated factor) homology (MATH) domain. In addition, we provide a detailed description of BPM gene expression patterns in different tissues and on abiotic stress treatments, as well as their subcellular localization. This work connects, for the first time, BPM proteins with ethylene response factor/Apetala2 family members, which is likely to represent a novel regulatory mechanism of transcriptional control.

  16. TIF1alpha: a possible link between KRAB zinc finger proteins and nuclear receptors

    DEFF Research Database (Denmark)

    Le Douarin, B; You, J; Nielsen, Anders Lade

    1998-01-01

    Ligand-induced gene activation by nuclear receptors (NRs) is thought to be mediated by transcriptional intermediary factors (TIFs), that interact with their ligand-dependent AF-2 activating domain. Included in the group of the putative AF-2 TIFs identified so far is TIF1alpha, a member of a new...... family of proteins which contains an N-terminal RBCC (RING finger-B boxes-coiled coil) motif and a C-terminal bromodomain preceded by a PHD finger. In addition to these conserved domains present in a number of transcriptional regulatory proteins, TIF1alpha was found to contain several protein......-protein interaction sites. Of these, one specifically interacts with NRs bound to their agonistic ligand and not with NR mutants that are defective in the AF-2 activity. Immediately adjacent to this 'NR box', TIF1alpha contains an interaction site for members of the chromatin organization modifier (chromo) family, HP...

  17. Isolation of three B-box zinc finger proteins that interact with STF1 and COP1 defines a HY5/COP1 interaction network involved in light control of development in soybean

    International Nuclear Information System (INIS)

    Shin, Su Young; Kim, Seong Hee; Kim, Hye Jin; Jeon, Su Jeong; Sim, Soon Ae; Ryu, Gyeong Ryul; Yoo, Cheol Min; Cheong, Yong Hwa; Hong, Jong Chan

    2016-01-01

    LONG HYPOCOTYL5 (HY5) and STF1 (Soybean TGACG-motif binding Factor 1) are two related bZIP transcription factors that play a positive role in photomorphogenesis and hormonal signaling. In this study, we compared full length STF1 and truncated STF1 overexpression lines and found that the C-terminal 133 amino acids (194–306) possess all the HY5-like function in Arabidopsis. The STF1-DC1 mutant (1–306), with a 20 amino acid deletion at the carboxy terminus, failed to complement the hy5 mutant phenotype, which suggests an intact C-terminus is required for STF1 function. To understand the role of the C-terminal domain in photomorphogenesis we used a yeast two-hybrid screen to isolate proteins that bind to the STF1 C-terminus. We isolated three soybean cDNAs encoding the zinc-finger proteins GmSTO, GmSTH, and GmSTH2, which interact with STF1. These proteins belong to a family of B-box zinc finger proteins that include Arabidopsis SALT TOLERANCE (STO) and STO HOMOLOG (STH) and STH2, which play a role in light-dependent development and gene expression. The C-terminal 63 amino acids of STF1, containing a leucine zipper and the two N-terminal B-boxes, contains the domain involved in interactions between STF1 and GmSTO. In addition, we identified an interaction between soybean COP1 (GmCOP1) and GmSTO and GmSTH, as well as STF1, which strongly suggests the presence of a similar regulatory circuit for light signaling in soybean as in Arabidopsis. This study shows that photomorphogenic control requires complex molecular interactions among several different classes of transcription factors such as bZIP, B-box factors, and COP1, a ubiquitin ligase. - Highlights: • STF1 interact with GmSTO, GmSTH and GmSTH2. • The bZIP transcription factor STF1 requires an intact C-terminal domain for STF1 function. • STF1 and GmSTO are nuclear proteins.

  18. Co(II) Coordination in Prokaryotic Zinc Finger Domains as Revealed by UV-Vis Spectroscopy

    Science.gov (United States)

    Sivo, Valeria; D'Abrosca, Gianluca; Russo, Luigi; Iacovino, Rosa; Pedone, Paolo Vincenzo; Fattorusso, Roberto

    2017-01-01

    Co(II) electronic configuration allows its use as a spectroscopic probe in UV-Vis experiments to characterize the metal coordination sphere that is an essential component of the functional structure of zinc-binding proteins and to evaluate the metal ion affinities of these proteins. Here, exploiting the capability of the prokaryotic zinc finger to use different combinations of residues to properly coordinate the structural metal ion, we provide the UV-Vis characterization of Co(II) addition to Ros87 and its mutant Ros87_C27D which bears an unusual CysAspHis2 coordination sphere. Zinc finger sites containing only one cysteine have been infrequently characterized. We show for the CysAspHis2 coordination an intense d-d transition band, blue-shifted with respect to the Cys2His2 sphere. These data complemented by NMR and CD data demonstrate that the tetrahedral geometry of the metal site is retained also in the case of a single-cysteine coordination sphere. PMID:29386985

  19. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases

    DEFF Research Database (Denmark)

    Chen, Fuqiang; Pruett-Miller, Shondra M; Huang, Yuping

    2011-01-01

    Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point...

  20. Modulation of Caenorhabditis elegans transcription factor activity by HIM-8 and the related Zinc-Finger ZIM proteins.

    Science.gov (United States)

    Sun, Hongliu; Nelms, Brian L; Sleiman, Sama F; Chamberlin, Helen M; Hanna-Rose, Wendy

    2007-10-01

    The previously reported negative regulatory activity of HIM-8 on the Sox protein EGL-13 is shared by the HIM-8-related ZIM proteins. Furthermore, mutation of HIM-8 can modulate the effects of substitution mutations in the DNA-binding domains of at least four other transcription factors, suggesting broad regulatory activity by HIM-8.

  1. Targeting Ligandable Pockets on Plant Homeodomain (PHD) Zinc Finger Domains by a Fragment-Based Approach.

    Science.gov (United States)

    Amato, Anastasia; Lucas, Xavier; Bortoluzzi, Alessio; Wright, David; Ciulli, Alessio

    2018-04-20

    Plant homeodomain (PHD) zinc fingers are histone reader domains that are often associated with human diseases. Despite this, they constitute a poorly targeted class of readers, suggesting low ligandability. Here, we describe a successful fragment-based campaign targeting PHD fingers from the proteins BAZ2A and BAZ2B as model systems. We validated a pool of in silico fragments both biophysically and structurally and solved the first crystal structures of PHD zinc fingers in complex with fragments bound to an anchoring pocket at the histone binding site. The best-validated hits were found to displace a histone H3 tail peptide in competition assays. This work identifies new chemical scaffolds that provide suitable starting points for future ligand optimization using structure-guided approaches. The demonstrated ligandability of the PHD reader domains could pave the way for the development of chemical probes to drug this family of epigenetic readers.

  2. Chemical Approach to Biological Safety: Molecular-Level Control of an Integrated Zinc Finger Nuclease

    DEFF Research Database (Denmark)

    Németh, Eszter; Asaka, Masamitsu N; Kato, Kohsuke

    2018-01-01

    circular dichroism spectroscopy, and nano-electrospray ionisation mass spectrometry. In situ intramolecular activation of the nuclease domain was observed, resulting in specific cleavage of DNA with moderate activity. This study represents a new approach to AN design through integrated nucleases consisting......Application of artificial nucleases (ANs) in genome editing is still hindered by their cytotoxicity related to off-target cleavages. This problem can be targeted by regulation of the nuclease domain. Here, we provide an experimental survey of computationally designed integrated zinc finger...... nucleases, constructed by linking the inactivated catalytic centre and the allosteric activator sequence of the colicin E7 nuclease domain to the two opposite termini of a zinc finger array. DNA specificity and metal binding were confirmed by electrophoretic mobility shift assays, synchrotron radiation...

  3. Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Wataru; Kezuka, Aki; Murakami, Yoshiyuki; Lee, Jinhee; Abe, Koichi [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588 (Japan); Motoki, Hiroaki; Matsuo, Takafumi; Shimura, Nobuaki [System Instruments Co., Ltd., 776-2 Komiya-cho, Hachioji, Tokyo 192-0031 (Japan); Noda, Mamoru; Igimi, Shizunobu [Division of Biomedical Food Research, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan); Ikebukuro, Kazunori, E-mail: ikebu@cc.tuat.ac.jp [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588 (Japan)

    2013-11-01

    Graphical abstract: -- Highlights: •Zif268 fused to luciferase was used for E. coli O157, Salmonella and coliform detection. •Artificial zinc finger protein fused to luciferase was constructed for Norovirus detection. •An analyzer that automatically detects PCR products by zinc finger protein fused to luciferase was developed. •Target pathogens were specifically detected by the automatic analyzer with zinc finger protein fused to luciferase. -- Abstract: An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268–luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF–luciferase fusion protein. By means of the automatic analyzer with ZF–luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0 × 10 to 1.0 × 10{sup 6} copies.

  4. Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase

    International Nuclear Information System (INIS)

    Yoshida, Wataru; Kezuka, Aki; Murakami, Yoshiyuki; Lee, Jinhee; Abe, Koichi; Motoki, Hiroaki; Matsuo, Takafumi; Shimura, Nobuaki; Noda, Mamoru; Igimi, Shizunobu; Ikebukuro, Kazunori

    2013-01-01

    Graphical abstract: -- Highlights: •Zif268 fused to luciferase was used for E. coli O157, Salmonella and coliform detection. •Artificial zinc finger protein fused to luciferase was constructed for Norovirus detection. •An analyzer that automatically detects PCR products by zinc finger protein fused to luciferase was developed. •Target pathogens were specifically detected by the automatic analyzer with zinc finger protein fused to luciferase. -- Abstract: An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268–luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF–luciferase fusion protein. By means of the automatic analyzer with ZF–luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0 × 10 to 1.0 × 10 6 copies

  5. Can AtTZF1 act as a transcriptional activator or repressor in plants?

    OpenAIRE

    Pomeranz, Marcelo; Zhang, Li; Finer, John; Jang, Jyan-Chyun

    2011-01-01

    In animals, Tandem CCCH Zinc Finger (TZF) proteins can affect gene expression at both transcriptional and post-transcriptional levels. In Arabidopsis thaliana, AtTZF1 is a member of the TZF family characterized by a plant-unique tandem zinc finger motif. AtTZF1 can bind both DNA and RNA in vitro, and it can traffic between the nucleus and cytoplasmic foci. However, no in vivo DNA/RNA targets have been identified so far, and little is known about the molecular mechanisms underlying AtTZF1's pr...

  6. SP and KLF Transcription Factors in Digestive Physiology and Diseases.

    Science.gov (United States)

    Kim, Chang-Kyung; He, Ping; Bialkowska, Agnieszka B; Yang, Vincent W

    2017-06-01

    Specificity proteins (SPs) and Krüppel-like factors (KLFs) belong to the family of transcription factors that contain conserved zinc finger domains involved in binding to target DNA sequences. Many of these proteins are expressed in different tissues and have distinct tissue-specific activities and functions. Studies have shown that SPs and KLFs regulate not only physiological processes such as growth, development, differentiation, proliferation, and embryogenesis, but pathogenesis of many diseases, including cancer and inflammatory disorders. Consistently, these proteins have been shown to regulate normal functions and pathobiology in the digestive system. We review recent findings on the tissue- and organ-specific functions of SPs and KLFs in the digestive system including the oral cavity, esophagus, stomach, small and large intestines, pancreas, and liver. We provide a list of agents under development to target these proteins. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

  7. A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions

    Science.gov (United States)

    Khalil, Ahmad S.; Lu, Timothy K.; Bashor, Caleb J.; Ramirez, Cherie L.; Pyenson, Nora C.; Joung, J. Keith; Collins, James J.

    2013-01-01

    SUMMARY Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks. PMID:22863014

  8. Genome-Wide Identification, Evolution and Expression Analysis of the Grape (Vitis vinifera L. Zinc Finger-Homeodomain Gene Family

    Directory of Open Access Journals (Sweden)

    Hao Wang

    2014-04-01

    Full Text Available Plant zinc finger-homeodomain (ZHD genes encode a family of transcription factors that have been demonstrated to play an important role in the regulation of plant growth and development. In this study, we identified a total of 13 ZHD genes (VvZHD in the grape genome that were further classified into at least seven groups. Genome synteny analysis revealed that a number of VvZHD genes were present in the corresponding syntenic blocks of Arabidopsis, indicating that they arose before the divergence of these two species. Gene expression analysis showed that the identified VvZHD genes displayed distinct spatiotemporal expression patterns, and were differentially regulated under various stress conditions and hormone treatments, suggesting that the grape VvZHDs might be also involved in plant response to a variety of biotic and abiotic insults. Our work provides insightful information and knowledge about the ZHD genes in grape, which provides a framework for further characterization of their roles in regulation of stress tolerance as well as other aspects of grape productivity.

  9. Transcription factor CgMTF-1 regulates CgZnT1 and CgMT expression in Pacific oyster (Crassostrea gigas) under zinc stress

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Jie; Zhang, Linlin [Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, Shandong (China); Li, Li, E-mail: lili@qdio.ac.cn [Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, Shandong (China); Li, Chunyan; Wang, Ting [Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, Shandong (China); University of Chinese Academy of Sciences, Beijing 100039 (China); Zhang, Guofan, E-mail: gfzhang@qdio.ac.cn [Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, Shandong (China)

    2015-08-15

    Highlights: • CgMTF-1 and CgZnT1 were first identified in oysters. • CgMTF-1 localized in cell nucleus under unstressed conditions. • CgMTF-1 proteins could bind with the typical MRE motif. • CgMTF-1 activated CgZnT1, CgMT1 and CgMT4 promoters and regulated their expressions under zinc exposure. - Abstract: Oysters accumulate zinc at high tissue concentrations, and the metal response element (MRE)-binding transcription factor (MTF) functions as the cellular zinc sensor that coordinates the expression of genes involved in zinc efflux and storage, as well as those that protect against metal toxicity. In this study, we cloned MTF-1 in oysters and examined its regulation mechanism for its classic target genes, including MTs and ZnT1 under zinc exposure conditions. We cloned CgMTF-1 and determined the subcellular locations of its protein product in HEK293 cells. CgMTF-1 has a 2826 bp open reading frame that encodes a predicted polypeptide with 707 amino acid residues, showing six well-conserved zinc finger domains that are required for metal binding. In HEK293 cell lines, CgMTF-1 primarily localizes in the cell nucleus under unstressed conditions and nuclear translocation was not critical for the activation of this gene. We searched for CgMTF-1-regulated genes in oysters using RNA interference. Decreased expression levels of CgMT1, CgMT4, and CgZnT1 were observed after CgMTF-1 interference (>70% inhibition) under zinc exposure, indicating the critical role of CgMTF-1 in the regulation of these genes. We searched for a direct regulation mechanism involving CgMTF-1 for CgMT1, CgMT4, and CgZnT1 in vitro. EMSA experiments indicated that CgMTF-1 can bind with the MREs found in the CgZnT1, CgMT1 and CgMT4 promoter regions. Additionally, luciferase reporter gene experiments indicated that CgMTF-1 could activate the CgMT1, CgMT4, and CgZnT1 promoters. Overall, our results suggest that CgMTF-1 directly coordinates the regulation of CgMTs and CgZnT1 expression and plays

  10. Transcription factor CgMTF-1 regulates CgZnT1 and CgMT expression in Pacific oyster (Crassostrea gigas) under zinc stress

    International Nuclear Information System (INIS)

    Meng, Jie; Zhang, Linlin; Li, Li; Li, Chunyan; Wang, Ting; Zhang, Guofan

    2015-01-01

    Highlights: • CgMTF-1 and CgZnT1 were first identified in oysters. • CgMTF-1 localized in cell nucleus under unstressed conditions. • CgMTF-1 proteins could bind with the typical MRE motif. • CgMTF-1 activated CgZnT1, CgMT1 and CgMT4 promoters and regulated their expressions under zinc exposure. - Abstract: Oysters accumulate zinc at high tissue concentrations, and the metal response element (MRE)-binding transcription factor (MTF) functions as the cellular zinc sensor that coordinates the expression of genes involved in zinc efflux and storage, as well as those that protect against metal toxicity. In this study, we cloned MTF-1 in oysters and examined its regulation mechanism for its classic target genes, including MTs and ZnT1 under zinc exposure conditions. We cloned CgMTF-1 and determined the subcellular locations of its protein product in HEK293 cells. CgMTF-1 has a 2826 bp open reading frame that encodes a predicted polypeptide with 707 amino acid residues, showing six well-conserved zinc finger domains that are required for metal binding. In HEK293 cell lines, CgMTF-1 primarily localizes in the cell nucleus under unstressed conditions and nuclear translocation was not critical for the activation of this gene. We searched for CgMTF-1-regulated genes in oysters using RNA interference. Decreased expression levels of CgMT1, CgMT4, and CgZnT1 were observed after CgMTF-1 interference (>70% inhibition) under zinc exposure, indicating the critical role of CgMTF-1 in the regulation of these genes. We searched for a direct regulation mechanism involving CgMTF-1 for CgMT1, CgMT4, and CgZnT1 in vitro. EMSA experiments indicated that CgMTF-1 can bind with the MREs found in the CgZnT1, CgMT1 and CgMT4 promoter regions. Additionally, luciferase reporter gene experiments indicated that CgMTF-1 could activate the CgMT1, CgMT4, and CgZnT1 promoters. Overall, our results suggest that CgMTF-1 directly coordinates the regulation of CgMTs and CgZnT1 expression and plays

  11. Nuclear import of transcription factor BR-C is mediated by its interaction with RACK1.

    Science.gov (United States)

    Cheng, Daojun; Qian, Wenliang; Wang, Yonghu; Meng, Meng; Wei, Ling; Li, Zhiqing; Kang, Lixia; Peng, Jian; Xia, Qingyou

    2014-01-01

    The transcription factor Broad Complex (BR-C) is an early ecdysone response gene in insects and contains two types of domains: two zinc finger domains for the activation of gene transcription and a Bric-a-brac/Tramtrack/Broad complex (BTB) domain for protein-protein interaction. Although the mechanism of zinc finger-mediated gene transcription is well studied, the partners interacting with the BTB domain of BR-C has not been elucidated until now. Here, we performed a yeast two-hybrid screen using the BTB domain of silkworm BR-C as bait and identified the receptor for activated C-kinase 1 (RACK1), a scaffolding/anchoring protein, as the novel partner capable of interacting with BR-C. The interaction between BR-C and RACK1 was further confirmed by far-western blotting and pull-down assays. Importantly, the disruption of this interaction, via RNAi against the endogenous RACK1 gene or deletion of the BTB domain, abolished the nuclear import of BR-C in BmN4 cells. In addition, RNAi against the endogenous PKC gene as well as phosphorylation-deficient mutation of the predicted PKC phosphorylation sites at either Ser373 or Thr406 in BR-C phenocopied RACK1 RNAi and altered the nuclear localization of BR-C. However, when BTB domain was deleted, phosphorylation mimics of either Ser373 or Thr406 had no effect on the nuclear import of BR-C. Moreover, mutating the PKC phosphorylation sites at Ser373 and Thr406 or deleting the BTB domain significantly decreased the transcriptional activation of a BR-C target gene. Given that RACK1 is necessary for recruiting PKC to close and phosphorylate target proteins, we suggest that the PKC-mediated phosphorylation and nuclear import of BR-C is determined by its interaction with RACK1. This novel finding will be helpful for further deciphering the mechanism underlying the role of BR-C proteins during insect development.

  12. Nuclear import of transcription factor BR-C is mediated by its interaction with RACK1.

    Directory of Open Access Journals (Sweden)

    Daojun Cheng

    Full Text Available The transcription factor Broad Complex (BR-C is an early ecdysone response gene in insects and contains two types of domains: two zinc finger domains for the activation of gene transcription and a Bric-a-brac/Tramtrack/Broad complex (BTB domain for protein-protein interaction. Although the mechanism of zinc finger-mediated gene transcription is well studied, the partners interacting with the BTB domain of BR-C has not been elucidated until now. Here, we performed a yeast two-hybrid screen using the BTB domain of silkworm BR-C as bait and identified the receptor for activated C-kinase 1 (RACK1, a scaffolding/anchoring protein, as the novel partner capable of interacting with BR-C. The interaction between BR-C and RACK1 was further confirmed by far-western blotting and pull-down assays. Importantly, the disruption of this interaction, via RNAi against the endogenous RACK1 gene or deletion of the BTB domain, abolished the nuclear import of BR-C in BmN4 cells. In addition, RNAi against the endogenous PKC gene as well as phosphorylation-deficient mutation of the predicted PKC phosphorylation sites at either Ser373 or Thr406 in BR-C phenocopied RACK1 RNAi and altered the nuclear localization of BR-C. However, when BTB domain was deleted, phosphorylation mimics of either Ser373 or Thr406 had no effect on the nuclear import of BR-C. Moreover, mutating the PKC phosphorylation sites at Ser373 and Thr406 or deleting the BTB domain significantly decreased the transcriptional activation of a BR-C target gene. Given that RACK1 is necessary for recruiting PKC to close and phosphorylate target proteins, we suggest that the PKC-mediated phosphorylation and nuclear import of BR-C is determined by its interaction with RACK1. This novel finding will be helpful for further deciphering the mechanism underlying the role of BR-C proteins during insect development.

  13. Structures of three members of Pfam PF02663 (FmdE) implicated in microbial methanogenesis reveal a conserved α+β core domain and an auxiliary C-terminal treble-clef zinc finger

    International Nuclear Information System (INIS)

    Axelrod, Herbert L.; Das, Debanu; Abdubek, Polat; Astakhova, Tamara; Bakolitsa, Constantina; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Farr, Carol L.; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Lam, Winnie W.; Marciano, David; McMullan, Daniel; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; Bedem, Henry van den; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    The first structures from the FmdE Pfam family (PF02663) reveal that some members of this family form tightly intertwined dimers consisting of two domains (N-terminal α+β core and C-terminal zinc-finger domains), whereas others contain only the core domain. The presence of the zinc-finger domain suggests that some members of this family may perform functions associated with transcriptional regulation, protein–protein interaction, RNA binding or metal-ion sensing. Examination of the genomic context for members of the FmdE Pfam family (PF02663), such as the protein encoded by the fmdE gene from the methanogenic archaeon Methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (EC 1.2.99.5), an enzyme that is involved in microbial methane production. Here, the first crystal structures from PF02663 are described, representing two bacterial and one archaeal species: B8FYU2-DESHY from the anaerobic dehalogenating bacterium Desulfitobacterium hafniense DCB-2, Q2LQ23-SYNAS from the syntrophic bacterium Syntrophus aciditrophicus SB and Q9HJ63-THEAC from the thermoacidophilic archaeon Thermoplasma acidophilum. Two of these proteins, Q9HJ63-THEAC and Q2LQ23-SYNAS, contain two domains: an N-terminal thioredoxin-like α+β core domain (NTD) consisting of a five-stranded, mixed β-sheet flanked by several α-helices and a C-terminal zinc-finger domain (CTD). B8FYU2-DESHY, on the other hand, is composed solely of the NTD. The CTD of Q9HJ63-THEAC and Q2LQ23-SYNAS is best characterized as a treble-clef zinc finger. Two significant structural differences between Q9HJ63-THEAC and Q2LQ23-SYNAS involve their metal binding. First, zinc is bound to the putative active site on the NTD of Q9HJ63-THEAC, but is absent from the NTD of Q2LQ23-SYNAS. Second, whereas the structure of the CTD of Q2LQ23-SYNAS shows four Cys side chains within coordination distance of the Zn atom, the structure

  14. Myocardial ischemic preconditioning upregulated protein 1(Mipu1):zinc finger protein 667 - a multifunctional KRAB/C{sub 2}H{sub 2} zinc finger protein

    Energy Technology Data Exchange (ETDEWEB)

    Han, D.; Zhang, C. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China); Fan, W.J. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China); The Second Affiliated Hospital, University of South China, Hengyang City, Hunan Province (China); Pan, W.J.; Feng, D.M.; Qu, S.L.; Jiang, Z.S. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China)

    2014-10-31

    Myocardial ischemic preconditioning upregulated protein 1 (Mipu1) is a newly discovered upregulated gene produced in rats during the myocardial ischemic preconditioning process. Mipu1 cDNA contains a 1824-base pair open reading frame and encodes a 608 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and classical zinc finger C{sub 2}H{sub 2} motifs in the C-terminus. Mipu1 protein is located in the cell nucleus. Recent studies found that Mipu1 has a protective effect on the ischemia-reperfusion injury of heart, brain, and other organs. As a nuclear factor, Mipu1 may perform its protective function through directly transcribing and repressing the expression of proapoptotic genes to repress cell apoptosis. In addition, Mipu1 also plays an important role in regulating the gene expression of downstream inflammatory mediators by inhibiting the activation of activator protein-1 and serum response element.

  15. Global and stage specific patterns of Krüppel-associated-box zinc finger protein gene expression in murine early embryonic cells.

    Directory of Open Access Journals (Sweden)

    Andrea Corsinotti

    Full Text Available Highly coordinated transcription networks orchestrate the self-renewal of pluripotent stem cell and the earliest steps of mammalian development. KRAB-containing zinc finger proteins represent the largest group of transcription factors encoded by the genomes of higher vertebrates including mice and humans. Together with their putatively universal cofactor KAP1, they have been implicated in events as diverse as the silencing of endogenous retroelements, the maintenance of imprinting and the pluripotent self-renewal of embryonic stem cells, although the genomic targets and specific functions of individual members of this gene family remain largely undefined. Here, we first generated a list of Ensembl-annotated KRAB-containing genes encoding the mouse and human genomes. We then defined the transcription levels of these genes in murine early embryonic cells. We found that the majority of KRAB-ZFP genes are expressed in mouse pluripotent stem cells and other early progenitors. However, we also identified distinctively cell- or stage-specific patterns of expression, some of which are pluripotency-restricted. Finally, we determined that individual KRAB-ZFP genes exhibit highly distinctive modes of expression, even when grouped in genomic clusters, and that these cannot be correlated with the presence of prototypic repressive or activating chromatin marks. These results pave the way to delineating the role of specific KRAB-ZFPs in early embryogenesis.

  16. Zinc Deficiency in Humans and its Amelioration

    OpenAIRE

    Yashbir Singh Shivay

    2015-01-01

    Zinc (Zn) deficiency in humans has recently received considerable attention. Global mortality in children under 5 years of age in 2004 due to Zn deficiency was estimated at 4,53,207 as against 6,66,771 for vitamin A deficiency; 20,854 for iron deficiency and 3,619 for iodine deficiency. In humans 2800-3000 proteins contain Zn prosthetic group and Zn is an integral component of zinc finger prints that regulate DNA transcription. Zinc is a Type-2 nutrient, which means that its concentration in ...

  17. Intrinsic and extrinsic connections of Tet3 dioxygenase with CXXC zinc finger modules.

    Directory of Open Access Journals (Sweden)

    Nan Liu

    Full Text Available Tet proteins are emerging as major epigenetic modulators of cell fate and plasticity. However, little is known about how Tet proteins are targeted to selected genomic loci in distinct biological contexts. Previously, a CXXC-type zinc finger domain in Tet1 was shown to bind CpG-rich DNA sequences. Interestingly, in human and mouse the Tet2 and Tet3 genes are adjacent to Cxxc4 and Cxxc10-1, respectively. The CXXC domains encoded by these loci, together with those in Tet1 and Cxxc5, identify a distinct homology group within the CXXC domain family. Here we provide evidence for alternative mouse Tet3 transcripts including the Cxxc10-1 sequence (Tet3(CXXC and for an interaction between Tet3 and Cxxc4. In vitro Cxxc4 and the isolated CXXC domains of Tet1 and Tet3(CXXC bind DNA substrates with similar preference towards the modification state of cytosine at a single CpG site. In vivo Tet1 and Tet3 isoforms with and without CXXC domain hydroxylate genomic 5-methylcytosine with similar activity. Relative transcript levels suggest that distinct ratios of Tet3(CXXC isoforms and Tet3-Cxxc4 complex may be present in adult tissues. Our data suggest that variable association with CXXC modules may contribute to context specific functions of Tet proteins.

  18. Complex Interdependence Regulates Heterotypic Transcription Factor Distribution and Coordinates Cardiogenesis.

    Science.gov (United States)

    Luna-Zurita, Luis; Stirnimann, Christian U; Glatt, Sebastian; Kaynak, Bogac L; Thomas, Sean; Baudin, Florence; Samee, Md Abul Hassan; He, Daniel; Small, Eric M; Mileikovsky, Maria; Nagy, Andras; Holloway, Alisha K; Pollard, Katherine S; Müller, Christoph W; Bruneau, Benoit G

    2016-02-25

    Transcription factors (TFs) are thought to function with partners to achieve specificity and precise quantitative outputs. In the developing heart, heterotypic TF interactions, such as between the T-box TF TBX5 and the homeodomain TF NKX2-5, have been proposed as a mechanism for human congenital heart defects. We report extensive and complex interdependent genomic occupancy of TBX5, NKX2-5, and the zinc finger TF GATA4 coordinately controlling cardiac gene expression, differentiation, and morphogenesis. Interdependent binding serves not only to co-regulate gene expression but also to prevent TFs from distributing to ectopic loci and activate lineage-inappropriate genes. We define preferential motif arrangements for TBX5 and NKX2-5 cooperative binding sites, supported at the atomic level by their co-crystal structure bound to DNA, revealing a direct interaction between the two factors and induced DNA bending. Complex interdependent binding mechanisms reveal tightly regulated TF genomic distribution and define a combinatorial logic for heterotypic TF regulation of differentiation. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Over-expression of a NAC 67 transcription factor from finger millet (Eleusine coracana L.) confers tolerance against salinity and drought stress in rice.

    Science.gov (United States)

    Rahman, Hifzur; Ramanathan, Valarmathi; Nallathambi, Jagedeeshselvam; Duraialagaraja, Sudhakar; Muthurajan, Raveendran

    2016-05-11

    NAC proteins (NAM (No apical meristem), ATAF (Arabidopsis transcription activation factor) and CUC (cup-shaped cotyledon)) are plant-specific transcription factors reported to be involved in regulating growth, development and stress responses. Salinity responsive transcriptome profiling in a set of contrasting finger millet genotypes through RNA-sequencing resulted in the identification of a NAC homolog (EcNAC 67) exhibiting differential salinity responsive expression pattern. Full length cDNA of EcNAC67 was isolated, characterized and validated for its role in abiotic stress tolerance through agrobacterium mediated genetic transformation in a rice cultivar ASD16. Bioinformatics analysis of putative NAC transcription factor (TF) isolated from a salinity tolerant finger millet showed its genetic relatedness to NAC67 family TFs in related cereals. Putative transgenic lines of rice over-expressing EcNAC67 were generated through Agrobacterium mediated transformation and presence/integration of transgene was confirmed through PCR and southern hybridization analysis. Transgenic rice plants harboring EcNAC67 showed enhanced tolerance against drought and salinity under greenhouse conditions. Transgenic rice plants were found to possess higher root and shoot biomass during stress and showed better revival ability upon relief from salinity stress. Upon drought stress, transgenic lines were found to maintain higher relative water content and lesser reduction in grain yield when compared to non-transgenic ASD16 plants. Drought induced spikelet sterility was found to be much lower in the transgenic lines than the non-transgenic ASD16. Results revealed the significant role of EcNAC67 in modulating responses against dehydration stress in rice. No detectable abnormalities in the phenotypic traits were observed in the transgenic plants under normal growth conditions. Results indicate that EcNAC67 can be used as a novel source for engineering tolerance against drought and salinity

  20. Regulation of trichome development in tobacco by JcZFP8, a C2H2 zinc finger protein gene from Jatropha curcas L.

    Science.gov (United States)

    Shi, Xiaodong; Gu, Yuxi; Dai, Tingwei; Wu, Yang; Wu, Peng; Xu, Ying; Chen, Fang

    2018-06-05

    Trichomes are epidermal outgrowths of plant tissues that can secrete or store large quantities of secondary metabolites, which contribute to plant defense responses against stress. The use of bioengineering methods for regulating the development of trichomes and metabolism is a widely researched topic. In the present study, we demonstrate that JcZFP8, a C2H2 zinc finger protein gene from Jatropha curcas L., can regulate trichome development in transgenic tobacco. To understand the underlying mechanisms, we performed transcriptome profiling of overexpression JcZFP8 transgenic plants and wild-type tobacco. Based on the analysis of differentially expressed genes, we determined that genes of the plant hormone signal transduction pathway was significantly enriched, suggesting that these pathways were modulated in the transgenic plants. In addition, the transcript levels of the known trichome-related genes in Arabidopsis were not significantly changed, whereas CycB2 and MYB genes were differentially expressed in the transgenic plants. Despite tobacco and Arabidopsis have different types of trichomes, all the pathways were associated with C2H2 zinc finger protein genes. Our findings help us to understand the regulation of multicellular trichome formation and suggest a new metabolic engineering method for the improvement of plants. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Targeted genome regulation via synthetic programmable transcriptional regulators

    KAUST Repository

    Piatek, Agnieszka Anna; Mahfouz, Magdy M.

    2016-01-01

    genes in linear and interacting pathways in a native context. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like proteins (TALE) are amenable to bioengineering to bind DNA target sequences of interest. As a result, ZF

  2. Zinc finger protein rotund deficiency affects development of the thoracic leg in Bombyx mori.

    Science.gov (United States)

    Zhou, Chun-Yan; Zha, Xing-Fu; Liu, Hua-Wei; Xia, Qing-You

    2017-06-01

    The insect limb develops from the imaginal disc or larval leg during metamorphosis. The molecular mechanisms involved in the development from the larval to the adult leg are poorly understood. Herein, we cloned the full length of a zinc finger gene rotund from Bombyx mori (Bmrn), which contained a 1419 bp open reading frame, and encoded a 473 amino acid protein. Reverse transcription polymerase chain reaction and Western blot analyses demonstrated that Bmrn was expressed at higher levels in the epidermis than in other tissues tested, and it showed a very high expression level during metamorphosis. Knock-down of Bmrn produced defects in the tarsus and pretarsus, including the fusion and reduction of tarsomeres, and the developmental arrest of pretarsus. Our data showed that Bmrn is involved in the formation of the tarsus and pretarsus, whereas its homologous gene in Drosophila has been shown to affect three tarsal segments (t2-t4), suggesting that the remodeling of the leg has involved changes in the patterning of gene regulation during evolution. © 2016 Institute of Zoology, Chinese Academy of Sciences.

  3. Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases.

    Science.gov (United States)

    Cai, Yujia; Bak, Rasmus O; Mikkelsen, Jacob Giehm

    2014-04-24

    Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in 'all-in-one' lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases.DOI: http://dx.doi.org/10.7554/eLife.01911.001. Copyright © 2014, Cai et al.

  4. The Zinc Finger Transcription Factor Sp9 Is Required for the Development of Striatopallidal Projection Neurons

    Directory of Open Access Journals (Sweden)

    Qiangqiang Zhang

    2016-08-01

    Full Text Available Striatal medium-sized spiny neurons (MSNs, composed of striatonigral and striatopallidal neurons, are derived from the lateral ganglionic eminence (LGE. We find that the transcription factor Sp9 is expressed in LGE progenitors that generate nearly all striatal MSNs and that Sp9 expression is maintained in postmitotic striatopallidal MSNs. Sp9-null mice lose most striatopallidal MSNs because of decreased proliferation of striatopallidal MSN progenitors and increased Bax-dependent apoptosis, whereas the development of striatonigral neurons is largely unaffected. ChIP qPCR provides evidence that Ascl1 directly binds the Sp9 promoter. RNA-seq and in situ hybridization reveal that Sp9 promotes expression of Adora2a, P2ry1, Gpr6, and Grik3 in the LGE and striatum. Thus, Sp9 is crucial for the generation, differentiation, and survival of striatopallidal MSNs.

  5. Supplementary data: Table 1. Identification numbers of sequences ...

    Indian Academy of Sciences (India)

    Lenovo

    . -. -. SP1. Stimulating protein 1, ubiquitous zinc finger transcription factor. +. +. GSH2. Homeodomain transcription factor Gsh-2. -. +. RUSH. SWI/SNF related nucleophosphoproteins with a RING finger DNA binding motif. +. +. TAL1. T-cell acute ...

  6. Phosphorylation of the leukemic oncoprotein EVI1 on serine 196 modulates DNA binding, transcriptional repression and transforming ability.

    Directory of Open Access Journals (Sweden)

    Daniel J White

    Full Text Available The EVI1 (ecotropic viral integration site 1 gene at 3q26 codes for a transcriptional regulator with an essential role in haematopoiesis. Overexpression of EVI1 in acute myeloid leukaemia (AML is frequently associated with 3q26 rearrangements and confers extremely poor prognosis. EVI1 mediates transcriptional regulation, signalling, and epigenetic modifications by interacting with DNA, proteins and protein complexes. To explore to what extent protein phosphorylation impacts on EVI1 functions, we analysed endogenous EVI1 protein from a high EVI1 expressing Fanconi anaemia (FA derived AML cell line. Mass spectrometric analysis of immunoprecipitated EVI1 revealed phosphorylation at serine 196 (S196 in the sixth zinc finger of the N-terminal zinc finger domain. Mutated EVI1 with an aspartate substitution at serine 196 (S196D, which mimics serine phosphorylation of this site, exhibited reduced DNA-binding and transcriptional repression from a gene promotor selectively targeted by the N-terminal zinc finger domain. Forced expression of the S196D mutant significantly reduced EVI1 mediated transformation of Rat1 fibroblasts. While EVI1-mediated serial replating of murine haematopoietic progenitors was maintained by EVI1-S196D, this was associated with significantly higher Evi1-trancript levels compared with WT-EVI1 or EVI1-S196A, mimicking S196 non-phosphorylated EVI1. These data suggest that EVI1 function is modulated by phosphorylation of the first zinc finger domain.

  7. Transcriptome-wide identification of Camellia sinensis WRKY transcription factors in response to temperature stress.

    Science.gov (United States)

    Wu, Zhi-Jun; Li, Xing-Hui; Liu, Zhi-Wei; Li, Hui; Wang, Yong-Xin; Zhuang, Jing

    2016-02-01

    Tea plant [Camellia sinensis (L.) O. Kuntze] is a leaf-type healthy non-alcoholic beverage crop, which has been widely introduced worldwide. Tea is rich in various secondary metabolites, which are important for human health. However, varied climate and complex geography have posed challenges for tea plant survival. The WRKY gene family in plants is a large transcription factor family that is involved in biological processes related to stress defenses, development, and metabolite synthesis. Therefore, identification and analysis of WRKY family transcription factors in tea plant have a profound significance. In the present study, 50 putative C. sinensis WRKY proteins (CsWRKYs) with complete WRKY domain were identified and divided into three Groups (Group I-III) on the basis of phylogenetic analysis results. The distribution of WRKY family transcription factors among plantae, fungi, and protozoa showed that the number of WRKY genes increased in higher plant, whereas the number of these genes did not correspond to the evolutionary relationships of different species. Structural feature and annotation analysis results showed that CsWRKY proteins contained WRKYGQK/WRKYGKK domains and C2H2/C2HC-type zinc-finger structure: D-X18-R-X1-Y-X2-C-X4-7-C-X23-H motif; CsWRKY proteins may be associated with the biological processes of abiotic and biotic stresses, tissue development, and hormone and secondary metabolite biosynthesis. Temperature stresses suggested that the candidate CsWRKY genes were involved in responses to extreme temperatures. The current study established an extensive overview of the WRKY family transcription factors in tea plant. This study also provided a global survey of CsWRKY transcription factors and a foundation of future functional identification and molecular breeding.

  8. Manipulation of flowering time and branching by overexpression of the tomato transcription factor SlZFP2.

    Science.gov (United States)

    Weng, Lin; Bai, Xiaodong; Zhao, Fangfang; Li, Rong; Xiao, Han

    2016-12-01

    Flowering of higher plants is orchestrated by complex regulatory networks through integration of various environmental signals such as photoperiod, temperature, light quality and developmental cues. In Arabidopsis, transcription of the flowering integrator gene FLOWERING LOCUS T (FT) that several flowering pathways converge to is directly regulated by more than ten transcription factors. However, very little is known about the transcriptional regulation of the FT homolog SINGLE FLOWER TRUESS (SFT) in the day-neutral plant tomato (Solanum lycopersicum). Previously, we showed that the zinc finger transcription factor SlZFP2 plays important roles in regulation of seed germination and fruit ripening in tomato and also found that overexpression of SlZFP2 impacted flowering and branching. Here, we characterized in detail the early flowering and high branching phenotypes by overexpression of this transcription factor. Our data showed that overexpression of SlZFP2 accelerated flowering in an SFT-dependent manner as demonstrated by elevated SFT expression in the leaves and the transcription factor's binding ability to SFT promoter in vitro and in vivo. Furthermore, overexpression of the SlZFP2 gene in the sft plants failed to rescue the mutant's late flowering. Through analysis of grafting phenotype, growth response of branches to auxin application and transcriptome profiling by RNA sequencing, we also showed that overexpression of SlZFP2 affected shoot apical dominance through multiple regulatory pathways. Our results suggest that the transcription factor SlZFP2 has potential applications in genetic modification of plant architecture and flowering time for tomato production and other crops as well. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  9. Rmt1 catalyzes zinc-finger independent arginine methylation of ribosomal protein Rps2 in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Lipson, Rebecca S.; Webb, Kristofor J.; Clarke, Steven G.

    2010-01-01

    Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here, we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These results are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells.

  10. Identification of novel transcription factors regulating secondary cell wall formation in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Hua eCassan-Wang

    2013-06-01

    Full Text Available The presence of lignin in secondary cell walls (SCW is a major factor preventing hydrolytic enzymes from gaining access to cellulose, thereby limiting the saccharification potential of plant biomass. To understand how lignification is regulated is a prerequisite for selecting plant biomass better adapted to bioethanol production. Because transcriptional regulation is a major mechanism controlling the expression of genes involved in lignin biosynthesis, our aim was to identify novel transcription factors dictating lignin profiles in the model plant Arabidopsis. To this end, we have developed a post-genomic approach by combining four independent in-house SCW-related transcriptome datasets obtained from (i the fiber cell wall-deficient wat1 Arabidopsis mutant, (ii Arabidopsis lines over-expressing either the master regulatory activator EgMYB2 or (iii the repressor EgMYB1 and finally (iv Arabidopsis orthologs of Eucalyptus xylem-expressed genes. This allowed us to identify 502 up- or down-regulated transcription factors. We preferentially selected those present in more than one dataset and further analyzed their in silico expression patterns as an additional selection criteria. This selection process led to 80 candidates. Notably, 16 of them were already proven to regulate SCW formation, thereby validating the overall strategy. Then, we phenotyped 43 corresponding mutant lines focusing on histological observations of xylem and interfascicular fibers. This phenotypic screen revealed six mutant lines exhibiting altered lignification patterns. Two of them (blh6 and a zinc finger transcription factor presented hypolignified SCW. Three others (myb52, myb-like TF, hb5 showed hyperlignified SCW whereas the last one (hb15 showed ectopic lignification. In addition, our meta-analyses highlighted a reservoir of new potential regulators adding to the gene network regulating SCW but also opening new avenues to ultimately improve SCW composition for biofuel

  11. Molecular cloning and expression analysis of a zebrafish novel zinc finger protein gene rnf141

    Directory of Open Access Journals (Sweden)

    Wenqian Deng

    2009-01-01

    Full Text Available ZNF230 is a novel zinc finger gene cloned by our laboratory. In order to understand the potential functions of this gene in vertebrate development, we cloned the zebrafish orthologue of human ZNF230, named rnf141. The cDNA fragment of rnf141 was obtained by rapid amplification of cDNA ends (RACE. The open reading frame (ORF encodes a polypeptide of 222 amino acids which shares 75.65% identity with the human ZNF230. RT-PCR analysis in zebrafish embryo and adult tissues revealed that rnf141 transcripts are maternally derived and that rnf141 mRNA has a broad distribution. Zygotic rnf141 message is strongly localized in the central nervous system, as shown by whole-mount in situ hybridization. Knockdown and over expression of rnf141 can induce abnormal phenotypes, including abnormal development of brain, as well as yolk sac and axis extendsion. Marker gene analysis showed that rnf141 may play a role in normal dorsoventral patterning of zebrafish embryos, suggesting that rnf141 may have a broad function during early development of vertebrates.

  12. Role of Bmznf-2, a Bombyx mori CCCH zinc finger gene, in masculinisation and differential splicing of Bmtra-2.

    Science.gov (United States)

    Gopinath, Gajula; Arunkumar, Kallare P; Mita, Kazuei; Nagaraju, Javaregowda

    2016-08-01

    Deciphering the regulatory factors involved in Bombyx mori sex determination has been a puzzle, challenging researchers for nearly a century now. The pre-mRNA of B. mori doublesex (Bmdsx), a master regulator gene of sexual differentiation, is differentially spliced, producing Bmdsxm and Bmdsxf transcripts in males and females respectively. The putative proteins encoded by these differential transcripts orchestrate antagonistic functions, which lead to sexual differentiation. A recent study in B. mori illustrated the role of a W-derived fem piRNA in conferring femaleness. In females, the fem piRNA was shown to suppress the activity of a Z-linked CCCH type zinc finger (znf) gene, Masculiniser (masc), which indirectly promotes the Bmdsxm type of splicing. In this study, we report a novel autosomal (Chr 25) CCCH type znf motif encoding gene Bmznf-2 as one of the potential factors in the Bmdsx sex specific differential splicing, and we also provide insights into its role in the alternative splicing of Bmtra2 by using ovary derived BmN cells. Over-expression of Bmznf-2 induced Bmdsxm type of splicing (masculinisation) with a correspondingly reduced expression of Bmdsxf type isoform in BmN cells. Further, the site-directed mutational studies targeting the tandem CCCH znf motifs revealed their indispensability in the observed phenotype of masculinisation. Additionally, the dual luciferase assays in BmN cells using 5' UTR region of the Bmznf-2 strongly implied the existence of a translational repression over this gene. From these findings, we propose Bmznf-2 to be one of the potential factors of masculinisation similar to Masc. From the growing number of Bmdsx splicing regulators, we assume that the sex determination cascade of B. mori is quite intricate in nature; hence, it has to be further investigated for its comprehensive understanding. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. The DOF transcription factor Dof5.1 influences leaf axial patterning by promoting Revoluta transcription in Arabidopsis

    KAUST Repository

    Kim, Hyungsae

    2010-10-05

    Dof proteins are transcription factors that have a conserved single zinc finger DNA-binding domain. In this study, we isolated an activation tagging mutant Dof5.1-D exhibiting an upward-curling leaf phenotype due to enhanced expression of the REV gene that is required for establishing adaxialabaxial polarity. Dof5.1-D plants also had reduced transcript levels for IAA6 and IAA19 genes, indicating an altered auxin biosynthesis in Dof5.1-D. An electrophoretic mobility shift assay using the Dof5.1 DNA-binding motif and the REV promoter region indicated that the DNA-binding domain of Dof5.1 binds to a TAAAGT motif located in the 5′-distal promoter region of the REV promoter. Further, transient and chromatin immunoprecipitation assays verified binding activity of the Dof5.1 DNA-binding motif with the REV promoter. Consistent with binding assays, constitutive over-expression of the Dof5.1 DNA-binding domain in wild-type plants caused a downward-curling phenotype, whereas crossing Dof5.1-D to a rev mutant reverted the upward-curling phenotype of the Dof5.1-D mutant leaf to the wild-type. These results suggest that the Dof5.1 protein directly binds to the REV promoter and thereby regulates adaxialabaxial polarity. © 2010 Blackwell Publishing Ltd.

  14. The DOF transcription factor Dof5.1 influences leaf axial patterning by promoting Revoluta transcription in Arabidopsis

    KAUST Repository

    Kim, Hyungsae; Kim, Sungjin; Abbasi, Nazia; Bressan, Ray Anthony; Yun, Daejin; Yoo, Sangdong; Kwon, SukYun; Choi, Sangbong

    2010-01-01

    Dof proteins are transcription factors that have a conserved single zinc finger DNA-binding domain. In this study, we isolated an activation tagging mutant Dof5.1-D exhibiting an upward-curling leaf phenotype due to enhanced expression of the REV gene that is required for establishing adaxialabaxial polarity. Dof5.1-D plants also had reduced transcript levels for IAA6 and IAA19 genes, indicating an altered auxin biosynthesis in Dof5.1-D. An electrophoretic mobility shift assay using the Dof5.1 DNA-binding motif and the REV promoter region indicated that the DNA-binding domain of Dof5.1 binds to a TAAAGT motif located in the 5′-distal promoter region of the REV promoter. Further, transient and chromatin immunoprecipitation assays verified binding activity of the Dof5.1 DNA-binding motif with the REV promoter. Consistent with binding assays, constitutive over-expression of the Dof5.1 DNA-binding domain in wild-type plants caused a downward-curling phenotype, whereas crossing Dof5.1-D to a rev mutant reverted the upward-curling phenotype of the Dof5.1-D mutant leaf to the wild-type. These results suggest that the Dof5.1 protein directly binds to the REV promoter and thereby regulates adaxialabaxial polarity. © 2010 Blackwell Publishing Ltd.

  15. Phylogenetic analysis of F-bZIP transcription factors indicates conservation of the zinc deficiency response across land plants

    DEFF Research Database (Denmark)

    Castro, Pedro Humberto Araújo R F; Lilay, Grmay Hailu; Muñoz-Mérida, Antonio

    2017-01-01

    Basic leucine zipper (bZIP) transcription factors control important developmental and physiological processes in plants. In Arabidopsis thaliana, the three gene F-bZIP subfamily has been associated with zinc deficiency and salt stress response. Benefiting from the present abundance of plant genomic...... data, we performed an evolutionary and structural characterization of plant F-bZIPs. We observed divergence during seed plant evolution, into two groups and inferred different selective pressures for each. Group 1 contains AtbZIP19 and AtbZIP23 and appears more conserved, whereas Group 2, containing...... of AtZIP4. A survey of AtZIP4 orthologs promoters across different plant taxa revealed an enrichment of the Zinc Deficiency Response Element (ZDRE) to which both AtbZIP19/23 bind. Overall, our results indicate that while the AtbZIP24 function in the regulation of the salt stress response may...

  16. Genome-wide analysis of the CCCH zinc finger family identifies tissue specific and stress responsive candidates in chickpea (Cicer arietinum L.).

    Science.gov (United States)

    Pradhan, Seema; Kant, Chandra; Verma, Subodh; Bhatia, Sabhyata

    2017-01-01

    The CCCH zinc finger is a group of proteins characterised by a typical motif consisting of three cysteine residues and one histidine residue. These proteins have been reported to play important roles in regulation of plant growth, developmental processes and environmental responses. In the present study, genome wide analysis of the CCCH zinc finger gene family was carried out in the available chickpea genome. Various bioinformatics tools were employed to predict 58 CCCH zinc finger genes in chickpea (designated CarC3H1-58), which were analysed for their physio-chemical properties. Phylogenetic analysis classified the proteins into 12 groups in which members of a particular group had similar structural organization. Further, the numbers as well as the types of CCCH motifs present in the CarC3H proteins were compared with those from Arabidopsis and Medicago truncatula. Synteny analysis revealed valuable information regarding the evolution of this gene family. Tandem and segmental duplication events were identified and their Ka/Ks values revealed that the CarC3H gene family in chickpea had undergone purifying selection. Digital, as well as real time qRT-PCR expression analysis was performed which helped in identification of several CarC3H members that expressed preferentially in specific chickpea tissues as well as during abiotic stresses (desiccation, cold, salinity). Moreover, molecular characterization of an important member CarC3H45 was carried out. This study provides comprehensive genomic information about the important CCCH zinc finger gene family in chickpea. The identified tissue specific and abiotic stress specific CCCH genes could be potential candidates for further characterization to delineate their functional roles in development and stress.

  17. Control of Hepatic Gluconeogenesis by the Promyelocytic Leukemia Zinc Finger Protein

    Science.gov (United States)

    Chen, Siyu; Qian, Jinchun; Shi, Xiaoli; Gao, Tingting; Liang, Tingming

    2014-01-01

    The promyelocytic leukemia zinc finger (PLZF) protein is involved in major biological processes including energy metabolism, although its role remains unknown. In this study, we demonstrated that hepatic PLZF expression was induced in fasted or diabetic mice. PLZF promoted gluconeogenic gene expression and hepatic glucose output, leading to hyperglycemia. In contrast, hepatic PLZF knockdown improved glucose homeostasis in db/db mice. Mechanistically, peroxisome proliferator-activated receptor γ coactivator 1α and the glucocorticoid receptor synergistically activated PLZF expression. We conclude that PLZF is a critical regulator of hepatic gluconeogenesis. PLZF manipulation may benefit the treatment of metabolic diseases associated with gluconeogenesis. PMID:25333514

  18. Predicting success of oligomerized pool engineering (OPEN for zinc finger target site sequences

    Directory of Open Access Journals (Sweden)

    Goodwin Mathew J

    2010-11-01

    Full Text Available Abstract Background Precise and efficient methods for gene targeting are critical for detailed functional analysis of genomes and regulatory networks and for potentially improving the efficacy and safety of gene therapies. Oligomerized Pool ENgineering (OPEN is a recently developed method for engineering C2H2 zinc finger proteins (ZFPs designed to bind specific DNA sequences with high affinity and specificity in vivo. Because generation of ZFPs using OPEN requires considerable effort, a computational method for identifying the sites in any given gene that are most likely to be successfully targeted by this method is desirable. Results Analysis of the base composition of experimentally validated ZFP target sites identified important constraints on the DNA sequence space that can be effectively targeted using OPEN. Using alternate encodings to represent ZFP target sites, we implemented Naïve Bayes and Support Vector Machine classifiers capable of distinguishing "active" targets, i.e., ZFP binding sites that can be targeted with a high rate of success, from those that are "inactive" or poor targets for ZFPs generated using current OPEN technologies. When evaluated using leave-one-out cross-validation on a dataset of 135 experimentally validated ZFP target sites, the best Naïve Bayes classifier, designated ZiFOpT, achieved overall accuracy of 87% and specificity+ of 90%, with an ROC AUC of 0.89. When challenged with a completely independent test set of 140 newly validated ZFP target sites, ZiFOpT performance was comparable in terms of overall accuracy (88% and specificity+ (92%, but with reduced ROC AUC (0.77. Users can rank potentially active ZFP target sites using a confidence score derived from the posterior probability returned by ZiFOpT. Conclusion ZiFOpT, a machine learning classifier trained to identify DNA sequences amenable for targeting by OPEN-generated zinc finger arrays, can guide users to target sites that are most likely to function

  19. Oncogenes Activate an Autonomous Transcriptional Regulatory Circuit That Drives Glioblastoma

    Directory of Open Access Journals (Sweden)

    Dinesh K. Singh

    2017-01-01

    Full Text Available Efforts to identify and target glioblastoma (GBM drivers have primarily focused on receptor tyrosine kinases (RTKs. Clinical benefits, however, have been elusive. Here, we identify an SRY-related box 2 (SOX2 transcriptional regulatory network that is independent of upstream RTKs and capable of driving glioma-initiating cells. We identified oligodendrocyte lineage transcription factor 2 (OLIG2 and zinc-finger E-box binding homeobox 1 (ZEB1, which are frequently co-expressed irrespective of driver mutations, as potential SOX2 targets. In murine glioma models, we show that different combinations of tumor suppressor and oncogene mutations can activate Sox2, Olig2, and Zeb1 expression. We demonstrate that ectopic co-expression of the three transcription factors can transform tumor-suppressor-deficient astrocytes into glioma-initiating cells in the absence of an upstream RTK oncogene. Finally, we demonstrate that the transcriptional inhibitor mithramycin downregulates SOX2 and its target genes, resulting in markedly reduced proliferation of GBM cells in vivo.

  20. Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL

    International Nuclear Information System (INIS)

    Do, To Uyen; Ho, Bay; Shih, Shyh-Jen; Vaughan, Andrew

    2012-01-01

    Highlights: ► A Zinc Finger Nuclease (ZFN) targeting a leukemogenic hot spot for rearrangement in MLL is created. ► The novel ZFN efficiently cleaves MLL exon 13. ► Despite MLL cleavage and evidence of mis-repair, no leukemogenic translocations were produced. ► MLL cleavage alone is insufficient to generate leukemogenic translocations. - Abstract: Radiation treatment or chemotherapy has been linked with a higher risk of secondary cancers such as therapy related Acute Myeloid Leukemia (tAML). Several of these cancers have been shown to be correlated to the introduction of double stranded breaks (DSB) and rearrangements within the Mixed Lineage Leukemia (MLL) gene. We used Zinc Finger Nucleases (ZFNs) to introduce precise cuts within MLL to examine how a single DNA DSB might lead to chromosomal rearrangements. A ZFN targeting exon 13 within the Breakpoint Cluster Region of MLL was transiently expressed in a human lymphoblast cell line originating from a CML patient. Although FISH analysis showed ZFN DSB at this region increased the rate of MLL fragmentation, we were unable to detect leukemogenic rearrangements or translocations via inverse PCR. Interestingly, gene fragmentation as well as small interstitial deletions, insertions and base substitutions increased with the inhibition of DNA-PK, suggesting repair of this particular DSB is linked to non-homologous end joining (NHEJ). Although mis-repair of DSBs may be necessary for the initiation of leukemogenic translocations, a MLL targeted DNA break alone is insufficient

  1. Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL

    Energy Technology Data Exchange (ETDEWEB)

    Do, To Uyen [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Ho, Bay; Shih, Shyh-Jen [Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Vaughan, Andrew, E-mail: Andrew.vaughan@ucdmc.ucdavis.edu [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States)

    2012-12-15

    Highlights: ► A Zinc Finger Nuclease (ZFN) targeting a leukemogenic hot spot for rearrangement in MLL is created. ► The novel ZFN efficiently cleaves MLL exon 13. ► Despite MLL cleavage and evidence of mis-repair, no leukemogenic translocations were produced. ► MLL cleavage alone is insufficient to generate leukemogenic translocations. - Abstract: Radiation treatment or chemotherapy has been linked with a higher risk of secondary cancers such as therapy related Acute Myeloid Leukemia (tAML). Several of these cancers have been shown to be correlated to the introduction of double stranded breaks (DSB) and rearrangements within the Mixed Lineage Leukemia (MLL) gene. We used Zinc Finger Nucleases (ZFNs) to introduce precise cuts within MLL to examine how a single DNA DSB might lead to chromosomal rearrangements. A ZFN targeting exon 13 within the Breakpoint Cluster Region of MLL was transiently expressed in a human lymphoblast cell line originating from a CML patient. Although FISH analysis showed ZFN DSB at this region increased the rate of MLL fragmentation, we were unable to detect leukemogenic rearrangements or translocations via inverse PCR. Interestingly, gene fragmentation as well as small interstitial deletions, insertions and base substitutions increased with the inhibition of DNA-PK, suggesting repair of this particular DSB is linked to non-homologous end joining (NHEJ). Although mis-repair of DSBs may be necessary for the initiation of leukemogenic translocations, a MLL targeted DNA break alone is insufficient.

  2. The membrane tethered transcription factor EcbZIP17 from finger millet promotes plant growth and enhances tolerance to abiotic stresses.

    Science.gov (United States)

    Ramakrishna, Chopperla; Singh, Sonam; Raghavendrarao, Sangala; Padaria, Jasdeep C; Mohanty, Sasmita; Sharma, Tilak Raj; Solanke, Amolkumar U

    2018-02-01

    The occurrence of various stresses, as the outcome of global climate change, results in the yield losses of crop plants. Prospecting of genes in stress tolerant plant species may help to protect and improve their agronomic performance. Finger millet (Eleusine coracana L.) is a valuable source of superior genes and alleles for stress tolerance. In this study, we isolated a novel endoplasmic reticulum (ER) membrane tethered bZIP transcription factor from finger millet, EcbZIP17. Transgenic tobacco plants overexpressing this gene showed better vegetative growth and seed yield compared with wild type (WT) plants under optimal growth conditions and confirmed upregulation of brassinosteroid signalling genes. Under various abiotic stresses, such as 250 mM NaCl, 10% PEG6000, 400 mM mannitol, water withdrawal, and heat stress, the transgenic plants showed higher germination rate, biomass, primary and secondary root formation, and recovery rate, compared with WT plants. The transgenic plants exposed to an ER stress inducer resulted in greater leaf diameter and plant height as well as higher expression of the ER stress-responsive genes BiP, PDIL, and CRT1. Overall, our results indicated that EcbZIP17 improves plant growth at optimal conditions through brassinosteroid signalling and provide tolerance to various environmental stresses via ER signalling pathways.

  3. An A20/AN1-type zinc finger protein modulates gibberellins and abscisic acid contents and increases sensitivity to abiotic stress in rice (Oryza sativa).

    Science.gov (United States)

    Zhang, Ye; Lan, Hongxia; Shao, Qiaolin; Wang, Ruqin; Chen, Hui; Tang, Haijuan; Zhang, Hongsheng; Huang, Ji

    2016-01-01

    The plant hormones gibberellins (GA) and abscisic acid (ABA) play important roles in plant development and stress responses. Here we report a novel A20/AN1-type zinc finger protein ZFP185 involved in GA and ABA signaling in the regulation of growth and stress response. ZFP185 was constitutively expressed in various rice tissues. Overexpression of ZFP185 in rice results in a semi-dwarfism phenotype, reduced cell size, and the decrease of endogenous GA3 content. By contrast, higher GA3 content was observed in RNAi plants. The application of exogenous GA3 can fully rescue the semi-dwarfism phenotype of ZFP185 overexpressing plants, suggesting the negative role of ZFP185 in GA biosynthesis. Besides GA, overexpression of ZFP185 decreased ABA content and expression of several ABA biosynthesis-related genes. Moreover, it was found that ZFP185, unlike previously known A20/AN1-type zinc finger genes, increases sensitivity to drought, cold, and salt stresses, implying the negative role of ZFP185 in stress tolerance. ZFP185 was localized in the cytoplasm and lacked transcriptional activation potential. Our study suggests that ZFP185 regulates plant growth and stress responses by affecting GA and ABA biosynthesis in rice. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  4. Development of novel metabolite-responsive transcription factors via transposon-mediated protein fusion.

    Science.gov (United States)

    Younger, Andrew K D; Su, Peter Y; Shepard, Andrea J; Udani, Shreya V; Cybulski, Thaddeus R; Tyo, Keith E J; Leonard, Joshua N

    2018-02-01

    Naturally evolved metabolite-responsive biosensors enable applications in metabolic engineering, ranging from screening large genetic libraries to dynamically regulating biosynthetic pathways. However, there are many metabolites for which a natural biosensor does not exist. To address this need, we developed a general method for converting metabolite-binding proteins into metabolite-responsive transcription factors-Biosensor Engineering by Random Domain Insertion (BERDI). This approach takes advantage of an in vitro transposon insertion reaction to generate all possible insertions of a DNA-binding domain into a metabolite-binding protein, followed by fluorescence activated cell sorting to isolate functional biosensors. To develop and evaluate the BERDI method, we generated a library of candidate biosensors in which a zinc finger DNA-binding domain was inserted into maltose binding protein, which served as a model well-studied metabolite-binding protein. Library diversity was characterized by several methods, a selection scheme was deployed, and ultimately several distinct and functional maltose-responsive transcriptional biosensors were identified. We hypothesize that the BERDI method comprises a generalizable strategy that may ultimately be applied to convert a wide range of metabolite-binding proteins into novel biosensors for applications in metabolic engineering and synthetic biology. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. The AT-Hook motif as a versatile minor groove anchor for promoting DNA binding of transcription factor fragments? ?Electronic supplementary information (ESI) available: Peptide synthesis, full experimental procedures and analytical data of the peptides and products obtained. See DOI: 10.1039/c5sc01415h Click here for additional data file.

    OpenAIRE

    Rodr?guez, J?ssica; Mosquera, Jes?s; Couceiro, Jose R.; V?zquez, M. Eugenio; Mascare?as, Jos? L.

    2015-01-01

    We report the development of chimeric DNA binding peptides comprising a DNA binding fragment of natural transcription factors (the basic region of a bZIP protein or a monomeric zinc finger module) and an AT-Hook peptide motif. The resulting peptide conjugates display high DNA affinity and excellent sequence selectivity. Furthermore, the AT-Hook motif also favors the cell internalization of the conjugates.

  6. Occupancy of a C2-C2 type 'zinc-finger' protein domain by copper. Direct observation by electrospray ionization mass spectrometry.

    Science.gov (United States)

    Hutchens, T W; Allen, M H; Li, C M; Yip, T T

    1992-09-07

    The metal ion specificity of most 'zinc-finger' metal binding domains is unknown. The human estrogen receptor protein contains two different C2-C2 type 'zinc-finger' sequences within its DNA-binding domain (ERDBD). Copper inhibits the function of this protein by mechanisms which remain unclear. We have used electrospray ionization mass spectrometry to evaluate directly the 71-residue ERDBD (K180-M250) in the absence and presence of Cu(II) ions. The ERDBD showed a high affinity for Cu and was completely occupied with 4 Cu bound; each Cu ion was evidently bound to only two ligand residues (net loss of only 2 Da per bound Cu). The Cu binding stoichiometry was confirmed by atomic absorption. These results (i) provide the first direct physical evidence for the ability of the estrogen receptor DNA-binding domain to bind Cu and (ii) document a twofold difference in the Zn- and Cu-binding capacity. Differences in the ERDBD domain structure with bound Zn and Cu are predicted. Given the relative intracellular contents of Zn and Cu, our findings demonstrate the need to investigate further the Cu occupancy of this and other zinc-finger domains both in vitro and in vivo.

  7. The DnaJ-like zinc finger domain protein PSA2 affects light acclimation and chloroplast development in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Yan-Wen eWang

    2016-03-01

    Full Text Available The biosynthesis of chlorophylls and carotenoids and the assembly of thylakoid membranes are critical for the photoautotrophic growth of plants. Different factors are involved in these two processes. In recent years, members of the DnaJ-like zinc finger domain proteins have been found to take part in the biogenesis and/or the maintenance of plastids. One member of this family of proteins, PSA2, was recently found to localize to the thylakoid lumen and regulate the accumulation of photosystem I. In this study, we report that the silencing of PSA2 in Arabidopsis thaliana resulted in variegated leaves and retarded growth. Although both chlorophylls and total carotenoids decreased in the psa2 mutant, violaxanthin and zeaxanthin accumulated in the mutant seedlings grown under growth condition. Lower levels of non-photochemical quenching and electron transport rate were also found in the psa2 mutant seedlings under growth condition compared with those of the wild-type plants, indicating an impaired capability to acclimate to normal light irradiance when PSA2 was silenced. Moreover, we also observed an abnormal assembly of grana thylakoids and poorly developed stroma thylakoids in psa2 chloroplasts. Taken together, our results demonstrate that PSA2 is a member of the DnaJ-like zinc finger domain protein family that affects light acclimation and chloroplast development.

  8. Protein, Calcium, Zinc, and Iron Contents of Finger Millet Grain Response to Varietal Differences and Phosphorus Application in Kenya

    Directory of Open Access Journals (Sweden)

    Wekha N. Wafula

    2018-02-01

    Full Text Available This study was carried out to investigate the influence of phosphorus fertilizers on the concentrations of nutrients, particularly calcium, protein, zinc, and iron in finger millet grains grown in different agro-ecologies in Kenya. The on-station experiments were carried out at Kiboko (Eastern Kenya, Kakamega, and Alupe (Western Kenya in 2015 during the short and long rainy seasons. The trials were laid out in a randomized complete block design (RCBD in a 4 × 3 factorial arrangement with three replicates. The treatments comprised of four levels of phosphorus (0, 12.5, 25.0 and 37.5 kg ha−1 P2O5 and three finger millet varieties (U-15, P-224 and a local variety. Application of phosphorus significantly (p ≤ 0.05 increased the protein content of finger millet grain in varieties in all the three sites. Variety U-15 had the highest protein content (11.0% at 25 kg ha−1 P2O5 with the control (zero P on variety P-224 eliciting the lowest (4.4% at Kiboko. At Kakamega, the 25 kg ha−1 P2O5 treatment with U-15 variety had the highest protein content (15.3% while the same variety at 12.5 kg ha−1 P2O5 rate elicited the highest protein content (15.0% at Alupe. Phosphorus application significantly enhanced the nutritional quality of finger millet grains specifically protein, calcium, iron, and zinc. Variety P-224 had the highest calcium content in all sites and highest iron content at Kakamega while the local varieties had the highest zinc content in all sites. The varieties responded differently to each quality component but generally, based on the protein content, the 25 kg ha−1 P2O5 is recommended.

  9. Reactive Oxygen Species Regulate the Inflammatory Function of NKT Cells through Promyelocytic Leukemia Zinc Finger.

    Science.gov (United States)

    Kim, Yeung-Hyen; Kumar, Ajay; Chang, Cheong-Hee; Pyaram, Kalyani

    2017-11-15

    Reactive oxygen species (ROS) are byproducts of aerobic metabolism and contribute to both physiological and pathological conditions as second messengers. ROS are essential for activation of T cells, but how ROS influence NKT cells is unknown. In the present study, we investigated the role of ROS in NKT cell function. We found that NKT cells, but not CD4 or CD8 T cells, have dramatically high ROS in the spleen and liver of mice but not in the thymus or adipose tissues. Accordingly, ROS-high NKT cells exhibited increased susceptibility and apoptotic cell death with oxidative stress. High ROS in the peripheral NKT cells were primarily produced by NADPH oxidases and not mitochondria. We observed that sorted ROS-high NKT cells were enriched in NKT1 and NKT17 cells, whereas NKT2 cells were dominant in ROS-low cells. Furthermore, treatment of NKT cells with antioxidants led to reduced frequencies of IFN-γ- and IL-17-expressing cells, indicating that ROS play a role in regulating the inflammatory function of NKT cells. The transcription factor promyelocytic leukemia zinc finger (PLZF) seemed to control the ROS levels. NKT cells from adipose tissues that do not express PLZF and those from PLZF haplodeficient mice have low ROS. Conversely, ROS were highly elevated in CD4 T cells from mice ectopically expressing PLZF. Thus, our findings demonstrate that PLZF controls ROS levels, which in turn governs the inflammatory function of NKT cells. Copyright © 2017 by The American Association of Immunologists, Inc.

  10. Isolation, Expression, and Promoter Analysis of GbWRKY2: A Novel Transcription Factor Gene from Ginkgo biloba

    Directory of Open Access Journals (Sweden)

    Yong-Ling Liao

    2015-01-01

    Full Text Available WRKY transcription factor is involved in multiple life activities including plant growth and development as well as biotic and abiotic responses. We identified 28 WRKY genes from transcriptome data of Ginkgo biloba according to conserved WRKY domains and zinc finger structure and selected three WRKY genes, which are GbWRKY2, GbWRKY16, and GbWRKY21, for expression pattern analysis. GbWRKY2 was preferentially expressed in flowers and strongly induced by methyl jasmonate. Here, we cloned the full-length cDNA and genomic DNA of GbWRKY2. The full-length cDNA of GbWRKY2 was 1,713 bp containing a 1,014 bp open reading frame encoding a polypeptide of 337 amino acids. The GbWRKY2 genomic DNA had one intron and two exons. The deduced GbWRKY2 contained one WRKY domain and one zinc finger motif. GbWRKY2 was classified into Group II WRKYs. Southern blot analysis revealed that GbWRKY2 was a single copy gene in G. biloba. Many cis-acting elements related to hormone and stress responses were identified in the 1,363 bp-length 5′-flanking sequence of GbWRKY2, including W-box, ABRE-motif, MYBCOREs, and PYRIMIDINE-boxes, revealing the molecular mechanism of upregulated expression of GbWRKY2 by hormone and stress treatments. Further functional characterizations in transiently transformed tobacco leaves allowed us to identify the region that can be considered as the minimal promoter.

  11. Molecular analysis of the interaction between the hematopoietic master transcription factors GATA-1 and PU.1

    DEFF Research Database (Denmark)

    Liew, Chu Wai; Rand, Kasper Dyrberg; Simpson, Raina J Y

    2006-01-01

    GATA-1 and PU.1 are transcription factors that control erythroid and myeloid development, respectively. The two proteins have been shown to function in an antagonistic fashion, with GATA-1 repressing PU.1 activity during erythropoiesis and PU.1 repressing GATA-1 function during myelopoiesis. It has...... also become clear that this functional antagonism involves direct interactions between the two proteins. However, the molecular basis for these interactions is not known, and a number of inconsistencies exist in the literature. We have used a range of biophysical methods to define the molecular details...... of the GATA-1-PU.1 interaction. A combination of NMR titration data and extensive mutagenesis revealed that the PU.1-Ets domain and the GATA-1 C-terminal zinc finger (CF) form a low affinity interaction in which specific regions of each protein are implicated. Surprisingly, the interaction cannot be disrupted...

  12. Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains

    NARCIS (Netherlands)

    Bird, L.E.; Ren, J.; Nettleship, J.E.; Folkers, G.E.|info:eu-repo/dai/nl/162277202; Owens, RJ; Stammers, D.K.

    2010-01-01

    Zhx1 to 3 (zinc-fingers and homeoboxes) form a set of paralogous genes encoding multi-domain proteins. ZHX proteins consist of two zinc fingers followed by five homeodomains. ZHXs have biological roles in cell cycle control by acting as co-repressors of the transcriptional regulator Nuclear Factor

  13. The C. elegans Tailless/TLX transcription factor nhr-67 controls neuronal identity and left/right asymmetric fate diversification.

    Science.gov (United States)

    Sarin, Sumeet; Antonio, Celia; Tursun, Baris; Hobert, Oliver

    2009-09-01

    An understanding of the molecular mechanisms of cell fate determination in the nervous system requires the elucidation of transcriptional regulatory programs that ultimately control neuron-type-specific gene expression profiles. We show here that the C. elegans Tailless/TLX-type, orphan nuclear receptor NHR-67 acts at several distinct steps to determine the identity and subsequent left/right (L/R) asymmetric subtype diversification of a class of gustatory neurons, the ASE neurons. nhr-67 controls several broad aspects of sensory neuron development and, in addition, triggers the expression of a sensory neuron-type-specific selector gene, che-1, which encodes a zinc-finger transcription factor. Subsequent to its induction of overall ASE fate, nhr-67 diversifies the fate of the two ASE neurons ASEL and ASER across the L/R axis by promoting ASER and inhibiting ASEL fate. This function is achieved through direct expression activation by nhr-67 of the Nkx6-type homeobox gene cog-1, an inducer of ASER fate, that is inhibited in ASEL through the miRNA lsy-6. Besides controlling bilateral and asymmetric aspects of ASE development, nhr-67 is also required for many other neurons of diverse lineage history and function to appropriately differentiate, illustrating the broad and diverse use of this type of transcription factor in neuronal development.

  14. Specific Labeling of Zinc Finger Proteins using Non-canonical Amino Acids and Copper-free Click Chemistry

    Science.gov (United States)

    Kim, Younghoon; Kim, Sung Hoon; Ferracane, Dean; Katzenellenbogen, John A.

    2012-01-01

    Zinc finger proteins (ZFPs) play a key role in transcriptional regulation and serve as invaluable tools for gene modification and genetic engineering. Development of efficient strategies for labeling metalloproteins such as ZFPs is essential for understanding and controlling biological processes. In this work, we engineered ZFPs containing cysteine-histidine (Cys2-His2) motifs by metabolic incorporation of the unnatural amino acid azidohomoalanine (AHA), followed by specific protein labeling via click chemistry. We show that cyclooctyne promoted [3 + 2] dipolar cycloaddition with azides, known as copper-free click chemistry, provides rapid and specific labeling of ZFPs at high yields as determined by mass spectrometry analysis. We observe that the DNA-binding activity of ZFPs labeled by conventional copper-mediated click chemistry was completely abolished, whereas ZFPs labeled by copper-free click chemistry retain their sequence-specific DNA-binding activity under native conditions, as determined by electrophoretic mobility shift assays, protein microarrays and kinetic binding assays based on Förster resonance energy transfer (FRET). Our work provides a general framework to label metalloproteins such as ZFPs by metabolic incorporation of unnatural amino acids followed by copper-free click chemistry. PMID:22871171

  15. Activation of Fetal γ-globin Gene Expression via Direct Protein Delivery of Synthetic Zinc-finger DNA-Binding Domains

    Directory of Open Access Journals (Sweden)

    Mir A Hossain

    2016-01-01

    Full Text Available Reactivation of γ-globin expression has been shown to ameliorate disease phenotypes associated with mutations in the adult β-globin gene, including sickle cell disease. Specific mutations in the promoter of the γ-globin genes are known to prevent repression of the genes in the adult and thus lead to hereditary persistence of fetal hemoglobin. One such hereditary persistence of fetal hemoglobin is associated with a sequence located 567 bp upstream of the Gγ-globin gene which assembles a GATA-containing repressor complex. We generated two synthetic zinc-finger DNA-binding domains (ZF-DBDs targeting this sequence. The -567Gγ ZF-DBDs associated with high affinity and specificity with the target site in the γ-globin gene promoter. We delivered the -567Gγ ZF-DBDs directly to primary erythroid cells. Exposure of these cells to the recombinant -567Gγ ZF-DBDs led to increased expression of the γ-globin gene. Direct protein delivery of ZF-DBDs that compete with transcription regulatory proteins will have broad implications for modulating gene expression in analytical or therapeutic settings.

  16. FYVE zinc-finger proteins in the plant model Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Jensen, R B; La Cour, T; Albrethsen, J

    2001-01-01

    Classic FYVE zinc-finger domains recognize the phosphoinositide signal PtdIns3P and share the basic (R/K)(1)(R/K)HHCR(6) (single-letter amino acid codes) consensus sequence. This domain is present in predicted PtdIns3P 5-kinases and lipases from Arabidopsis thaliana. Other Arabidopsis proteins......) of the basic motif. Dot-blot and liposome-binding assays were used in vitro to examine the phospholipid-binding ability of isolated PRAF domains. Whereas the PH domain preferentially bound PtdIns(4,5)P(2), the variant FYVE domain showed a weaker charge-dependent binding of phosphoinositides. In contrast....... A biochemical function for PRAF was indicated by its ability to catalyse guanine nucleotide exchange on some of the small GTPases of the Rab family, permitting a discussion of the biological roles of plant FYVE proteins and their regulation by phosphoinositides....

  17. A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.

    Science.gov (United States)

    Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

    2015-05-18

    An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes.

  18. A Mini Zinc-Finger Protein (MIF from Gerbera hybrida Activates the GASA Protein Family Gene, GEG, to Inhibit Ray Petal Elongation

    Directory of Open Access Journals (Sweden)

    Meixiang Han

    2017-09-01

    Full Text Available Petal appearance is an important horticultural trail that is generally used to evaluate the ornamental value of plants. However, knowledge of the molecular regulation of petal growth is mostly derived from analyses of Arabidopsis thaliana, and relatively little is known about this process in ornamental plants. Previously, GEG (Gerbera hybrida homolog of the gibberellin [GA]–stimulated transcript 1 [GAST1] from tomato, a gene from the GA stimulated Arabidopsis (GASA family, was reported to be an inhibitor of ray petal growth in the ornamental species, G. hybrida. To explore the molecular regulatory mechanism of GEG in petal growth inhibition, a mini zinc-finger protein (MIF was identified using yeast one-hybrid (Y1H screen. The direct binding of GhMIF to the GEG promoter was verified by using an electrophoretic mobility shift assay and a dual-luciferase assay. A yeast two-hybrid (Y2H revealed that GhMIF acts as a transcriptional activator. Transient transformation assay indicated that GhMIF is involved in inhibiting ray petal elongation by activating the expression of GEG. Spatiotemporal expression analyses and hormone treatment assay showed that the expression of GhMIF and GEG is coordinated during petal development. Taken together, these results suggest that GhMIF acts as a direct transcriptional activator of GEG, a gene from the GASA protein family to regulate the petal elongation.

  19. Specificity protein 1-zinc finger protein 179 pathway is involved in the attenuation of oxidative stress following brain injury

    Directory of Open Access Journals (Sweden)

    Jian-Ying Chuang

    2017-04-01

    Full Text Available After sudden traumatic brain injuries, secondary injuries may occur during the following days or weeks, which leads to the accumulation of reactive oxygen species (ROS. Since ROS exacerbate brain damage, it is important to protect neurons against their activity. Zinc finger protein 179 (Znf179 was shown to act as a neuroprotective factor, but the regulation of gene expression under oxidative stress remains unknown. In this study, we demonstrated an increase in Znf179 protein levels in both in vitro model of hydrogen peroxide (H2O2-induced ROS accumulation and animal models of traumatic brain injury. Additionally, we examined the sub-cellular localization of Znf179, and demonstrated that oxidative stress increases Znf179 nuclear shuttling and its interaction with specificity protein 1 (Sp1. Subsequently, the positive autoregulation of Znf179 expression, which is Sp1-dependent, was further demonstrated using luciferase reporter assay and green fluorescent protein (GFP-Znf179-expressing cells and transgenic mice. The upregulation of Sp1 transcriptional activity induced by the treatment with nerve growth factor (NGF led to an increase in Znf179 levels, which further protected cells against H2O2-induced damage. However, Sp1 inhibitor, mithramycin A, was shown to inhibit NGF effects, leading to a decrease in Znf179 expression and lower cellular protection. In conclusion, the results obtained in this study show that Znf179 autoregulation through Sp1-dependent mechanism plays an important role in neuroprotection, and NGF-induced Sp1 signaling may help attenuate more extensive (ROS-induced damage following brain injury.

  20. Genome-wide binding of transcription factor ZEB1 in triple-negative breast cancer cells.

    Science.gov (United States)

    Maturi, Varun; Enroth, Stefan; Heldin, Carl-Henrik; Moustakas, Aristidis

    2018-05-10

    Zinc finger E-box binding homeobox 1 (ZEB1) is a transcriptional regulator involved in embryonic development and cancer progression. ZEB1 induces epithelial-mesenchymal transition (EMT). Triple-negative human breast cancers express high ZEB1 mRNA levels and exhibit features of EMT. In the human triple-negative breast cancer cell model Hs578T, ZEB1 associates with almost 2,000 genes, representing many cellular functions, including cell polarity regulation (DLG2 and FAT3). By introducing a CRISPR-Cas9-mediated 30 bp deletion into the ZEB1 second exon, we observed reduced migratory and anchorage-independent growth capacity of these tumor cells. Transcriptomic analysis of control and ZEB1 knockout cells, revealed 1,372 differentially expressed genes. The TIMP metallopeptidase inhibitor 3 and the teneurin transmembrane protein 2 genes showed increased expression upon loss of ZEB1, possibly mediating pro-tumorigenic actions of ZEB1. This work provides a resource for regulators of cancer progression that function under the transcriptional control of ZEB1. The data confirm that removing a single EMT transcription factor, such as ZEB1, is not sufficient for reverting the triple-negative mesenchymal breast cancer cells into more differentiated, epithelial-like clones, but can reduce tumorigenic potential, suggesting that not all pro-tumorigenic actions of ZEB1 are linked to the EMT. © 2018 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

  1. Human ribosomal protein L37 has motifs predicting serine/threonine phosphorylation and a zinc-finger domain.

    Science.gov (United States)

    Barnard, G F; Staniunas, R J; Puder, M; Steele, G D; Chen, L B

    1994-08-02

    Ribosomal protein L37 mRNA is overexpressed in colon cancer. The nucleotide sequences of human L37 from several tumor and normal, colon and liver cDNA sources were determined to be identical. L37 mRNA was approximately 375 nucleotides long encoding 97 amino acids with M(r) = 11,070, pI = 12.6, multiple potential serine/threonine phosphorylation sites and a zinc-finger domain. The human sequence is compared to other species.

  2. Design of a colicin E7 based chimeric zinc-finger nuclease

    Science.gov (United States)

    Németh, Eszter; Schilli, Gabriella K.; Nagy, Gábor; Hasenhindl, Christoph; Gyurcsik, Béla; Oostenbrink, Chris

    2014-08-01

    Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity—offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region.

  3. The Effect of Salts in Promoting Specific and Competitive Interactions between Zinc Finger Proteins and Metals

    Science.gov (United States)

    Li, Gongyu; Yuan, Siming; Zheng, Shihui; Chen, Yuting; Zheng, Zhen; Liu, Yangzhong; Huang, Guangming

    2017-12-01

    Specific protein-metal interactions (PMIs) fulfill essential functions in cells and organic bodies, and activation of these functions in vivo are mostly modulated by the complex environmental factors, including pH value, small biomolecules, and salts. Specifically, the role of salts in promoting specific PMIs and their competition among various metals has remained untapped mainly due to the difficulty to distinguish nonspecific PMIs from specific PMIs by classic spectroscopic techniques. Herein, we report Hofmeister salts differentially promote the specific PMIs by combining nanoelectrospray ionization mass spectrometry and spectroscopic techniques (fluorescence measurement and circular dichroism). Furthermore, to explore the influence of salts in competitive binding between metalloproteins and various metals, we designed a series of competitive experiments and applied to a well-defined model system, the competitive binding of zinc (II) and arsenic (III) to holo-promyelocytic leukemia protein (PML). These experiments not only provided new insights at the molecular scale as complementary to previous NMR and spectroscopic results, but also deduced the relative binding ability between zinc finger proteins and metals at the molecular scale, which avoids the mass spectrometric titration-based determination of binding constants that is frequently affected and often degraded by variable solution conditions including salt contents. [Figure not available: see fulltext.

  4. Transcriptional regulation of epithelial-mesenchymal transition in melanoma

    International Nuclear Information System (INIS)

    Wels, C.

    2010-01-01

    The downregulation of epithelial markers followed by upregulation of mesenchymal characteristics is an important step in melanoma development. This process goes along with gains in cell proliferation and motility, depolarization and detachment from neighbouring cells, finally enabling melanoma cells to leave the primary site of tumor growth and to circulate through the blood or lymphatic system. The entirety of these events is referred to as epithelial-mesenchymal transition (EMT). Changes during EMT are accomplished by a set of transcription factors which share the same DNA binding site called E-box. These E-box binding transcription factors are subsumed as epithelial-mesenchymal transitions regulators (EMTRs). In this thesis, I studied the interplay of the zinc-finger transcription factors Slug and ZEB1 and the basic helix-loop-helix transcription factor Twist during melanoma progression. I demonstrate for the first time the direct and specific transcriptional upregulation of one EMTR, ZEB1, by another, Slug, using gene silencing and overexpression studies together with mobility shift and luciferase assays. The two transcription factors cooperate in repressing the epithelial adhesion molecule E-cadherin which is supposed to be a crucial step during early EMT. Further, they show additive effects in promoting detachment from neighbouring cells and cell migration. Conceptually, Slug and ZEB1 are supported by Twist, a transcription factor that might be less pivotal for E-cadherin repression but rather for inducing the expression of the mesenchymal marker N-cadherin, enabling adhesion to mesenchymal cells, thereby promoting migration and invasion of melanoma cells.Taken together, I provide a model of a hierarchical organization of EMT transcription factors, with Slug as a transcriptional activator of ZEB1, leading to cooperative effects on detachment and migration and, together with Twist, leading to EMT in melanoma. (author) [de

  5. WRKY transcription factor genes in wild rice Oryza nivara.

    Science.gov (United States)

    Xu, Hengjian; Watanabe, Kenneth A; Zhang, Liyuan; Shen, Qingxi J

    2016-08-01

    The WRKY transcription factor family is one of the largest gene families involved in plant development and stress response. Although many WRKY genes have been studied in cultivated rice (Oryza sativa), the WRKY genes in the wild rice species Oryza nivara, the direct progenitor of O. sativa, have not been studied. O. nivara shows abundant genetic diversity and elite drought and disease resistance features. Herein, a total of 97 O. nivara WRKY (OnWRKY) genes were identified. RNA-sequencing demonstrates that OnWRKY genes were generally expressed at higher levels in the roots of 30-day-old plants. Bioinformatic analyses suggest that most of OnWRKY genes could be induced by salicylic acid, abscisic acid, and drought. Abundant potential MAPK phosphorylation sites in OnWRKYs suggest that activities of most OnWRKYs can be regulated by phosphorylation. Phylogenetic analyses of OnWRKYs support a novel hypothesis that ancient group IIc OnWRKYs were the original ancestors of only some group IIc and group III WRKYs. The analyses also offer strong support that group IIc OnWRKYs containing the HVE sequence in their zinc finger motifs were derived from group Ia WRKYs. This study provides a solid foundation for the study of the evolution and functions of WRKY genes in O. nivara. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  6. DPL-1 DP, LIN-35 Rb and EFL-1 E2F act with the MCD-1 zinc-finger protein to promote programmed cell death in Caenorhabditis elegans.

    Science.gov (United States)

    Reddien, Peter W; Andersen, Erik C; Huang, Michael C; Horvitz, H Robert

    2007-04-01

    The genes egl-1, ced-9, ced-4, and ced-3 play major roles in programmed cell death in Caenorhabditis elegans. To identify genes that have more subtle activities, we sought mutations that confer strong cell-death defects in a genetically sensitized mutant background. Specifically, we screened for mutations that enhance the cell-death defects caused by a partial loss-of-function allele of the ced-3 caspase gene. We identified mutations in two genes not previously known to affect cell death, dpl-1 and mcd-1 (modifier of cell death). dpl-1 encodes the C. elegans homolog of DP, the human E2F-heterodimerization partner. By testing genes known to interact with dpl-1, we identified roles in cell death for four additional genes: efl-1 E2F, lin-35 Rb, lin-37 Mip40, and lin-52 dLin52. mcd-1 encodes a novel protein that contains one zinc finger and that is synthetically required with lin-35 Rb for animal viability. dpl-1 and mcd-1 act with efl-1 E2F and lin-35 Rb to promote programmed cell death and do so by regulating the killing process rather than by affecting the decision between survival and death. We propose that the DPL-1 DP, MCD-1 zinc finger, EFL-1 E2F, LIN-35 Rb, LIN-37 Mip40, and LIN-52 dLin52 proteins act together in transcriptional regulation to promote programmed cell death.

  7. The novel BTB/POZ and zinc finger factor Zbtb45 is essential for proper glial differentiation of neural and oligodendrocyte progenitor cells

    DEFF Research Database (Denmark)

    Södersten, Erik; Lilja, Tobias; Hermanson, Ola

    2010-01-01

    Understanding the regulatory mechanisms controlling the fate decisions of neural stem cells (NSCs) is a crucial issue to shed new light on mammalian central nervous system (CNS) development in health and disease. We have investigated a possible role for the previously uncharacterized BTB/POZ-doma......Understanding the regulatory mechanisms controlling the fate decisions of neural stem cells (NSCs) is a crucial issue to shed new light on mammalian central nervous system (CNS) development in health and disease. We have investigated a possible role for the previously uncharacterized BTB....../POZ-domain containing zinc finger factor Zbtb45 in the differentiation of NSCs and postnatal oligodendrocyte precursors. In situ hybridization histochemistry and RT-qPCR analysis revealed that Zbtb45 mRNA was ubiquitously expressed in the developing CNS in mouse embryos at embryonic day (E) 12.5 and 14.5. Zbtb45 m......RNA knockdown in embryonic forebrain NSCs by siRNA resulted in a rapid decrease in the expression of oligodendrocyte-characteristic genes after mitogen (FGF2) withdrawal, whereas the expression of astrocyte-associated genes such as CD44 and GFAP increased compared to control. Accordingly, the number...

  8. The electrostatic role of the Zn-Cys2His2 complex in binding of operator DNA with transcription factors: mouse EGR-1 from the Cys2His2 family.

    Science.gov (United States)

    Chirgadze, Y N; Boshkova, E A; Polozov, R V; Sivozhelezov, V S; Dzyabchenko, A V; Kuzminsky, M B; Stepanenko, V A; Ivanov, V V

    2018-01-07

    The mouse factor Zif268, known also as early growth response protein EGR-1, is a classical representative for the Cys2His2 transcription factor family. It is required for binding the RNA polymerase with operator dsDNA to initialize the transcription process. We have shown that only in this family of total six Zn-finger protein families the Zn complex plays a significant role in the protein-DNA binding. Electrostatic feature of this complex in the binding of factor Zif268 from Mus musculus with operator DNA has been considered. The factor consists of three similar Zn-finger units which bind with triplets of coding DNA. Essential contacts of the factor with the DNA phosphates are formed by three conservative His residues, one in each finger. We describe here the results of calculations of the electrostatic potentials for the Zn-Cys2His2 complex, Zn-finger unit 1, and the whole transcription factor. The potential of Zif268 has a positive area on the factor surface, and it corresponds exactly to the binding sites of each of Zn-finger units. The main part of these areas is determined by conservative His residues, which form contacts with the DNA phosphate groups. Our result shows that the electrostatic positive potential of this histidine residue is enhanced due to the Zn complex. The other contacts of the Zn-finger with DNA are related to nucleotide bases, and they are responsible for the sequence-specific binding with DNA. This result may be extended to all other members of the Cys2His2 transcription factor family.

  9. Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases.

    Directory of Open Access Journals (Sweden)

    Craig B Wilen

    2011-04-01

    Full Text Available HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5 virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4 in place of or in addition to CCR5 (R5X4 remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.

  10. Zinc-finger antiviral protein inhibits XMRV infection.

    Directory of Open Access Journals (Sweden)

    Xinlu Wang

    Full Text Available BACKGROUND: The zinc-finger antiviral protein (ZAP is a host factor that specifically inhibits the replication of certain viruses, including Moloney murine leukemia virus (MoMLV, HIV-1, and certain alphaviruses and filoviruses. ZAP binds to specific viral mRNAs and recruits cellular mRNA degradation machinery to degrade the target RNA. The common features of ZAP-responsive RNA sequences remain elusive and thus whether a virus is susceptible to ZAP can only be determined experimentally. Xenotropic murine leukemia virus-related virus (XMRV is a recently identified γ-retrovirus that was originally thought to be involved in prostate cancer and chronic fatigue syndrome but recently proved to be a laboratory artefact. Nonetheless, XMRV as a new retrovirus has been extensively studied. Since XMRV and MoMLV share only 67.9% sequence identity in the 3'UTRs, which is the target sequence of ZAP in MoMLV, whether XMRV is susceptible to ZAP remains to be determined. FINDINGS: We constructed an XMRV-luc vector, in which the coding sequences of Gag-Pol and part of Env were replaced with luciferase-coding sequence. Overexpression of ZAP potently inhibited the expression of XMRV-luc in a ZAP expression-level-dependent manner, while downregulation of endogenous ZAP rendered cells more sensitive to infection. Furthermore, ZAP inhibited the spreading of replication-competent XMRV. Consistent with the previously reported mechanisms by which ZAP inhibits viral infection, ZAP significantly inhibited the accumulation of XMRV-luc mRNA in the cytoplasm. The ZAP-responsive element in XMRV mRNA was mapped to the 3'UTR. CONCLUSIONS: ZAP inhibits XMRV replication by preventing the accumulation of viral mRNA in the cytoplasm. Documentation of ZAP inhibiting XMRV helps to broaden the spectrum of ZAP's antiviral activity. Comparison of the target sequences of ZAP in XMRV and MoMLV helps to better understand the features of ZAP-responsive elements.

  11. ZFP226 is a novel artificial transcription factor for selective activation of tumor suppressor KIBRA.

    Science.gov (United States)

    Schelleckes, Katrin; Schmitz, Boris; Lenders, Malte; Mewes, Mirja; Brand, Stefan-Martin; Brand, Eva

    2018-03-09

    KIBRA has been suggested as a key regulator of the hippo pathway, regulating organ size, cell contact inhibition as well as tissue regeneration and tumorigenesis. Recently, alterations of KIBRA expression caused by promotor methylation have been reported for several types of cancer. Our current study aimed to design an artificial transcription factor capable of re-activating expression of the tumor suppressor KIBRA and the hippo pathway. We engineered a new gene named 'ZFP226' encoding for a ~23 kDa fusion protein. ZFP226 belongs to the Cys2-His2 zinc finger type and recognizes a nine base-pair DNA sequence 5'-GGC-GGC-GGC-3' in the KIBRA core promoter P1a. ZFP226 showed nuclear localization in human immortalized kidney epithelial cells and activated the KIBRA core promoter (p < 0.001) resulting in significantly increased KIBRA mRNA and protein levels (p < 0.001). Furthermore, ZFP226 led to activation of hippo signaling marked by elevated YAP and LATS phosphorylation. In Annexin V flow cytometry assays ZFP226 overexpression showed strong pro-apoptotic capacity on MCF-7 breast cancer cells (p < 0.01 early-, p < 0.001 late-apoptotic cells). We conclude that the artificial transcription factor ZFP226 can be used for target KIBRA and hippo pathway activation. This novel molecule may represent a molecular tool for the development of future applications in cancer treatment.

  12. Gata4 expression in lateral mesoderm is downstream of BMP4 and isactivated directly by Forkhead and GATA transcription factors through adistal enhancer element

    Energy Technology Data Exchange (ETDEWEB)

    Rojas, Anabel; De Val, Sarah; Heidt, Analeah B.; Xu, Shan-Mei; Bristow, James; Black, Brian L.

    2005-05-20

    The GATA family of zinc-finger transcription factors plays key roles in the specification and differentiation of multiple cell types during development. GATA4 is an early regulator of gene expression during the development of endoderm and mesoderm, and genetic studies in mice have demonstrated that GATA4 is required for embryonic development.Despite the importance of GATA4 in tissue specification and differentiation, the mechanisms by which Gata4 expression is activated and the transcription factor pathways upstream of GATA4 remain largely undefined. To identify transcriptional regulators of Gata4 in the mouse,we screened conserved noncoding sequences from the mouse Gata4 gene for enhancer activity in transgenic embryos. Here, we define the regulation of a distal enhancer element from Gata4 that is sufficient to direct expression throughout the lateral mesoderm, beginning at 7.5 days of mouse embryonic development. The activity of this enhancer is initially broad but eventually becomes restricted to the mesenchyme surrounding the liver. We demonstrate that the function of this enhancer in transgenic embryos is dependent upon highly conserved Forkhead and GATA transcription factor binding sites, which are bound by FOXF1 and GATA4,respectively. Furthermore, the activity of the Gata4 lateral mesoderm enhancer is attenuated by the BMP antagonist Noggin, and the enhancer is not activated in Bmp4-null embryos. Thus, these studies establish that Gata4 is a direct transcriptional target of Forkhead and GATA transcription factors in the lateral mesoderm, and demonstrate that Gata4lateral mesoderm enhancer activation requires BMP4, supporting a model in which GATA4 serves as a downstream effector of BMP signaling in the lateral mesoderm.

  13. A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking

    Science.gov (United States)

    Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

    2015-01-01

    An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes. DOI: http://dx.doi.org/10.7554/eLife.06041.001 PMID:25985087

  14. Expression of a finger millet transcription factor, EcNAC1, in tobacco confers abiotic stress-tolerance.

    Directory of Open Access Journals (Sweden)

    Venkategowda Ramegowda

    Full Text Available NAC (NAM, ATAF1-2, and CUC2 proteins constitute one of the largest families of plant-specific transcription factors and have been shown to be involved in diverse plant processes including plant growth, development, and stress-tolerance. In this study, a stress-responsive NAC gene, EcNAC1, was isolated from the subtracted stress cDNA library generated from a drought adapted crop, finger millet, and characterized for its role in stress-tolerance. The expression analysis showed that EcNAC1 was highly induced during water-deficit and salt stress. EcNAC1 shares high amino acid similarity with rice genes that have been phylogenetically classified into stress-related NAC genes. Our results demonstrated that tobacco transgenic plants expressing EcNAC1 exhibit tolerance to various abiotic stresses like simulated osmotic stress, by polyethylene glycol (PEG and mannitol, and salinity stress. The transgenic plants also showed enhanced tolerance to methyl-viologen (MV induced oxidative stress. Reduced levels of reactive oxygen species (ROS and ROS-induced damage were noticed in pot grown transgenic lines under water-deficit and natural high light conditions. Root growth under stress and recovery growth after stress alleviation was more in transgenic plants. Many stress-responsive genes were found to be up-regulated in transgenic lines expressing EcNAC1. Our results suggest that EcNAC1 overexpression confers tolerance against abiotic stress in susceptible species, tobacco.

  15. Arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Xi; Zhou, Xixi [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Du, Libo [Center for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Liu, Wenlan [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Liu, Yang [Center for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Hudson, Laurie G. [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Liu, Ke Jian, E-mail: kliu@salud.unm.edu [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States)

    2014-01-15

    Inhibition of DNA repair is a recognized mechanism for arsenic enhancement of ultraviolet radiation-induced DNA damage and carcinogenesis. Poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger DNA repair protein, has been identified as a sensitive molecular target for arsenic. The zinc finger domains of PARP-1 protein function as a critical structure in DNA recognition and binding. Since cellular poly(ADP-ribosyl)ation capacity has been positively correlated with zinc status in cells, we hypothesize that arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair. To test this hypothesis, we compared the effects of arsenite exposure with zinc deficiency, created by using the membrane-permeable zinc chelator TPEN, on 8-OHdG formation, PARP-1 activity and zinc binding to PARP-1 in HaCat cells. Our results show that arsenite exposure and zinc deficiency had similar effects on PARP-1 protein, whereas supplemental zinc reversed these effects. To investigate the molecular mechanism of zinc loss induced by arsenite, ICP-AES, near UV spectroscopy, fluorescence, and circular dichroism spectroscopy were utilized to examine arsenite binding and occupation of a peptide representing the first zinc finger of PARP-1. We found that arsenite binding as well as zinc loss altered the conformation of zinc finger structure which functionally leads to PARP-1 inhibition. These findings suggest that arsenite binding to PARP-1 protein created similar adverse biological effects as zinc deficiency, which establishes the molecular mechanism for zinc supplementation as a potentially effective treatment to reverse the detrimental outcomes of arsenic exposure. - Highlights: • Arsenite binding is equivalent to zinc deficiency in reducing PARP-1 function. • Zinc reverses arsenic inhibition of PARP-1 activity and enhancement of DNA damage. • Arsenite binding and zinc loss alter the conformation of zinc finger

  16. Arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair

    International Nuclear Information System (INIS)

    Sun, Xi; Zhou, Xixi; Du, Libo; Liu, Wenlan; Liu, Yang; Hudson, Laurie G.; Liu, Ke Jian

    2014-01-01

    Inhibition of DNA repair is a recognized mechanism for arsenic enhancement of ultraviolet radiation-induced DNA damage and carcinogenesis. Poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger DNA repair protein, has been identified as a sensitive molecular target for arsenic. The zinc finger domains of PARP-1 protein function as a critical structure in DNA recognition and binding. Since cellular poly(ADP-ribosyl)ation capacity has been positively correlated with zinc status in cells, we hypothesize that arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair. To test this hypothesis, we compared the effects of arsenite exposure with zinc deficiency, created by using the membrane-permeable zinc chelator TPEN, on 8-OHdG formation, PARP-1 activity and zinc binding to PARP-1 in HaCat cells. Our results show that arsenite exposure and zinc deficiency had similar effects on PARP-1 protein, whereas supplemental zinc reversed these effects. To investigate the molecular mechanism of zinc loss induced by arsenite, ICP-AES, near UV spectroscopy, fluorescence, and circular dichroism spectroscopy were utilized to examine arsenite binding and occupation of a peptide representing the first zinc finger of PARP-1. We found that arsenite binding as well as zinc loss altered the conformation of zinc finger structure which functionally leads to PARP-1 inhibition. These findings suggest that arsenite binding to PARP-1 protein created similar adverse biological effects as zinc deficiency, which establishes the molecular mechanism for zinc supplementation as a potentially effective treatment to reverse the detrimental outcomes of arsenic exposure. - Highlights: • Arsenite binding is equivalent to zinc deficiency in reducing PARP-1 function. • Zinc reverses arsenic inhibition of PARP-1 activity and enhancement of DNA damage. • Arsenite binding and zinc loss alter the conformation of zinc finger

  17. Structural and functional organization of the HF.10 human zinc finger gene (ZNF35) located on chromosome 3p21-p22

    DEFF Research Database (Denmark)

    Lanfrancone, L; Pengue, G; Pandolfi, P P

    1992-01-01

    We report the structural and functional characterization of the HF.10 zinc finger gene (ZNF35) in normal human cells, as well as a processed pseudogene. The HF.10 gene spans about 13 kb and it is interrupted by three introns. All 11 zinc finger DNA-binding domains are contiguously encoded within...... and partial nucleotide sequencing of the HF.10 pseudogene indicated that it has arisen by retroposition of spliced HF.10 mRNA. In situ hybridization experiments revealed that both the functional locus and the pseudogene map to chromosome 3p21p22, a region that is frequently deleted in small cell lung...... and renal carcinomas. Hybridization of the HF.10 gene and the HF.10 pseudogene DNA probes to metaphases from a small cell lung carcinoma cell line with the 3p deletion revealed that both loci are part of the deleted chromosome region....

  18. Promoter of CaZF, a chickpea gene that positively regulates growth and stress tolerance, is activated by an AP2-family transcription factor CAP2.

    Directory of Open Access Journals (Sweden)

    Deepti Jain

    Full Text Available Plants respond to different forms of stresses by inducing transcription of a common and distinct set of genes by concerted actions of a cascade of transcription regulators. We previously reported that a gene, CaZF encoding a C2H2-zinc finger family protein from chickpea (Cicer arietinum imparted high salinity tolerance when expressed in tobacco plants. We report here that in addition to promoting tolerance against dehydration, salinity and high temperature, the CaZF overexpressing plants exhibited similar phenotype of growth and development like the plants overexpressing CAP2, encoding an AP2-family transcription factor from chickpea. To investigate any relationship between these two genes, we performed gene expression analysis in the overexpressing plants, promoter-reporter analysis and chromatin immunoprecipitation. A number of transcripts that exhibited enhanced accumulation upon expression of CAP2 or CaZF in tobacco plants were found common. Transient expression of CAP2 in chickpea leaves resulted in increased accumulation of CaZF transcript. Gel mobility shift and transient promoter-reporter assays suggested that CAP2 activates CaZF promoter by interacting with C-repeat elements (CRTs in CaZF promoter. Chromatin immunoprecipitation (ChIP assay demonstrated an in vivo interaction of CAP2 protein with CaZF promoter.

  19. Knockout of Myostatin by Zinc-finger Nuclease in Sheep Fibroblasts and Embryos

    Directory of Open Access Journals (Sweden)

    Xuemei Zhang

    2016-10-01

    Full Text Available Myostatin (MSTN can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause “double-muscling” trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos.

  20. Proliferation and osteo/odontogenic differentiation of stem cells from apical papilla regulated by Zinc fingers and homeoboxes 2: An in vitro study.

    Science.gov (United States)

    Wan, Fang; Gao, Lifen; Lu, Yating; Ma, Hongxin; Wang, Hongxing; Liang, Xiaohong; Wang, Yan; Ma, Chunhong

    2016-01-15

    In the process of tooth root development, stem cells from the apical papilla (SCAPs) can differentiate into odontoblasts and form root dentin, however, molecules regulating SCAPs differentiation have not been elucidated. Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. It is reported to modulate the development of nerve cells, liver cells, B cells, red blood cells, and so on. However, the role of ZHX2 in tooth root development remains unclear. In this study, we explored the potential role of ZHX2 in the process of SCAPs differentiation. The results showed that overexpression of ZHX2 upregulated the expression of osteo/odontogenic related genes and ALP activity, inhibited the proliferation of SCAPs. Consistently, ZHX2 knockdown reduced SCAPs mineralization and promoted SCAPs proliferation. These results indicated that ZHX2 plays a critical role in the proliferation and osteo/odontogenic differentiation of SCAPs. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. 20-hydroxyecdysone enhances the expression of the chitinase 5 via Broad-Complex Zinc-Finger 4 during metamorphosis in silkworm, Bombyx mori.

    Science.gov (United States)

    Zhang, X; Zheng, S

    2017-04-01

    Insect chitinases are hydrolytic enzymes required for the degradation of chitin. They are essential for insect moulting and metamorphosis. In this study, the regulation mechanism of a chitinase gene, Bombyx mori chitinase 5 (BmCHT5), was studied. Quantitative reverse transcription PCR (qRT-PCR) analysis showed that BmCHT5 was up-regulated during the larval-larval and larval-pupa transitions and notably induced by 20-hydroxyecdysone (20E). Analysis of the BmCHT5 promoter revealed the presence of one Bombyx mori Broad-Complex Zinc-Finger Isoform 4 (BR-C Z4), two BR-C Z2 and two ecdysone-induced protein 74A (E74A) cis-regulatory elements (CREs) that are related to 20E. qRT-PCR showed that the expression of both BmBR-C Z4 and BmBR-C Z2 during metamorphosis, and when induced by 20E, was anastomotic with the variations in BmCHT5 mRNA level. In contrast, BmE74A did not follow this trend. An electrophoretic mobility shift assay did not retrieve a binding partner for the two BR-C Z2 CREs in the BmN cell line nuclear extract, whereas BR-C Z4 CRE specifically bound to BmBR-C Z4. Besides, luciferase activity analysis confirmed that BmBR-C Z4 could enhance the activity of the BmCHT5 promoter with BR-C Z4 CRE and could not enhance the promoter activity by mutating BR-C Z4 CRE. Taken together, these data suggest that the transcription factor BmBR-C Z4 enhances the expression of BmCHT5 during metamorphosis. © 2016 The Royal Entomological Society.

  2. Promiscuous and specific phospholipid binding by domains in ZAC, a membrane-associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain

    DEFF Research Database (Denmark)

    Jensen, R B; Lykke-Andersen, K; Frandsen, G I

    2000-01-01

    domain are separated by a region without homology to other known proteins. Zac promoter/beta-glucuronidase reporter assays revealed highest expression levels in flowering tissue, rosettes and roots. ZAC protein was immuno-detected mainly in association with membranes and fractionated with Golgi...... and plasma membrane marker proteins. ZAC membrane association was confirmed in assays by a fusion between ZAC and the green fluorescence protein and prompted an analysis of the in vitro phospholipid-binding ability of ZAC. Phospholipid dot-blot and liposome-binding assays indicated that fusion proteins...... zinc finger motif, but proteins containing only the zinc finger domain (residues 1-105) did not bind PI-3-P. Recombinant ZAC possessed GTPase-activating activity on Arabidopsis ARF proteins. These data identify a novel PI-3-P-binding protein region and thereby provide evidence...

  3. A novel human AP endonuclease with conserved zinc-finger-like motifs involved in DNA strand break responses

    OpenAIRE

    Kanno, Shin-ichiro; Kuzuoka, Hiroyuki; Sasao, Shigeru; Hong, Zehui; Lan, Li; Nakajima, Satoshi; Yasui, Akira

    2007-01-01

    DNA damage causes genome instability and cell death, but many of the cellular responses to DNA damage still remain elusive. We here report a human protein, PALF (PNK and APTX-like FHA protein), with an FHA (forkhead-associated) domain and novel zinc-finger-like CYR (cysteine–tyrosine–arginine) motifs that are involved in responses to DNA damage. We found that the CYR motif is widely distributed among DNA repair proteins of higher eukaryotes, and that PALF, as well as a Drosophila protein with...

  4. Krüppel-like factor 4, a novel transcription factor regulates microglial activation and subsequent neuroinflammation

    Directory of Open Access Journals (Sweden)

    Das Sulagna

    2010-10-01

    Full Text Available Abstract Background Activation of microglia, the resident macrophages of the central nervous system (CNS, is the hallmark of neuroinflammation in neurodegenerative diseases and other pathological conditions associated with CNS infection. The activation of microglia is often associated with bystander neuronal death. Nuclear factor-κB (NF-κB is one of the important transcription factors known to be associated with microglial activation which upregulates the expression of inducible nitric oxide synthase (iNOS, cyclooxygenase-2 (Cox-2 and other pro-inflammatory cytokines. Recent studies have focused on the role of Krüppel-like factor 4 (Klf4, one of the zinc-finger transcription factors, in mediating inflammation. However, these studies were limited to peripheral system and its role in CNS is not understood. Our studies focused on the possible role of Klf4 in mediating CNS inflammation. Methods For in vitro studies, mouse microglial BV-2 cell lines were treated with 500 ng/ml Salmonella enterica lipopolysacchride (LPS. Brain tissues were isolated from BALB/c mice administered with 5 mg/kg body weight of LPS. Expressions of Klf4, Cox-2, iNOS and pNF-κB were evaluated using western blotting, quantitative real time PCR, and reverse transcriptase polymerase chain reactions (RT-PCRs. Klf4 knockdown was carried out using SiRNA specific for Klf4 mRNA and luciferase assays and electromobility shift assay (EMSA were performed to study the interaction of Klf4 to iNOS promoter elements in vitro. Co-immunoprecipitation of Klf4 and pNF-κB was done in order to study a possible interaction between the two transcription factors. Results LPS stimulation increased Klf4 expression in microglial cells in a time- and dose-dependent manner. Knockdown of Klf4 resulted in decreased levels of the pro-inflammatory cytokines TNF-α, MCP-1 and IL-6, along with a significant decrease in iNOS and Cox-2 expression. NO production also decreased as a result of Klf4 knockdown

  5. The Fdb3 transcription factor of the Fusarium Detoxification of Benzoxazolinone gene cluster is required for MBOA but not BOA degradation in Fusarium pseudograminearum.

    Science.gov (United States)

    Kettle, Andrew J; Carere, Jason; Batley, Jacqueline; Manners, John M; Kazan, Kemal; Gardiner, Donald M

    2016-03-01

    A number of cereals produce the benzoxazolinone class of phytoalexins. Fusarium species pathogenic towards these hosts can typically degrade these compounds via an aminophenol intermediate, and the ability to do so is encoded by a group of genes found in the Fusarium Detoxification of Benzoxazolinone (FDB) cluster. A zinc finger transcription factor encoded by one of the FDB cluster genes (FDB3) has been proposed to regulate the expression of other genes in the cluster and hence is potentially involved in benzoxazolinone degradation. Herein we show that Fdb3 is essential for the ability of Fusarium pseudograminearum to efficiently detoxify the predominant wheat benzoxazolinone, 6-methoxy-benzoxazolin-2-one (MBOA), but not benzoxazoline-2-one (BOA). Furthermore, additional genes thought to be part of the FDB gene cluster, based upon transcriptional response to benzoxazolinones, are regulated by Fdb3. However, deletion mutants for these latter genes remain capable of benzoxazolinone degradation, suggesting that they are not essential for this process. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

  6. The POU proteins Brn-2 and Oct-6 share important functions in Schwann cell development.

    NARCIS (Netherlands)

    M.M. Jaegle (Martine); M. Ghazvini (Mehrnaz); W.J. Mandemakers (Wim); M. Piirsoo (Marko); S. Driegen (Siska); F. Levavasseur (Francoise); S. Raghoenath; F.G. Grosveld (Frank); D. Meijer (Daniëlle)

    2003-01-01

    textabstractThe genetic hierarchy that controls myelination of peripheral nerves by Schwann cells includes the POU domain Oct-6/Scip/Tst-1and the zinc-finger Krox-20/Egr2 transcription factors. These pivotal transcription factors act to control the onset of myelination during

  7. The C. elegans Spalt-like protein SEM-4 functions through the SoxC transcription factor SEM-2 to promote a proliferative blast cell fate in the postembryonic mesoderm.

    Science.gov (United States)

    Shen, Qinfang; Shi, Herong; Tian, Chenxi; Ghai, Vikas; Liu, Jun

    2017-09-01

    Proper development of a multicellular organism relies on well-coordinated regulation of cell fate specification, cell proliferation and cell differentiation. The C. elegans postembryonic mesoderm provides a useful system for uncovering factors involved in these processes and for further dissecting their regulatory relationships. The single Spalt-like zinc finger containing protein SEM-4/SALL is known to be involved in specifying the proliferative sex myoblast (SM) fate. We have found that SEM-4/SALL is sufficient to promote the SM fate and that it does so in a cell autonomous manner. We further showed that SEM-4/SALL acts through the SoxC transcription factor SEM-2 to promote the SM fate. SEM-2 is known to promote the SM fate by inhibiting the expression of two BWM-specifying transcription factors. In light of recent findings in mammals showing that Sall4, one of the mammalian homologs of SEM-4, contributes to pluripotency regulation by inhibiting differentiation, our work suggests that the function of SEM-4/SALL proteins in regulating pluripotency versus differentiation appears to be evolutionarily conserved. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Targeted genome regulation via synthetic programmable transcriptional regulators

    KAUST Repository

    Piatek, Agnieszka Anna

    2016-04-19

    Regulation of gene transcription controls cellular functions and coordinates responses to developmental, physiological and environmental cues. Precise and efficient molecular tools are needed to characterize the functions of single and multiple genes in linear and interacting pathways in a native context. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like proteins (TALE) are amenable to bioengineering to bind DNA target sequences of interest. As a result, ZF and TALE proteins were used to develop synthetic programmable transcription factors. However, these systems are limited by the requirement to re-engineer proteins for each new target sequence. The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9) genome editing tool was recently repurposed for targeted transcriptional regulation by inactivation of the nuclease activity of Cas9. Due to the facile engineering, simplicity, precision and amenability to library construction, the CRISPR/Cas9 system is poised to revolutionize the functional genomics field across diverse eukaryotic species. In this review, we discuss the development of synthetic customizable transcriptional regulators and provide insights into their current and potential applications, with special emphasis on plant systems, in characterization of gene functions, elucidation of molecular mechanisms and their biotechnological applications. © 2016 Informa UK Limited, trading as Taylor & Francis Group

  9. Arabidopsis KHZ1 and KHZ2, two novel non-tandem CCCH zinc-finger and K-homolog domain proteins, have redundant roles in the regulation of flowering and senescence.

    Science.gov (United States)

    Yan, Zongyun; Jia, Jianheng; Yan, Xiaoyuan; Shi, Huiying; Han, Yuzhen

    2017-12-01

    The two novel CCCH zinc-finger and K-homolog (KH) proteins, KHZ1 and KHZ2, play important roles in regulating flowering and senescence redundantly in Arabidopsis. The CCCH zinc-finger proteins and K-homolog (KH) proteins play important roles in plant development and stress responses. However, the biological functions of many CCCH zinc-finger proteins and KH proteins remain uncharacterized. In Arabidopsis, KHZ1 and KHZ2 are characterized as two novel CCCH zinc-finger and KH domain proteins which belong to subfamily VII in CCCH family. We obtained khz1, khz2 mutants and khz1 khz2 double mutants, as well as overexpression (OE) lines of KHZ1 and KHZ2. Compared with the wild type (WT), the khz2 mutants displayed no defects in growth and development, and the khz1 mutants were slightly late flowering, whereas the khz1 khz2 double mutants showed a pronounced late flowering phenotype. In contrast, artificially overexpressing KHZ1 and KHZ2 led to the early flowering. Consistent with the late flowering phenotype, the expression of flowering repressor gene FLC was up-regulated, while the expression of flowering integrator and floral meristem identity (FMI) genes were down-regulated significantly in khz1 khz2. In addition, we also observed that the OE plants of KHZ1 and KHZ2 showed early leaf senescence significantly, whereas the khz1 khz2 double mutants showed delayed senescence of leaf and the whole plant. Both KHZ1 and KHZ2 were ubiquitously expressed throughout the tissues of Arabidopsis. KHZ1 and KHZ2 were localized to the nucleus, and possessed both transactivation activities and RNA-binding abilities. Taken together, we conclude that KHZ1 and KHZ2 have redundant roles in the regulation of flowering and senescence in Arabidopsis.

  10. Zinc coordination is required for and regulates transcription activation by Epstein-Barr nuclear antigen 1.

    Directory of Open Access Journals (Sweden)

    Siddhesh Aras

    2009-06-01

    Full Text Available Epstein-Barr Nuclear Antigen 1 (EBNA1 is essential for Epstein-Barr virus to immortalize naïve B-cells. Upon binding a cluster of 20 cognate binding-sites termed the family of repeats, EBNA1 transactivates promoters for EBV genes that are required for immortalization. A small domain, termed UR1, that is 25 amino-acids in length, has been identified previously as essential for EBNA1 to activate transcription. In this study, we have elucidated how UR1 contributes to EBNA1's ability to transactivate. We show that zinc is necessary for EBNA1 to activate transcription, and that UR1 coordinates zinc through a pair of essential cysteines contained within it. UR1 dimerizes upon coordinating zinc, indicating that EBNA1 contains a second dimerization interface in its amino-terminus. There is a strong correlation between UR1-mediated dimerization and EBNA1's ability to transactivate cooperatively. Point mutants of EBNA1 that disrupt zinc coordination also prevent self-association, and do not activate transcription cooperatively. Further, we demonstrate that UR1 acts as a molecular sensor that regulates the ability of EBNA1 to activate transcription in response to changes in redox and oxygen partial pressure (pO(2. Mild oxidative stress mimicking such environmental changes decreases EBNA1-dependent transcription in a lymphoblastoid cell-line. Coincident with a reduction in EBNA1-dependent transcription, reductions are observed in EBNA2 and LMP1 protein levels. Although these changes do not affect LCL survival, treated cells accumulate in G0/G1. These findings are discussed in the context of EBV latency in body compartments that differ strikingly in their pO(2 and redox potential.

  11. Mesenchymal cells reactivate Snail1 expression to drive three-dimensional invasion programs

    DEFF Research Database (Denmark)

    Rowe, R.G.; Li, X.Y.; Hu, Y.

    2009-01-01

    Epithelial-mesenchymal transition (EMT) is required for mesodermal differentiation during development. The zinc-finger transcription factor, Snail1, can trigger EMT and is sufficient to transcriptionally reprogram epithelial cells toward a mesenchymal phenotype during neoplasia and fibrosis. Whet...

  12. Generation of knockout rabbits using transcription activator-like effector nucleases

    OpenAIRE

    Wang, Yu; Fan, Nana; Song, Jun; Zhong, Juan; Guo, Xiaogang; Tian, Weihua; Zhang, Quanjun; Cui, Fenggong; Li, Li; Newsome, Philip N; Frampton, Jon; Esteban, Miguel A; Lai, Liangxue

    2014-01-01

    Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large ...

  13. Tumor necrosis factor receptor-associated factor 6 (TRAF6) participates in anti-lipopolysaccharide factors (ALFs) gene expression in mud crab.

    Science.gov (United States)

    Sun, Wan-Wei; Zhang, Xin-Xu; Wan, Wei-Song; Wang, Shu-Qi; Wen, Xiao-Bo; Zheng, Huai-Ping; Zhang, Yue-Ling; Li, Sheng-Kang

    2017-02-01

    Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key cytoplasm signal adaptor that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp TRAF6 (Sp-TRAF6) from Scylla paramamosain contains 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp-TRAf6 has highest identity of 88% with Pt-TRAF6 from Portunus trituberculatus, while the similarity of Sp-TRAF6 with other crustacean sequences was 54-55%. RT-PCR analysis indicated that Sp-TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Moreover, Sp-TRAF6 transcripts were significantly up-regulated after Vibrio parahemolyticus and LPS challenges. RNA interference assay was carried out used by siRNA to investigate the genes expression patterns regulated by Sp-TRAF6. The qRT-PCR results showed that silencing Sp-TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while inhibit SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 expression in hepatopancreas. Taken together, the acute-phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp-TRAF6 plays an important role in host defense against pathogen invasions via regulation of ALF gene expression in S. paramamosain. Copyright © 2016. Published by Elsevier Ltd.

  14. Targeting artificial transcription factors to the utrophin A promoter: effects on dystrophic pathology and muscle function.

    Science.gov (United States)

    Lu, Yifan; Tian, Chai; Danialou, Gawiyou; Gilbert, Rénald; Petrof, Basil J; Karpati, George; Nalbantoglu, Josephine

    2008-12-12

    Duchenne muscular dystrophy is caused by a genetic defect in the dystrophin gene. The absence of dystrophin results in muscle fiber necrosis and regeneration, leading to progressive muscle fiber loss. Utrophin is a close analogue of dystrophin. A substantial, ectopic expression of utrophin in the extrasynaptic sarcolemma of dystrophin-deficient muscle fibers can prevent deleterious effects of dystrophin deficiency. An alternative approach for the extrasynaptic up-regulation of utrophin involves the augmentation of utrophin transcription via the endogenous utrophin A promoter using custom-designed transcriptional activator proteins with zinc finger (ZFP) motifs. We tested a panel of custom-designed ZFP for their ability to activate the utrophin A promoter. Expression of one such ZFP efficiently increased, in a time-dependent manner, utrophin transcript and protein levels both in vitro and in vivo. In dystrophic mouse (mdx) muscles, administration of adenoviral vectors expressing this ZFP led to significant enhancement of muscle function with decreased necrosis, restoration of the dystrophin-associated proteins, and improved resistance to eccentric contractions. These studies provide evidence that specifically designed ZFPs can act as strong transcriptional activators of the utrophin A promoter. These may thus serve as attractive therapeutic agents for dystrophin deficiency states such as Duchenne muscular dystrophy.

  15. RNF11 is a multifunctional modulator of growth factor receptor signalling and transcriptional regulation.

    Science.gov (United States)

    Azmi, Peter; Seth, Arun

    2005-11-01

    Our laboratory has found that the 154aa RING finger protein 11 (RNF11), has modular domains and motifs including a RING-H2 finger domain, a PY motif, an ubiquitin interacting motif (UIM), a 14-3-3 binding sequence and an AKT phosphorylation site. RNF11 represents a unique protein with no other known immediate family members yet described. Comparative genetic analysis has shown that RNF11 is highly conserved throughout evolution. This may indicate a conserved and non-redundant role for the RNF11 protein. Molecular binding assays using RNF11 have shown that RNF11 has important roles in growth factor signalling, ubiquitination and transcriptional regulation. RNF11 has been shown to interact with HECT-type E3 ubiquitin ligases Nedd4, AIP4, Smurf1 and Smurf2, as well as with Cullin1, the core protein in the multi-subunit SCF E3 ubiquitin ligase complex. Work done in our laboratory has shown that RNF11 is capable of antagonizing Smurf2-mediated inhibition of TGFbeta signalling. Furthermore, RNF11 is capable of degrading AMSH, a positive regulator of both TGFbeta and EGFR signalling pathways. Recently, we have found that RNF11 can directly enhance TGFbeta signalling through a direct association with Smad4, the common signal transducer and transcription factor in the TGFbeta, BMP, and Activin pathways. Through its association with Smad4 and other transcription factors, RNF11 may have a role in direct transcriptional regulation. Our laboratory and others have found nearly 80 protein interactions for RNF11, placing RNF11 at the cross-roads of cell signalling and transcriptional regulation. RNF11 is highly expressed in breast tumours. Deregulation of RNF11 function may prove to be harmful to patient therapeutic outcomes. RNF11 may therefore provide a novel target for cancer therapeutics. The purpose of this review is to discuss the role of RNF11 in cell signalling and transcription factor modulation with special attention given to the ubiquitin-proteasomal pathway, TGFbeta

  16. A conserved PHD finger protein and endogenous RNAi modulate insulin signaling in Caenorhabditis elegans.

    Science.gov (United States)

    Mansisidor, Andres R; Cecere, Germano; Hoersch, Sebastian; Jensen, Morten B; Kawli, Trupti; Kennedy, Lisa M; Chavez, Violeta; Tan, Man-Wah; Lieb, Jason D; Grishok, Alla

    2011-09-01

    Insulin signaling has a profound effect on longevity and the oxidative stress resistance of animals. Inhibition of insulin signaling results in the activation of DAF-16/FOXO and SKN-1/Nrf transcription factors and increased animal fitness. By studying the biological functions of the endogenous RNA interference factor RDE-4 and conserved PHD zinc finger protein ZFP-1 (AF10), which regulate overlapping sets of genes in Caenorhabditis elegans, we identified an important role for these factors in the negative modulation of transcription of the insulin/PI3 signaling-dependent kinase PDK-1. Consistently, increased expression of pdk-1 in zfp-1 and rde-4 mutants contributed to their reduced lifespan and sensitivity to oxidative stress and pathogens due to the reduction in the expression of DAF-16 and SKN-1 targets. We found that the function of ZFP-1 in modulating pdk-1 transcription was important for the extended lifespan of the age-1(hx546) reduction-of-function PI3 kinase mutant, since the lifespan of the age-1; zfp-1 double mutant strain was significantly shorter compared to age-1(hx546). We further demonstrate that overexpression of ZFP-1 caused an increased resistance to oxidative stress in a DAF-16-dependent manner. Our findings suggest that epigenetic regulation of key upstream signaling components in signal transduction pathways through chromatin and RNAi may have a large impact on the outcome of signaling and expression of numerous downstream genes.

  17. The Saccharomyces cerevisiae RAD18 gene encodes a protein that contains potential zinc finger domains for nucleic acid binding and a putative nucleotide binding sequence

    Energy Technology Data Exchange (ETDEWEB)

    Jones, J.S.; Prakash, L. (Univ. of Rochester School of Medicine, NY (USA)); Weber, S. (Kodak Research Park, Rochester, NY (USA))

    1988-07-25

    The RAD18 gene of Saccharomyces cerevisiae is required for postreplication repair of UV damaged DNA. The authors have isolated the RAD18 gene, determined its nucleotide sequence and examined if deletion mutations of this gene show different or more pronounced phenotypic effects than the previously described point mutations. The RAD18 gene open reading frame encodes a protein of 487 amino acids, with a calculated molecular weight of 55,512. The RAD18 protein contains three potential zinc finger domains for nucleic acid binding, and a putative nucleotide binding sequence that is present in many proteins that bind and hydrolyze ATP. The DNA binding and nucleotide binding activities could enable the RAD18 protein to bind damaged sites in the template DNA with high affinity. Alternatively, or in addition, RAD18 protein may be a transcriptional regulator. The RAD18 deletion mutation resembles the previously described point mutations in its effects on viability, DNA repair, UV mutagenesis, and sporulation.

  18. Genome-wide investigation of transcription factors provides insights into transcriptional regulation in Plutella xylostella.

    Science.gov (United States)

    Zhao, Qian; Ma, Dongna; Huang, Yuping; He, Weiyi; Li, Yiying; Vasseur, Liette; You, Minsheng

    2018-04-01

    Transcription factors (TFs), which play a vital role in regulating gene expression, are prevalent in all organisms and characterization of them may provide important clues for understanding regulation in vivo. The present study reports a genome-wide investigation of TFs in the diamondback moth, Plutella xylostella (L.), a worldwide pest of crucifers. A total of 940 TFs distributed among 133 families were identified. Phylogenetic analysis of insect species showed that some of these families were found to have expanded during the evolution of P. xylostella or Lepidoptera. RNA-seq analysis showed that some of the TF families, such as zinc fingers, homeobox, bZIP, bHLH, and MADF_DNA_bdg genes, were highly expressed in certain tissues including midgut, salivary glands, fat body, and hemocytes, with an obvious sex-biased expression pattern. In addition, a number of TFs showed significant differences in expression between insecticide susceptible and resistant strains, suggesting that these TFs play a role in regulating genes related to insecticide resistance. Finally, we identified an expansion of the HOX cluster in Lepidoptera, which might be related to Lepidoptera-specific evolution. Knockout of this cluster using CRISPR/Cas9 showed that the egg cannot hatch, indicating that this cluster may be related to egg development and maturation. This is the first comprehensive study on identifying and characterizing TFs in P. xylostella. Our results suggest that some TF families are expanded in the P. xylostella genome, and these TFs may have important biological roles in growth, development, sexual dimorphism, and resistance to insecticides. The present work provides a solid foundation for understanding regulation via TFs in P. xylostella and insights into the evolution of the P. xylostella genome.

  19. AcEST: DK943548 [AcEST

    Lifescience Database Archive (English)

    Full Text Available astX Result : Swiss-Prot sp_hit_id Q9Y2K1 Definition sp|Q9Y2K1|ZBTB1_HUMAN Zinc finger and BTB domain-contai......done Score E Sequences producing significant alignments: (bits) Value sp|Q9Y2K1|ZBTB1_HUMAN Zinc finger a...6.3 sp|Q9DDT5|SPT5H_DANRE Transcription elongation factor SPT5 OS=Da... 29 8.2 >sp|Q9Y2K1|ZBTB1_HUMAN Zinc f

  20. Functional and DNA-protein binding studies of WRKY transcription factors and their expression analysis in response to biotic and abiotic stress in wheat (Triticum aestivum L.).

    Science.gov (United States)

    Satapathy, Lopamudra; Kumar, Dhananjay; Kumar, Manish; Mukhopadhyay, Kunal

    2018-01-01

    WRKY, a plant-specific transcription factor family, plays vital roles in pathogen defense, abiotic stress, and phytohormone signalling. Little is known about the roles and function of WRKY transcription factors in response to rust diseases in wheat. In the present study, three TaWRKY genes encoding complete protein sequences were cloned. They belonged to class II and III WRKY based on the number of WRKY domains and the pattern of zinc finger structures. Twenty-two DNA-protein binding docking complexes predicted stable interactions of WRKY domain with W-box. Quantitative real-time-PCR using wheat near-isogenic lines with or without Lr28 gene revealed differential up- or down-regulation in response to biotic and abiotic stress treatments which could be responsible for their functional divergence in wheat. TaWRKY62 was found to be induced upon treatment with JA, MJ, and SA and reduced after ABA treatments. Maximum induction of six out of seven genes occurred at 48 h post inoculation due to pathogen inoculation. Hence, TaWRKY (49, 50 , 52 , 55 , 57, and 62 ) can be considered as potential candidate genes for further functional validation as well as for crop improvement programs for stress resistance. The results of the present study will enhance knowledge towards understanding the molecular basis of mode of action of WRKY transcription factor genes in wheat and their role during leaf rust pathogenesis in particular.

  1. Zinc finger arrays binding human papillomavirus types 16 and 18 genomic DNA: precursors of gene-therapeutics for in-situ reversal of associated cervical neoplasia

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2012-07-01

    Full Text Available Abstract Background Human papillomavirus (HPV types 16 and 18 are the high-risk, sexually transmitted infectious causes of most cervical intraepithelial neoplasias (CIN or cancers. While efficacious vaccines to reduce the sexual acquisition of these high-risk HPVs have recently been introduced, no virus-targeted therapies exist for those already exposed and infected. Considering the oncogenic role of the transforming (E6 and E7 genes of high-risk HPVs in the slow pathogenesis of cervical cancer, we hypothesize that timely disruption or abolition of HPV genome expression within pre-cancerous lesions identified at screening may reverse neoplasia. We aimed to derive model zinc finger nucleases (ZFNs for mutagenesis of the genomes of two high-risk HPV (types 16 & 18. Methods and results Using ZiFiT software and the complete genomes of HPV types16 and 18, we computationally generated the consensus amino acid sequences of the DNA-binding domains (F1, F2, & F3 of (i 296 & 327 contextually unpaired (or single three zinc-finger arrays (sZFAs and (ii 9 & 13 contextually paired (left and right three- zinc-finger arrays (pZFAs that bind genomic DNA of HPV-types 16 and 18 respectively, inclusive of the E7 gene (s/pZFAHpV/E7. In the absence of contextually paired three-zinc-finger arrays (pZFAs that bind DNA corresponding to the genomic context of the E6 gene of either HPV type, we derived the DNA binding domains of another set of 9 & 14 contextually unpaired E6 gene-binding ZFAs (sZFAE6 to aid the future quest for paired ZFAs to target E6 gene sequences in both HPV types studied (pZFAE6. This paper presents models for (i synthesis of hybrid ZFNs that cleave within the genomic DNA of either HPV type, by linking the gene sequences of the DNA-cleavage domain of the FokI endonuclease FN to the gene sequences of a member of the paired-HPV-binding ZFAs (pZFAHpV/E7 + FN, and (ii delivery of the same into precancerous lesions using HPV-derived viral plasmids or

  2. CFLAP1 and CFLAP2 Are Two bHLH Transcription Factors Participating in Synergistic Regulation of AtCFL1-Mediated Cuticle Development in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Shibai Li

    2016-01-01

    Full Text Available The cuticle is a hydrophobic lipid layer covering the epidermal cells of terrestrial plants. Although many genes involved in Arabidopsis cuticle development have been identified, the transcriptional regulation of these genes is largely unknown. Previously, we demonstrated that AtCFL1 negatively regulates cuticle development by interacting with the HD-ZIP IV transcription factor HDG1. Here, we report that two bHLH transcription factors, AtCFL1 associated protein 1 (CFLAP1 and CFLAP2, are also involved in AtCFL1-mediated regulation of cuticle development. CFLAP1 and CFLAP2 interact with AtCFL1 both in vitro and in vivo. Overexpression of either CFLAP1 or CFLAP2 led to expressional changes of genes involved in fatty acids, cutin and wax biosynthesis pathways and caused multiple cuticle defective phenotypes such as organ fusion, breakage of the cuticle layer and decreased epicuticular wax crystal loading. Functional inactivation of CFLAP1 and CFLAP2 by chimeric repression technology caused opposite phenotypes to the CFLAP1 overexpressor plants. Interestingly, we find that, similar to the transcription factor HDG1, the function of CFLAP1 in cuticle development is dependent on the presence of AtCFL1. Furthermore, both HDG1 and CFLAP1/2 interact with the same C-terminal C4 zinc finger domain of AtCFL1, a domain that is essential for AtCFL1 function. These results suggest that AtCFL1 may serve as a master regulator in the transcriptional regulation of cuticle development, and that CFLAP1 and CFLAP2 are involved in the AtCFL1-mediated regulation pathway, probably through competing with HDG1 to bind to AtCFL1.

  3. Genome-wide analysis of the Dof transcription factor gene family reveals soybean-specific duplicable and functional characteristics.

    Directory of Open Access Journals (Sweden)

    Yong Guo

    Full Text Available The Dof domain protein family is a classic plant-specific zinc-finger transcription factor family involved in a variety of biological processes. There is great diversity in the number of Dof genes in different plants. However, there are only very limited reports on the characterization of Dof transcription factors in soybean (Glycine max. In the present study, 78 putative Dof genes were identified from the whole-genome sequence of soybean. The predicted GmDof genes were non-randomly distributed within and across 19 out of 20 chromosomes and 97.4% (38 pairs were preferentially retained duplicate paralogous genes located in duplicated regions of the genome. Soybean-specific segmental duplications contributed significantly to the expansion of the soybean Dof gene family. These Dof proteins were phylogenetically clustered into nine distinct subgroups among which the gene structure and motif compositions were considerably conserved. Comparative phylogenetic analysis of these Dof proteins revealed four major groups, similar to those reported for Arabidopsis and rice. Most of the GmDofs showed specific expression patterns based on RNA-seq data analyses. The expression patterns of some duplicate genes were partially redundant while others showed functional diversity, suggesting the occurrence of sub-functionalization during subsequent evolution. Comprehensive expression profile analysis also provided insights into the soybean-specific functional divergence among members of the Dof gene family. Cis-regulatory element analysis of these GmDof genes suggested diverse functions associated with different processes. Taken together, our results provide useful information for the functional characterization of soybean Dof genes by combining phylogenetic analysis with global gene-expression profiling.

  4. OVO homologue-like 1 (Ovol1) transcription factor: a novel target of neurogenin-3 in rodent pancreas.

    Science.gov (United States)

    Vetere, A; Li, W-C; Paroni, F; Juhl, K; Guo, L; Nishimura, W; Dai, X; Bonner-Weir, S; Sharma, A

    2010-01-01

    The basic helix-loop-helix transcription factor neurogenin-3 (NGN3) commits the fates of pancreatic progenitors to endocrine cell types, but knowledge of the mechanisms regulating the choice between proliferation and differentiation of these progenitors is limited. Using a chromatin immunoprecipitation cloning approach, we searched for direct targets of NGN3 and identified a zinc-finger transcription factor, OVO homologue-like 1 (OVOL1). Transactivation experiments were carried out to elucidate the functional role of NGN3 in Ovol1 gene expression. Embryonic and adult rodents pancreases were immunostained for OVOL1, Ki67 and NGN3. We showed that NGN3 negatively regulates transcription of Ovol1 in an E-box-dependent fashion. The presence of either NGN3 or NEUROD1, but not MYOD, reduced endogenous Ovol1 mRNA. OVOL1 was detected in pancreatic tissue around embryonic day 15.5, after which OVOL1 levels dramatically increased. In embryonic pancreas, OVOL1 protein levels were low in NGN3(+) or Ki67(+) cells, but high in quiescent differentiated cells. OVOL1 presence was maintained in adult pancreas, where it was detected in islets, pancreatic ducts and some acinar cells. Additionally OVOL1 presence was lacking in proliferating ductules in regenerating pancreas and induced in cells as they began to acquire their differentiated phenotype. The timing of OVOL1 appearance in pancreas and its increased levels in differentiated cells suggest that OVOL1 promotes the transition of cells from a proliferating, less-differentiated state to a quiescent more-differentiated state. We conclude that OVOL1, a downstream target of NGN3, may play an important role in regulating the balance between proliferation and differentiation of pancreatic cells.

  5. Role of LRF/Pokemon in lineage fate decisions

    Science.gov (United States)

    Lunardi, Andrea; Guarnerio, Jlenia; Wang, Guocan

    2013-01-01

    In the human genome, 43 different genes are found that encode proteins belonging to the family of the POK (poxvirus and zinc finger and Krüppel)/ZBTB (zinc finger and broad complex, tramtrack, and bric à brac) factors. Generally considered transcriptional repressors, several of these genes play fundamental roles in cell lineage fate decision in various tissues, programming specific tasks throughout the life of the organism. Here, we focus on functions of leukemia/lymphoma-related factor/POK erythroid myeloid ontogenic factor, which is probably one of the most exciting and yet enigmatic members of the POK/ZBTB family. PMID:23396304

  6. Proviral HIV-genome-wide and pol-gene specific Zinc Finger Nucleases: Usability for targeted HIV gene therapy

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2011-07-01

    Full Text Available Abstract Background Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that ex-vivo or even in-vivo abolition or disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively. However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i to assemble and construct zinc finger arrays and nucleases (ZFN with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii to advance a model for pre-clinically testing lentiviral vectors (LV that deliver and transduce either ZFN genotype. Methods and Results First, we computationally generated the consensus sequences of (a 114 dsDNA-binding zinc finger (Zif arrays (ZFAs or ZifHIV-pol and (b two zinc-finger nucleases (ZFNs which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (ZifHIV-polFN. Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (ZifHIV-1FN. Second, a model for constructing lentiviral vectors (LVs that deliver and transduce a diploid copy of either ZifHIV-polFN or ZifHIV-1FN chimeric genes (termed LV- 2xZifHIV-polFN and LV- 2xZifHIV-1FN, respectively is proposed. Third, two preclinical models for controlled testing of

  7. Proviral HIV-genome-wide and pol-gene specific zinc finger nucleases: usability for targeted HIV gene therapy.

    Science.gov (United States)

    Wayengera, Misaki

    2011-07-22

    Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that ex-vivo or even in-vivo abolition or disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases) AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively). However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX) at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i) to assemble and construct zinc finger arrays and nucleases (ZFN) with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii) to advance a model for pre-clinically testing lentiviral vectors (LV) that deliver and transduce either ZFN genotype. First, we computationally generated the consensus sequences of (a) 114 dsDNA-binding zinc finger (Zif) arrays (ZFAs or ZifHIV-pol) and (b) two zinc-finger nucleases (ZFNs) which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (ZifHIV-polFN). Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (ZifHIV-1FN). Second, a model for constructing lentiviral vectors (LVs) that deliver and transduce a diploid copy of either ZifHIV-polFN or ZifHIV-1FN chimeric genes (termed LV- 2xZifHIV-polFN and LV- 2xZifHIV-1FN, respectively) is proposed. Third, two preclinical models for controlled testing of the safety and efficacy of either of these

  8. GATA-2 and GATA-3 regulate trophoblast-specific gene expression in vivo.

    NARCIS (Netherlands)

    G.T. Ma (Grace); M.E. Roth (Matthew); J.C. Groskopf (John); F.G. Grosveld (Frank); J.D. Engel (Douglas); D.I.H. Linzer (Daniel); F.Y. Tsai (Fong-Ying); S.H. Orkin (Stuart)

    1997-01-01

    textabstractWe previously demonstrated that the zinc finger transcription factors GATA-2 and GATA-3 are expressed in trophoblast giant cells and that they regulate transcription from the mouse placental lactogen I gene promoter in a transfected trophoblast cell line. We present evidence here that

  9. An engineered split M.HhaI-zinc finger fusion lacks the intended methyltransferase specificity

    International Nuclear Information System (INIS)

    Meister, Glenna E.; Chandrasegaran, Srinivasan; Ostermeier, Marc

    2008-01-01

    The ability to site-specifically methylate DNA in vivo would have wide applicability to the study of basic biomedical problems as well as enable studies on the potential of site-specific DNA methylation as a therapeutic strategy for the treatment of diseases. Natural DNA methyltransferases lack the specificity required for these applications. Nomura and Barbas [W. Nomura, C.F. Barbas 3rd, In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase, J. Am. Chem. Soc. 129 (2007) 8676-8677] have reported that an engineered DNA methyltransferase comprised of fragments of M.HhaI methyltransferase and zinc finger proteins has very high specificity for the chosen target site. Our analysis of this engineered enzyme shows that the fusion protein methylates target and non-target sites with similar efficiency

  10. The ZEB1 transcription factor is a novel repressor of adiposity in female mice.

    Directory of Open Access Journals (Sweden)

    Jessica N Saykally

    Full Text Available BACKGROUND: Four genome-wide association studies mapped an "obesity" gene to human chromosome 10p11-12. As the zinc finger E-box binding homeobox 1 (ZEB1 transcription factor is encoded by the TCF8 gene located in that region, and as it influences the differentiation of various mesodermal lineages, we hypothesized that ZEB1 might also modulate adiposity. The goal of these studies was to test that hypothesis in mice. METHODOLOGY/PRINCIPAL FINDINGS: To ascertain whether fat accumulation affects ZEB1 expression, female C57BL/6 mice were fed a regular chow diet (RCD ad libitum or a 25% calorie-restricted diet from 2.5 to 18.3 months of age. ZEB1 mRNA levels in parametrial fat were six to ten times higher in the obese mice. To determine directly whether ZEB1 affects adiposity, wild type (WT mice and mice heterozygous for TCF8 (TCF8+/- were fed an RCD or a high-fat diet (HFD (60% calories from fat. By two months of age on an HFD and three months on an RCD, TCF8+/- mice were heavier than WT controls, which was attributed by Echo MRI to increased fat mass (at three months on an HFD: 0.517+/-0.081 total fat/lean mass versus 0.313+/-0.036; at three months on an RCD: 0.175+/-0.013 versus 0.124+/-0.012. No differences were observed in food uptake or physical activity, suggesting that the genotypes differ in some aspect of their metabolic activity. ZEB1 expression also increases during adipogenesis in cell culture. CONCLUSION/SIGNIFICANCE: These results show for the first time that the ZEB1 transcription factor regulates the accumulation of adipose tissue. Furthermore, they corroborate the genome-wide association studies that mapped an "obesity" gene at chromosome 10p11-12.

  11. Phylogenetic and comparative gene expression analysis of barley (Hordeum vulgare)WRKY transcription factor family reveals putatively retained functions betweenmonocots and dicots

    Energy Technology Data Exchange (ETDEWEB)

    Mangelsen, Elke; Kilian, Joachim; Berendzen, Kenneth W.; Kolukisaoglu, Uner; Harter, Klaus; Jansson, Christer; Wanke, Dierk

    2008-02-01

    WRKY proteins belong to the WRKY-GCM1 superfamily of zinc finger transcription factors that have been subject to a large plant-specific diversification. For the cereal crop barley (Hordeum vulgare), three different WRKY proteins have been characterized so far, as regulators in sucrose signaling, in pathogen defense, and in response to cold and drought, respectively. However, their phylogenetic relationship remained unresolved. In this study, we used the available sequence information to identify a minimum number of 45 barley WRKY transcription factor (HvWRKY) genes. According to their structural features the HvWRKY factors were classified into the previously defined polyphyletic WRKY subgroups 1 to 3. Furthermore, we could assign putative orthologs of the HvWRKY proteins in Arabidopsis and rice. While in most cases clades of orthologous proteins were formed within each group or subgroup, other clades were composed of paralogous proteins for the grasses and Arabidopsis only, which is indicative of specific gene radiation events. To gain insight into their putative functions, we examined expression profiles of WRKY genes from publicly available microarray data resources and found group specific expression patterns. While putative orthologs of the HvWRKY transcription factors have been inferred from phylogenetic sequence analysis, we performed a comparative expression analysis of WRKY genes in Arabidopsis and barley. Indeed, highly correlative expression profiles were found between some of the putative orthologs. HvWRKY genes have not only undergone radiation in monocot or dicot species, but exhibit evolutionary traits specific to grasses. HvWRKY proteins exhibited not only sequence similarities between orthologs with Arabidopsis, but also relatedness in their expression patterns. This correlative expression is indicative for a putative conserved function of related WRKY proteins in mono- and dicot species.

  12. Engineering customized TALE nucleases (TALENs) and TALE transcription factors by fast ligation-based automatable solid-phase high-throughput (FLASH) assembly.

    Science.gov (United States)

    Reyon, Deepak; Maeder, Morgan L; Khayter, Cyd; Tsai, Shengdar Q; Foley, Jonathan E; Sander, Jeffry D; Joung, J Keith

    2013-07-01

    Customized DNA-binding domains made using transcription activator-like effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in various organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or downregulate expression of endogenous genes in human cells and plants. This unit describes a detailed protocol for the recently described fast ligation-based automatable solid-phase high-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multichannel pipet. Using the automated approach, a single researcher can construct up to 96 DNA fragments encoding TALE repeat arrays of various lengths in a single day, and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in a week or less. Plasmids required for FLASH are available by request from the Joung lab (http://eGenome.org). This unit also describes improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) web server (http://ZiFiT.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high throughput. © 2013 by John Wiley & Sons, Inc.

  13. Bioaccessible mineral content of malted finger millet (Eleusine coracana), wheat (Triticum aestivum), and barley (Hordeum vulgare).

    Science.gov (United States)

    Platel, Kalpana; Eipeson, Sushma W; Srinivasan, Krishnapura

    2010-07-14

    Malted grains are extensively used in weaning and geriatric foods. Malting generally improves the nutrient content and digestibility of foods. The present investigation examined the influence of malting of finger millet, wheat, and barley on the bioaccessibility of iron, zinc, calcium, copper, and manganese. Malting increased the bioaccessibility of iron by >3-fold from the two varieties of finger millet and by >2-fold from wheat, whereas such a beneficial influence was not seen in barley. The bioaccessibility of zinc from wheat and barley increased to an extent of 234 and 100%, respectively, as a result of malting. However, malting reduced the bioaccessibility of zinc from finger millet. Malting marginally increased the bioaccessibility of calcium from white finger millet and wheat. Whereas malting did not exert any influence on bioaccessibility of copper from finger millet and wheat, it significantly decreased (75%) the same from barley. Malting did increase the bioaccessibility of manganese from brown finger millet (17%) and wheat (42%). Thus, malting could be an appropriate food-based strategy to derive iron and other minerals maximally from food grains.

  14. Revisiting and re-engineering the classical zinc finger peptide: consensus peptide-1 (CP-1).

    Science.gov (United States)

    Besold, Angelique N; Widger, Leland R; Namuswe, Frances; Michalek, Jamie L; Michel, Sarah L J; Goldberg, David P

    2016-04-01

    Zinc plays key structural and catalytic roles in biology. Structural zinc sites are often referred to as zinc finger (ZF) sites, and the classical ZF contains a Cys2His2 motif that is involved in coordinating Zn(II). An optimized Cys2His2 ZF, named consensus peptide 1 (CP-1), was identified more than 20 years ago using a limited set of sequenced proteins. We have reexamined the CP-1 sequence, using our current, much larger database of sequenced proteins that have been identified from high-throughput sequencing methods, and found the sequence to be largely unchanged. The CCHH ligand set of CP-1 was then altered to a CAHH motif to impart hydrolytic activity. This ligand set mimics the His2Cys ligand set of peptide deformylase (PDF), a hydrolytically active M(II)-centered (M = Zn or Fe) protein. The resultant peptide [CP-1(CAHH)] was evaluated for its ability to coordinate Zn(II) and Co(II) ions, adopt secondary structure, and promote hydrolysis. CP-1(CAHH) was found to coordinate Co(II) and Zn(II) and a pentacoordinate geometry for Co(II)-CP-1(CAHH) was implicated from UV-vis data. This suggests a His2Cys(H2O)2 environment at the metal center. The Zn(II)-bound CP-1(CAHH) was shown to adopt partial secondary structure by 1-D (1)H NMR spectroscopy. Both Zn(II)-CP-1(CAHH) and Co(II)-CP-1(CAHH) show good hydrolytic activity toward the test substrate 4-nitrophenyl acetate, exhibiting faster rates than most active synthetic Zn(II) complexes.

  15. Expression and Functional Analysis of WRKY Transcription Factors in Chinese Wild Hazel, Corylus heterophylla Fisch.

    Science.gov (United States)

    Zhao, Tian-Tian; Zhang, Jin; Liang, Li-Song; Ma, Qing-Hua; Chen, Xin; Zong, Jian-Wei; Wang, Gui-Xi

    2015-01-01

    Plant WRKY transcription factors are known to regulate various biotic and abiotic stress responses. In this study we identified a total of 30 putative WRKY unigenes in a transcriptome dataset of the Chinese wild Hazel, Corylus heterophylla, a species that is noted for its cold tolerance. Thirteen full-length of these ChWRKY genes were cloned and found to encode complete protein sequences, and they were divided into three groups, based on the number of WRKY domains and the pattern of zinc finger structures. Representatives of each of the groups, Unigene25835 (group I), Unigene37641 (group II) and Unigene20441 (group III), were transiently expressed as fusion proteins with yellow fluorescent fusion protein in Nicotiana benthamiana, where they were observed to accumulate in the nucleus, in accordance with their predicted roles as transcriptional activators. An analysis of the expression patterns of all 30 WRKY genes revealed differences in transcript abundance profiles following exposure to cold, drought and high salinity conditions. Among the stress-inducible genes, 23 were up-regulated by all three abiotic stresses and the WRKY genes collectively exhibited four different patterns of expression in flower buds during the overwintering period from November to April. The organ/tissue related expression analysis showed that 18 WRKY genes were highly expressed in stem but only 2 (Unigene9262 and Unigene43101) were greatest in male anthotaxies. The expression of Unigene37641, a member of the group II WRKY genes, was substantially up-regulated by cold, drought and salinity treatments, and its overexpression in Arabidopsis thaliana resulted in better seedling growth, compared with wild type plants, under cold treatment conditions. The transgenic lines also had exhibited higher soluble protein content, superoxide dismutase and peroxidase activiety and lower levels of malondialdehyde, which collectively suggets that Unigene37641 expression promotes cold tolerance.

  16. A novel zinc finger protein 219-like (ZNF219L) is involved in the regulation of collagen type 2 alpha 1a (col2a1a) gene expression in zebrafish notochord.

    Science.gov (United States)

    Lien, Huang-Wei; Yang, Chung-Hsiang; Cheng, Chia-Hsiung; Hung, Chin-Chun; Liao, Wei-Hao; Hwang, Pung-Pung; Han, Yu-San; Huang, Chang-Jen

    2013-01-01

    The notochord is required for body plan patterning in vertebrates, and defects in notochord development during embryogenesis can lead to diseases affecting the adult. It is therefore important to elucidate the gene regulatory mechanism underlying notochord formation. In this study, we cloned the zebrafish zinc finger 219-like (ZNF219L) based on mammalian ZNF219, which contains nine C2H2-type zinc finger domains. Through whole-mount in situ hybridization, we found that znf219L mRNA is mainly expressed in the zebrafish midbrain-hindbrain boundary, hindbrain, and notochord during development. The znf219L morpholino knockdown caused partial abnormal notochord phenotype and reduced expression of endogenous col2a1a in the notochord specifically. In addition, ZNF219L could recognize binding sites with GGGGG motifs and trigger augmented activity of the col2a1a promoter in a luciferase assay. Furthermore, in vitro binding experiments revealed that ZNF219L recognizes the GGGGG motifs in the promoter region of the zebrafish col2a1a gene through its sixth and ninth zinc finger domains. Taken together, our results reveal that ZNF219L is involved in regulating the expression of col2a1a in zebrafish notochord specifically.

  17. Transcription factor ART1 mediates starch hydrolysis and mycotoxin production in Fusarium graminearum and F. verticillioides.

    Science.gov (United States)

    Oh, Mira; Son, Hokyoung; Choi, Gyung Ja; Lee, Chanhui; Kim, Jin-Cheol; Kim, Hun; Lee, Yin-Won

    2016-06-01

    Molecular mechanisms underlying the responses to environmental factors, such as nitrogen, carbon and pH, involve components that regulate the production of secondary metabolites, including mycotoxins. In this study, we identified and characterized a gene in the FGSG_02083 locus, designated as FgArt1, which was predicted to encode a Zn(II)2 Cys6 zinc finger transcription factor. An FgArt1 deletion mutant of Fusarium graminearum exhibited impaired starch hydrolysis as a result of significantly reduced α-amylase gene expression. The deletion strain was unable to produce trichothecenes and exhibited low Tri5 and Tri6 expression levels, whereas the complemented strain showed a similar ability to produce trichothecenes as the wild-type strain. In addition, FgArt1 deletion resulted in impairment of germination in starch liquid medium and reduced pathogenicity on flowering wheat heads. To investigate the roles of the FgArt1 homologue in F. verticillioides, we deleted the FVEG_02083 gene, and the resulting strain showed defects in starch hydrolysis, similar to the FgArt1 deletion strain, and produced no detectable level of fumonisin B1 . Fum1 and Fum12 expression levels were undetectable in the deletion strain. However, when the FvArt1-deleted F. verticillioides strain was complemented with FgArt1, the resulting strain was unable to recover the production of fumonisin B1 , although FgArt1 expression and starch hydrolysis were induced. Thus, our results suggest that there are different regulatory pathways governed by each ART1 transcription factor in trichothecene and fumonisin biosynthesis. Taken together, we suggest that ART1 plays an important role in both trichothecene and fumonisin biosynthesis by the regulation of genes involved in starch hydrolysis. © 2015 BSPP AND JOHN WILEY & SONS LTD.

  18. Paradoxical role of an Egr transcription factor family member, Egr2/Krox20, in learning and memory

    Directory of Open Access Journals (Sweden)

    Roseline Poirier

    2007-12-01

    Full Text Available It is well established that Egr1/zif268, a member of the Egr family of transcription factors, is critical for the consolidation of several forms of memories. Recently, the Egr3 family member has also been implicated in learning and memory. Because Egr family members encode closely related zinc-finger transcription factors sharing a highly homologous DNA binding domain that recognises the same DNA sequence, they may have related functions in brain. Another Egr family member expressed in brain, Egr2/Krox20 is known to be crucial for normal hindbrain development and has been implicated in several inherited peripheral neuropathies; however, due to Egr2-null mice perinatal lethality, its potential role in cognitive functions in the adult has not been yet explored. Here, we generated Egr2 conditional mutant mice allowing postnatal, forebrain-specific Cre-mediated Egr2 excision and tested homozygous, heterozygous and control littermates on a battery of behavioural tasks to evaluate motor capacity, exploratory behaviour, emotional reactivity and learning and memory performance in spatial and non-spatial tasks. Egr2-deficient mice had no sign of locomotor, exploratory or anxiety disturbances. Surprisingly, they also had no impairment in spatial learning and memory, taste aversion memory or fear memory using a trace conditioning paradigm. On the contrary, Egr2-deficient mice had improved performance in motor learning on a rotarod, and in object recognition memory. These results clearly do not extend the phenotypic consequences resulting from either Egr1 or Egr3 loss-of-function to Egr2. In contrast, they indicate that Egr family members may have different, and in certain circumstances antagonistic functions in the adult brain.

  19. Systematic analysis and comparison of the PHD-Finger gene family in Chinese pear (Pyrus bretschneideri) and its role in fruit development.

    Science.gov (United States)

    Cao, Yunpeng; Han, Yahui; Meng, Dandan; Abdullah, Muhammad; Li, Dahui; Jin, Qing; Lin, Yi; Cai, Yongping

    2018-04-20

    PHD-finger proteins, which belongs to the type of zinc finger family, and that play an important role in the regulation of both transcription and the chromatin state in eukaryotes. Currently, PHD-finger proteins have been well studied in animals, while few studies have been carried out on their function in plants. In the present study, 129 non-redundant PHD-finger genes were identified from 5 Rosaceae species (pear, apple, strawberry, mei, and peach); among them, 31 genes were identified in pear. Subsequently, we carried out a bioinformatics analysis of the PHD-finger genes. Thirty-one PbPHD genes were divided into 7 subfamilies based on the phylogenetic analysis, which are consistent with the intron-exon and conserved motif analyses. In addition, we identified five segmental duplication events, implying that the segmental duplications might be a crucial role in the expansion of the PHD-finger gene family in pear. The microsynteny analysis of five Rosaceae species showed that there were independent duplication events in addition to the genome-wide duplication of the pear genome. Subsequently, ten expressed PHD-finger genes of pear fruit were identified using qRT-PCR, and one of these genes, PbPHD10, was identified as an important candidate gene for the regulation of lignin synthesis. Our research provides useful information for the further analysis of the function of PHD-finger gene family in pear.

  20. A novel zinc-finger-like gene from Tamarix hispida is involved in salt and osmotic tolerance.

    Science.gov (United States)

    An, Yan; Wang, Yucheng; Lou, Lingling; Zheng, Tangchun; Qu, Guan-Zheng

    2011-11-01

    In the present study, a zinc-finger-like cDNA (ThZFL) was cloned from the Tamarix hispida. Northern blot analysis showed that the expression of ThZFL can be induced by salt, osmotic stress and ABA treatment. Overexpression of the ThZFL confers salt and osmotic stress tolerance in both yeast Saccharomyces cerevisiae and tobacco. Furthermore, MDA levels in ThZFL transformed tobacco were significantly decreased compared with control plants under salt and osmotic stress, suggesting ThZFL may confer stress tolerance by decreasing membrane lipid peroxidation. Subcellular localization analysis showed the ThZFL protein is localized in the cell wall. Our results indicated the ThZFL gene is an excellent candidate for genetic engineering to improve salt and osmotic tolerance in agricultural plants.

  1. Expression of RIZ1 protein (Retinoblastoma-interacting zinc-finger protein 1) in prostate cancer epithelial cells changes with cancer grade progression and is modulated in vitro by DHT and E2.

    Science.gov (United States)

    Rossi, Valentina; Staibano, Stefania; Abbondanza, Ciro; Pasquali, Daniela; De Rosa, Caterina; Mascolo, Massimo; Bellastella, Giuseppe; Visconti, Daniela; De Bellis, Annamaria; Moncharmont, Bruno; De Rosa, Gaetano; Puca, Giovanni Alfredo; Bellastella, Antonio; Sinisi, Antonio Agostino

    2009-12-01

    The nuclear protein methyl-transferase Retinoblastoma-interacting zinc-finger protein 1 (RIZ1) is considered to be a downstream effector of estrogen action in target tissues. Silencing of RIZ1 expression is common in many tumors. We analyzed RIZ1 expression in normal and malignant prostate tissue and evaluated whether estradiol (E2) or dihydrotestosterone (DHT) treatment modulated RIZ1 in cultured prostate epithelial cells (PEC). Moreover, we studied the possible involvement of RIZ1 in estrogen action on the EPN prostate cell line, constitutively expressing both estrogen receptor (ER)-alpha and beta. RIZ1 protein, found in the nucleus of normal PECs by immunohistochemistry, was progressively lost in cancer tissues as the Gleason score increased and was only detected in the cytoplasmic compartment. RIZ1 transcript levels, as assayed by semi-quantitative RT-PCR in primary PEC cultures, were significantly reduced in cancer cells (P DHT treatment significantly increased RIZ1 transcript and protein levels (P DHT or E2 treatment in vitro. Furthermore, the E2 effects on ER-expressing prostate cells involve RIZ1, which confirms a possible role for ER-mediated pathways in a non-classic E(2)-target tissue.

  2. Zinc in Infection and Inflammation.

    Science.gov (United States)

    Gammoh, Nour Zahi; Rink, Lothar

    2017-06-17

    Micronutrient homeostasis is a key factor in maintaining a healthy immune system. Zinc is an essential micronutrient that is involved in the regulation of the innate and adaptive immune responses. The main cause of zinc deficiency is malnutrition. Zinc deficiency leads to cell-mediated immune dysfunctions among other manifestations. Consequently, such dysfunctions lead to a worse outcome in the response towards bacterial infection and sepsis. For instance, zinc is an essential component of the pathogen-eliminating signal transduction pathways leading to neutrophil extracellular traps (NET) formation, as well as inducing cell-mediated immunity over humoral immunity by regulating specific factors of differentiation. Additionally, zinc deficiency plays a role in inflammation, mainly elevating inflammatory response as well as damage to host tissue. Zinc is involved in the modulation of the proinflammatory response by targeting Nuclear Factor Kappa B (NF-κB), a transcription factor that is the master regulator of proinflammatory responses. It is also involved in controlling oxidative stress and regulating inflammatory cytokines. Zinc plays an intricate function during an immune response and its homeostasis is critical for sustaining proper immune function. This review will summarize the latest findings concerning the role of this micronutrient during the course of infections and inflammatory response and how the immune system modulates zinc depending on different stimuli.

  3. An estimation of finger-tapping rates and load capacities and the effects of various factors.

    Science.gov (United States)

    Ekşioğlu, Mahmut; İşeri, Ali

    2015-06-01

    The aim of this study was to estimate the finger-tapping rates and finger load capacities of eight fingers (excluding thumbs) for a healthy adult population and investigate the effects of various factors on tapping rate. Finger-tapping rate, the total number of finger taps per unit of time, can be used as a design parameter of various products and also as a psychomotor test for evaluating patients with neurologic problems. A 1-min tapping task was performed by 148 participants with maximum volitional tempo for each of eight fingers. For each of the tapping tasks, the participant with the corresponding finger tapped the associated key in the standard position on the home row of a conventional keyboard for touch typing. The index and middle fingers were the fastest fingers for both hands, and little fingers the slowest. All dominant-hand fingers, except little finger, had higher tapping rates than the fastest finger of the nondominant hand. Tapping rate decreased with age and smokers tapped faster than nonsmokers. Tapping duration and exercise had also significant effect on tapping rate. Normative data of tapping rates and load capacities of eight fingers were estimated for the adult population. In designs of psychomotor tests that require the use of tapping rate or finger load capacity data, the effects of finger, age, smoking, and tapping duration need to be taken into account. The findings can be used for ergonomic designs requiring finger-tapping capacity and also as a reference in psychomotor tests. © 2015, Human Factors and Ergonomics Society.

  4. Generation of knockout rabbits using transcription activator-like effector nucleases.

    Science.gov (United States)

    Wang, Yu; Fan, Nana; Song, Jun; Zhong, Juan; Guo, Xiaogang; Tian, Weihua; Zhang, Quanjun; Cui, Fenggong; Li, Li; Newsome, Philip N; Frampton, Jon; Esteban, Miguel A; Lai, Liangxue

    2014-01-01

    Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

  5. Identification, molecular characterization, and analysis of the ...

    Indian Academy of Sciences (India)

    Qiyan Du. E-mail: 041019@htu.edu.cn. Abstract. Sox7, Sox17, and Sox18 are members of the ... chondrogenesis, in endoderm development, and in sex determination and ...... Stimulating protein 1, ubiquitous zinc finger transcription factor. +.

  6. Haematopoietic malignancies caused by dysregulation of a chromatin-binding PHD finger.

    Science.gov (United States)

    Wang, Gang G; Song, Jikui; Wang, Zhanxin; Dormann, Holger L; Casadio, Fabio; Li, Haitao; Luo, Jun-Li; Patel, Dinshaw J; Allis, C David

    2009-06-11

    Histone H3 lysine 4 methylation (H3K4me) has been proposed as a critical component in regulating gene expression, epigenetic states, and cellular identities1. The biological meaning of H3K4me is interpreted by conserved modules including plant homeodomain (PHD) fingers that recognize varied H3K4me states. The dysregulation of PHD fingers has been implicated in several human diseases, including cancers and immune or neurological disorders. Here we report that fusing an H3K4-trimethylation (H3K4me3)-binding PHD finger, such as the carboxy-terminal PHD finger of PHF23 or JARID1A (also known as KDM5A or RBBP2), to a common fusion partner nucleoporin-98 (NUP98) as identified in human leukaemias, generated potent oncoproteins that arrested haematopoietic differentiation and induced acute myeloid leukaemia in murine models. In these processes, a PHD finger that specifically recognizes H3K4me3/2 marks was essential for leukaemogenesis. Mutations in PHD fingers that abrogated H3K4me3 binding also abolished leukaemic transformation. NUP98-PHD fusion prevented the differentiation-associated removal of H3K4me3 at many loci encoding lineage-specific transcription factors (Hox(s), Gata3, Meis1, Eya1 and Pbx1), and enforced their active gene transcription in murine haematopoietic stem/progenitor cells. Mechanistically, NUP98-PHD fusions act as 'chromatin boundary factors', dominating over polycomb-mediated gene silencing to 'lock' developmentally critical loci into an active chromatin state (H3K4me3 with induced histone acetylation), a state that defined leukaemia stem cells. Collectively, our studies represent, to our knowledge, the first report that deregulation of the PHD finger, an 'effector' of specific histone modification, perturbs the epigenetic dynamics on developmentally critical loci, catastrophizes cellular fate decision-making, and even causes oncogenesis during mammalian development.

  7. Expression Profiling and Functional Implications of a Set of Zinc Finger Proteins, ZNF423, ZNF470, ZNF521, and ZNF780B, in Primary Osteoarthritic Articular Chondrocytes

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    Maria Mesuraca

    2014-01-01

    Full Text Available Articular chondrocytes are responsible for the maintenance of healthy articulations; indeed, dysregulation of their functions, including the production of matrix proteins and matrix-remodeling proteases, may result in fraying of the tissue and development of osteoarthritis (OA. To explore transcriptional mechanisms that contribute to the regulation of chondrocyte homeostasis and may be implicated in OA development, we compared the gene expression profile of a set of zinc finger proteins potentially linked to the control of chondrocyte differentiation and/or functions (ZNF423, ZNF470, ZNF521, and ZNF780B in chondrocytes from patients affected by OA and from subjects not affected by OA. This analysis highlighted a significantly lower expression of the transcript encoding ZNF423 in chondrocytes from OA, particularly in elderly patients. Interestingly, this decrease was mirrored by the similarly reduced expression of PPARγ, a known target of ZNF423 with anti-inflammatory and chondroprotective properties. The ZNF521 mRNA instead was abundant in all primary chondrocytes studied; the RNAi-mediated silencing of this gene significantly altered the COL2A/COL1 expression ratio, associated with the maintenance of the differentiated phenotype, in chondrocytes cultivated in alginate beads. These results suggest a role for ZNF423 and ZNF521 in the regulation of chondrocyte homeostasis and warrant further investigations to elucidate their mechanism of action.

  8. Molecular and functional characterization of pigeon (Columba livia) tumor necrosis factor receptor-associated factor 3.

    Science.gov (United States)

    Zhou, Yingying; Kang, Xilong; Xiong, Dan; Zhu, Shanshan; Zheng, Huijuan; Xu, Ying; Guo, Yaxin; Pan, Zhiming; Jiao, Xinan

    2017-04-01

    Tumor necrosis factor receptor-associated factor 3 (TRAF3) plays a key antiviral role by promoting type I interferon production. We cloned the pigeon TRAF3 gene (PiTRAF3) according to its predicted mRNA sequence to investigate its function. The 1704-bp full-length open reading frame encodes a 567-amino acid protein. One Ring finger, two TRAF-type Zinc fingers, one Coiled coil, and one MATH domain were inferred. RT-PCR showed that PiTRAF3 was expressed in all tissues, with relatively weak expression in the heart and liver. In HEK293T cells, over-expression of wild-type, △Ring, △Zinc finger, and △Coiled coil PiTRAF3, but not a △MATH form, significantly increased IFN-β promoter activity. Zinc finger and Coiled coil domains were essential for NF-κB activation. In chicken HD11 cells, PiTRAF3 increased IFN-β promoter activity and four domains were all contributing. R848 stimulation of pigeon peripheral blood mononuclear cells and splenocytes significantly increased expression of PiTRAF3 and the inflammatory cytokine genes CCL5, IL-8, and IL-10. These data demonstrate TRAF3's innate immune function and improve understanding of its involvement in poultry antiviral defense. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Gold finger formation studied by high-resolution mass spectrometry and in silico methods

    NARCIS (Netherlands)

    Laskay, Ü.A.; Garino, C.; Tsybin, Y.O.; Salassa, L.; Casini, A.

    2015-01-01

    High-resolution mass spectrometry and quantum mechanics/molecular mechanics studies were employed for characterizing the formation of two gold finger (GF) domains from the reaction of zinc fingers (ZF) with gold complexes. The influence of both the gold oxidation state and the ZF coordination sphere

  10. Intrinsic functional defects of type 2 innate lymphoid cells impair innate allergic inflammation in promyelocytic leukemia zinc finger (PLZF)-deficient mice.

    Science.gov (United States)

    Verhoef, Philip A; Constantinides, Michael G; McDonald, Benjamin D; Urban, Joseph F; Sperling, Anne I; Bendelac, Albert

    2016-02-01

    The transcription factor promyelocytic leukemia zinc finger (PLZF) is transiently expressed during development of type 2 innate lymphoid cells (ILC2s) but is not present at the mature stage. We hypothesized that PLZF-deficient ILC2s have functional defects in the innate allergic response and represent a tool for studying innate immunity in a mouse with a functional adaptive immune response. We determined the consequences of PLZF deficiency on ILC2 function in response to innate and adaptive immune stimuli by using PLZF(-/-) mice and mixed wild-type:PLZF(-/-) bone marrow chimeras. PLZF(-/-) mice, wild-type littermates, or mixed bone marrow chimeras were treated with the protease allergen papain or the cytokines IL-25 and IL-33 or infected with the helminth Nippostrongylus brasiliensis to induce innate type 2 allergic responses. Mice were sensitized with intraperitoneal ovalbumin-alum, followed by intranasal challenge with ovalbumin alone, to induce adaptive TH2 responses. Lungs were analyzed for immune cell subsets, and alveolar lavage fluid was analyzed for ILC2-derived cytokines. In addition, ILC2s were stimulated ex vivo for their capacity to release type 2 cytokines. PLZF-deficient lung ILC2s exhibit a cell-intrinsic defect in the secretion of IL-5 and IL-13 in response to innate stimuli, resulting in defective recruitment of eosinophils and goblet cell hyperplasia. In contrast, the adaptive allergic inflammatory response to ovalbumin and alum was unimpaired. PLZF expression at the innate lymphoid cell precursor stage has a long-range effect on the functional properties of mature ILC2s and highlights the importance of these cells for innate allergic responses in otherwise immunocompetent mice. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. All rights reserved.

  11. In Vivo Chromatin Targets of the Transcription Factor Yin Yang 2 in Trophoblast Stem Cells

    Science.gov (United States)

    Pérez-Palacios, Raquel; Macías-Redondo, Sofía; Climent, María; Contreras-Moreira, Bruno; Muniesa, Pedro; Schoorlemmer, Jon

    2016-01-01

    Background Yin Yang 2 (YY2) is a zinc finger protein closely related to the well-characterized Yin Yang 1 (YY1). YY1 is a DNA-binding transcription factor, with defined functions in multiple developmental processes, such as implantation, cell differentiation, X inactivation, imprinting and organogenesis. Yy2 has been treated as a largely immaterial duplication of Yy1, as they share high homology in the Zinc Finger-region and similar if not identical in vitro binding sites. In contrast to these similarities, gene expression alterations in HeLa cells with attenuated levels of either Yy1 or Yy2 were to some extent gene-specific. Moreover, the chromatin binding sites for YY2, except for its association with transposable retroviral elements (RE) and Endogenous Retroviral Elements (ERVs), remain to be identified. As a first step towards defining potential Yy2 functions matching or complementary to Yy1, we considered in vivo DNA binding sites of YY2 in trophoblast stem (TS) cells. Results We report the presence of YY2 protein in mouse-derived embryonic stem (ES) and TS cell lines. Following up on our previous report on ERV binding by YY2 in TS cells, we investigated the tissue-specificity of REX1 and YY2 binding and confirm binding to RE/ERV targets in both ES cells and TS cells. Because of the higher levels of expression, we chose TS cells to understand the role of Yy2 in gene and chromatin regulation. We used in vivo YY2 association as a measure to identify potential target genes. Sequencing of chromatin obtained in chromatin-immunoprecipitation (ChIP) assays carried out with αYY2 serum allowed us to identify a limited number of chromatin targets for YY2. Some putative binding sites were validated in regular ChIP assays and gene expression of genes nearby was altered in the absence of Yy2. Conclusions YY2 binding to ERVs is not confined to TS cells. In vivo binding sites share the presence of a consensus binding motif. Selected sites were uniquely bound by YY2 as

  12. WRKY transcription factors

    Science.gov (United States)

    Bakshi, Madhunita; Oelmüller, Ralf

    2014-01-01

    WRKY transcription factors are one of the largest families of transcriptional regulators found exclusively in plants. They have diverse biological functions in plant disease resistance, abiotic stress responses, nutrient deprivation, senescence, seed and trichome development, embryogenesis, as well as additional developmental and hormone-controlled processes. WRKYs can act as transcriptional activators or repressors, in various homo- and heterodimer combinations. Here we review recent progress on the function of WRKY transcription factors in Arabidopsis and other plant species such as rice, potato, and parsley, with a special focus on abiotic, developmental, and hormone-regulated processes. PMID:24492469

  13. The B-subdomain of the Xenopus laevis XFIN KRAB-AB domain is responsible for its weaker transcriptional repressor activity compared to human ZNF10/Kox1.

    Science.gov (United States)

    Born, Nadine; Thiesen, Hans-Jürgen; Lorenz, Peter

    2014-01-01

    The Krüppel-associated box (KRAB) domain interacts with the nuclear hub protein TRIM28 to initiate or mediate chromatin-dependent processes like transcriptional repression, imprinting or suppression of endogenous retroviruses. The prototype KRAB domain initially identified in ZNF10/KOX1 encompasses two subdomains A and B that are found in hundreds of zinc finger transcription factors studied in human and murine genomes. Here we demonstrate for the first time transcriptional repressor activity of an amphibian KRAB domain. After sequence correction, the updated KRAB-AB domain of zinc finger protein XFIN from the frog Xenopus laevis was found to confer transcriptional repression in reporter assays in Xenopus laevis A6 kidney cells as well as in human HeLa, but not in the minnow Pimephales promelas fish cell line EPC. Binding of the XFIN KRAB-AB domain to human TRIM28 was demonstrated in a classical co-immunoprecipitation approach and visualized in a single-cell compartmentalization assay. XFIN-AB displayed reduced potency in repression as well as lower strength of interaction with TRIM28 compared to ZNF10 KRAB-AB. KRAB-B subdomain swapping between the two KRAB domains indicated that it was mainly the KRAB-B subdomain of XFIN that was responsible for its lower capacity in repression and binding to human TRIM28. In EPC fish cells, ZNF10 and XFIN KRAB repressor activity could be partially restored to low levels by adding exogenous human TRIM28. In contrast to XFIN, we did not find any transcriptional repression activity for the KRAB-like domain of human PRDM9 in HeLa cells. PRDM9 is thought to harbor an evolutionary older domain related to KRAB whose homologs even occur in invertebrates. Our results support the notion that functional bona fide KRAB domains which confer transcriptional repression and interact with TRIM28 most likely co-evolved together with TRIM28 at the beginning of tetrapode evolution.

  14. Zinc in Infection and Inflammation

    Directory of Open Access Journals (Sweden)

    Nour Zahi Gammoh

    2017-06-01

    Full Text Available Micronutrient homeostasis is a key factor in maintaining a healthy immune system. Zinc is an essential micronutrient that is involved in the regulation of the innate and adaptive immune responses. The main cause of zinc deficiency is malnutrition. Zinc deficiency leads to cell-mediated immune dysfunctions among other manifestations. Consequently, such dysfunctions lead to a worse outcome in the response towards bacterial infection and sepsis. For instance, zinc is an essential component of the pathogen-eliminating signal transduction pathways leading to neutrophil extracellular traps (NET formation, as well as inducing cell-mediated immunity over humoral immunity by regulating specific factors of differentiation. Additionally, zinc deficiency plays a role in inflammation, mainly elevating inflammatory response as well as damage to host tissue. Zinc is involved in the modulation of the proinflammatory response by targeting Nuclear Factor Kappa B (NF-κB, a transcription factor that is the master regulator of proinflammatory responses. It is also involved in controlling oxidative stress and regulating inflammatory cytokines. Zinc plays an intricate function during an immune response and its homeostasis is critical for sustaining proper immune function. This review will summarize the latest findings concerning the role of this micronutrient during the course of infections and inflammatory response and how the immune system modulates zinc depending on different stimuli.

  15. The ZEB1 Transcription Factor Is a Novel Repressor of Adiposity in Female Mice

    Science.gov (United States)

    Saykally, Jessica N.; Dogan, Soner; Cleary, Margot P.; Sanders, Michel M.

    2009-01-01

    Background Four genome-wide association studies mapped an “obesity” gene to human chromosome 10p11–12. As the zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is encoded by the TCF8 gene located in that region, and as it influences the differentiation of various mesodermal lineages, we hypothesized that ZEB1 might also modulate adiposity. The goal of these studies was to test that hypothesis in mice. Methodology/Principal Findings To ascertain whether fat accumulation affects ZEB1 expression, female C57BL/6 mice were fed a regular chow diet (RCD) ad libitum or a 25% calorie-restricted diet from 2.5 to 18.3 months of age. ZEB1 mRNA levels in parametrial fat were six to ten times higher in the obese mice. To determine directly whether ZEB1 affects adiposity, wild type (WT) mice and mice heterozygous for TCF8 (TCF8+/−) were fed an RCD or a high-fat diet (HFD) (60% calories from fat). By two months of age on an HFD and three months on an RCD, TCF8+/− mice were heavier than WT controls, which was attributed by Echo MRI to increased fat mass (at three months on an HFD: 0.517±0.081 total fat/lean mass versus 0.313±0.036; at three months on an RCD: 0.175±0.013 versus 0.124±0.012). No differences were observed in food uptake or physical activity, suggesting that the genotypes differ in some aspect of their metabolic activity. ZEB1 expression also increases during adipogenesis in cell culture. Conclusion/Significance These results show for the first time that the ZEB1 transcription factor regulates the accumulation of adipose tissue. Furthermore, they corroborate the genome-wide association studies that mapped an “obesity” gene at chromosome 10p11–12. PMID:20041147

  16. Identification and molecular characterization of 48 kDa calcium binding protein as calreticulin from finger millet (Eleusine coracana) using peptide mass fingerprinting and transcript profiling.

    Science.gov (United States)

    Singh, Manoj; Metwal, Mamta; Kumar, Vandana A; Kumar, Anil

    2016-01-30

    Attempts were made to identify and characterize the calcium binding proteins (CaBPs) in grain filling stages of finger millet using proteomics, bioinformatics and molecular approaches. A distinctly observed blue color band of 48 kDa stained by Stains-all was eluted and analyzed as calreticulin (CRT) using nano liquid chromatography-tandem mass spectrometry (nano LC-MS). Based on the top hits of peptide mass fingerprinting results, conserved primers were designed for isolation of the CRT gene from finger millet using calreticulin sequences of different cereals. The deduced nucleotide sequence analysis of 600 bp amplicon showed up to 91% similarity with CRT gene(s) of rice and other plant species and designated as EcCRT1. Transcript profiling of EcCRT1 showed different levels of relative expression at different stages of developing spikes. The higher expression of EcCRT1 transcripts and protein were observed in later stages of developing spikes which might be due to greater translational synthesis of EcCRT1 protein during seed maturation in finger millet. Preferentially higher synthesis of this CaBP during later stages of grain filling may be responsible for the sequestration of calcium in endoplasmic reticulum of finger millet grains. © 2015 Society of Chemical Industry.

  17. The ThPOK transcription factor differentially affects the development and function of self-specific CD8(+) T cells and regulatory CD4(+) T cells.

    Science.gov (United States)

    Twu, Yuh-Ching; Teh, Hung-Sia

    2014-03-01

    The zinc finger transcription factor ThPOK plays a crucial role in CD4 T-cell development and CD4/CD8 lineage decision. In ThPOK-deficient mice, developing T cells expressing MHC class II-restricted T-cell receptors are redirected into the CD8 T-cell lineage. In this study, we investigated whether the ThPOK transgene affected the development and function of two additional types of T cells, namely self-specific CD8 T cells and CD4(+) FoxP3(+) T regulatory cells. Self-specific CD8 T cells are characterized by high expression of CD44, CD122, Ly6C, 1B11 and proliferation in response to either IL-2 or IL-15. The ThPOK transgene converted these self-specific CD8 T cells into CD4 T cells. The converted CD4(+) T cells are no longer self-reactive, lose the characteristics of self-specific CD8 T cells, acquire the properties of conventional CD4 T cells and survive poorly in peripheral lymphoid organs. By contrast, the ThPOK transgene promoted the development of CD4(+) FoxP3(+) regulatory T cells resulting in an increased recovery of CD4(+) FoxP3(+) regulatory T cells that expressed higher transforming growth factor-β-dependent suppressor activity. These studies indicate that the ThPOK transcription factor differentially affects the development and function of self-specific CD8 T cells and CD4(+) FoxP3(+) regulatory T cells. © 2013 John Wiley & Sons Ltd.

  18. The transcriptional profiling of human in vivo-generated plasma cells identifies selective imbalances in monoclonal gammopathies.

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    Luis M Valor

    Full Text Available Plasma cells (PC represent the heterogeneous final stage of the B cells (BC differentiation process. To characterize the transition of BC into PC, transcriptomes from human naïve BC were compared to those of three functionally-different subsets of human in vivo-generated PC: i tonsil PC, mainly consisting of early PC; ii PC released to the blood after a potent booster-immunization (mostly cycling plasmablasts; and, iii bone marrow CD138+ PC that represent highly mature PC and include the long-lived PC compartment. This transcriptional transition involves subsets of genes related to key processes for PC maturation: the already known protein processing, apoptosis and homeostasis, and of new discovery including histones, macromolecule assembly, zinc-finger transcription factors and neuromodulation. This human PC signature is partially reproduced in vitro and is conserved in mouse. Moreover, the present study identifies genes that define PC subtypes (e.g., proliferation-associated genes for circulating PC and transcriptional-related genes for tonsil and bone marrow PC and proposes some putative transcriptional regulators of the human PC signatures (e.g., OCT/POU, XBP1/CREB, E2F, among others. Finally, we also identified a restricted imbalance of the present PC transcriptional program in monoclonal gammopathies that correlated with PC malignancy.

  19. Zinc finger protein 139 expression in gastric cancer and its clinical significance.

    Science.gov (United States)

    Li, Yong; Zhao, Qun; Fan, Li-Qiao; Wang, Li-Li; Tan, Bi-Bo; Leng, Yan-Li; Liu, Yu; Wang, Dong

    2014-12-28

    To investigate the expression of zinc finger protein 139 (ZNF139) in gastric cancer (GC), and to analyze its clinical significance. A total of 108 patients who were diagnosed with GC and underwent surgery between January 2005 and March 2007 were enrolled in this study. Gastric tumor specimens and paired tumor-adjacent tissues were collected and paraffin-embedded, and the clinicopathologic characteristics and prognosis were recorded. The expression of ZNF139, Bcl-2, Bax, and caspase-3 were determined by immunohistochemistry, and apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. SPSS 13.0 software was used for data processing and analyses, and significance was determined at P stage, lymphatic metastasis, and blood vessel invasion (all Ps < 0.05). Patients with overexpression of ZNF139 had a poorer prognosis (P < 0.01), and overexpression of ZNF139 was an independent factor for the prognosis of GC patients by a Cox survival analysis (P = 0.02). A negative relationship between ZNF139 and the apoptosis index was observed (r = -0.686; P < 0.01). The expression of Bcl-2 in GC was stronger than in tumor-adjacent tissues (66.67% vs 41.67%), whereas the expression levels of Bax and caspase-3 were lower in primary tumors (54.63% and 47.22%, respectively) than in tumor-adjacent tissues (73.15% and 73.15%, respectively) (all Ps < 0.05). The expression of ZNF139 negatively correlated with caspase-3 (r = -0.370; P < 0.01). The expressions of Bcl-2 and Bax were also negatively correlated (r = -0.231; P = 0.02). The expressions of caspase-3 and Bax protein were positively correlated (r = 0.217; P = 0.024). ZNF139 is related to clinicopathologic characteristics and prognosis of GC. Furthermore, it is overexpressed and involved in apoptosis in GC tissues by regulating caspase-3.

  20. Generation of knockout rabbits using transcription activator-like effector nucleases

    Directory of Open Access Journals (Sweden)

    Yu Wang

    2014-01-01

    Full Text Available Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

  1. Molecular and clinical analyses of Greig cephalopolysyndactyly and Pallister-Hall syndromes: Robust phenotype prediction from the type and position of GLI3 mutations

    NARCIS (Netherlands)

    Johnston, Jennifer J.; Olivos-Glander, Isabelle; Killoran, Christina; Elson, Emma; Turner, Joyce T.; Peters, Kathryn F.; Abbott, Margaret H.; Aughton, David J.; Aylsworth, Arthur S.; Bamshad, Michael J.; Booth, Carol; Curry, Cynthia J.; David, Albert; Dinulos, Mary Beth; Flannery, David B.; Fox, Michelle A.; Graham, John M.; Grange, Dorothy K.; Guttmacher, Alan E.; Hannibal, Mark C.; Henn, Wolfram; Hennekam, Raoul C. M.; Holmes, Lewis B.; Hoyme, H. Eugene; Leppig, Kathleen A.; Lin, Angela E.; Macleod, Patrick; Manchester, David K.; Marcelis, Carlo; Mazzanti, Laura; McCann, Emma; McDonald, Marie T.; Mendelsohn, Nancy J.; Moeschler, John B.; Moghaddam, Billur; Neri, Giovanni; Newbury-Ecob, Ruth; Pagon, Roberta A.; Phillips, John A.; Sadler, Laurie S.; Stoler, Joan M.; Tilstra, David; Walsh Vockley, Catherine M.; Zackai, Elaine H.; Zadeh, Touran M.; Brueton, Louise; Black, Graeme Charles M.; Biesecker, Leslie G.

    2005-01-01

    Mutations in the GLI3 zinc-finger transcription factor gene cause Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS), which are variable but distinct clinical entities. We hypothesized that GLI3 mutations that predict a truncated functional repressor protein cause PHS and

  2. The LSD1-Type Zinc Finger Motifs of Pisum sativa LSD1 Are a Novel Nuclear Localization Signal and Interact with Importin Alpha

    OpenAIRE

    He, Shanping; Huang, Kuowei; Zhang, Xu; Yu, Xiangchun; Huang, Ping; An, Chengcai

    2011-01-01

    BACKGROUND: Genetic studies of the Arabidopsis mutant lsd1 highlight the important role of LSD1 in the negative regulation of plant programmed cell death (PCD). Arabidopsis thaliana LSD1 (AtLSD1) contains three LSD1-type zinc finger motifs, which are involved in the protein-protein interaction. METHODOLOGY/PRINCIPAL FINDINGS: To further understand the function of LSD1, we have analyzed cellular localization and functional localization domains of Pisum sativa LSD1 (PsLSD1), which is a homolog ...

  3. Kaiso Directs the Transcriptional Corepressor MTG16 to the Kaiso Binding Site in Target Promoters

    Science.gov (United States)

    Barrett, Caitlyn W.; Smith, J. Joshua; Lu, Lauren C.; Markham, Nicholas; Stengel, Kristy R.; Short, Sarah P.; Zhang, Baolin; Hunt, Aubrey A.; Fingleton, Barbara M.; Carnahan, Robert H.; Engel, Michael E.; Chen, Xi; Beauchamp, R. Daniel; Wilson, Keith T.; Hiebert, Scott W.; Reynolds, Albert B.; Williams, Christopher S.

    2012-01-01

    Myeloid translocation genes (MTGs) are transcriptional corepressors originally identified in acute myelogenous leukemia that have recently been linked to epithelial malignancy with non-synonymous mutations identified in both MTG8 and MTG16 in colon, breast, and lung carcinoma in addition to functioning as negative regulators of WNT and Notch signaling. A yeast two-hybrid approach was used to discover novel MTG binding partners. This screen identified the Zinc fingers, C2H2 and BTB domain containing (ZBTB) family members ZBTB4 and ZBTB38 as MTG16 interacting proteins. ZBTB4 is downregulated in breast cancer and modulates p53 responses. Because ZBTB33 (Kaiso), like MTG16, modulates Wnt signaling at the level of TCF4, and its deletion suppresses intestinal tumorigenesis in the ApcMin mouse, we determined that Kaiso also interacted with MTG16 to modulate transcription. The zinc finger domains of Kaiso as well as ZBTB4 and ZBTB38 bound MTG16 and the association with Kaiso was confirmed using co-immunoprecipitation. MTG family members were required to efficiently repress both a heterologous reporter construct containing Kaiso binding sites (4×KBS) and the known Kaiso target, Matrix metalloproteinase-7 (MMP-7/Matrilysin). Moreover, chromatin immunoprecipitation studies placed MTG16 in a complex occupying the Kaiso binding site on the MMP-7 promoter. The presence of MTG16 in this complex, and its contributions to transcriptional repression both required Kaiso binding to its binding site on DNA, establishing MTG16-Kaiso binding as functionally relevant in Kaiso-dependent transcriptional repression. Examination of a large multi-stage CRC expression array dataset revealed patterns of Kaiso, MTG16, and MMP-7 expression supporting the hypothesis that loss of either Kaiso or MTG16 can de-regulate a target promoter such as that of MMP-7. These findings provide new insights into the mechanisms of transcriptional control by ZBTB family members and broaden the scope of co

  4. Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica

    Science.gov (United States)

    Martinez-Vazquez, Azul; Gonzalez-Hernandez, Angelica; Domínguez, Ángel; Rachubinski, Richard; Riquelme, Meritxell; Cuellar-Mata, Patricia; Guzman, Juan Carlos Torres

    2013-01-01

    The dimorphic yeast Yarrowia lipolytica is used as a model to study fungal differentiation because it grows as yeast-like cells or forms hyphal cells in response to changes in environmental conditions. Here, we report the isolation and characterization of a gene, ZNC1, involved in the dimorphic transition in Y. lipolytica. The ZNC1 gene encodes a 782 amino acid protein that contains a Zn(II)2C6 fungal-type zinc finger DNA-binding domain and a leucine zipper domain. ZNC1 transcription is elevated during yeast growth and decreases during the formation of mycelium. Cells in which ZNC1 has been deleted show increased hyphal cell formation. Znc1p-GFP localizes to the nucleus, but mutations within the leucine zipper domain of Znc1p, and to a lesser extent within the Zn(II)2C6 domain, result in a mislocalization of Znc1p to the cytoplasm. Microarrays comparing gene expression between znc1::URA3 and wild-type cells during both exponential growth and the induction of the yeast-to-hypha transition revealed 1,214 genes whose expression was changed by 2-fold or more under at least one of the conditions analyzed. Our results suggest that Znc1p acts as a transcription factor repressing hyphal cell formation and functions as part of a complex network regulating mycelial growth in Y. lipolytica. PMID:23826133

  5. Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress

    Directory of Open Access Journals (Sweden)

    Muhammed eJamsheer K

    2015-09-01

    Full Text Available Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1 signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response towards energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response.

  6. PLZF regulates fibroblast growth factor responsiveness and maintenance of neural progenitors.

    Science.gov (United States)

    Gaber, Zachary B; Butler, Samantha J; Novitch, Bennett G

    2013-10-01

    Distinct classes of neurons and glial cells in the developing spinal cord arise at specific times and in specific quantities from spatially discrete neural progenitor domains. Thus, adjacent domains can exhibit marked differences in their proliferative potential and timing of differentiation. However, remarkably little is known about the mechanisms that account for this regional control. Here, we show that the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF) plays a critical role shaping patterns of neuronal differentiation by gating the expression of Fibroblast Growth Factor (FGF) Receptor 3 and responsiveness of progenitors to FGFs. PLZF elevation increases FGFR3 expression and STAT3 pathway activity, suppresses neurogenesis, and biases progenitors towards glial cell production. In contrast, PLZF loss reduces FGFR3 levels, leading to premature neuronal differentiation. Together, these findings reveal a novel transcriptional strategy for spatially tuning the responsiveness of distinct neural progenitor groups to broadly distributed mitogenic signals in the embryonic environment.

  7. Nucleocytoplasmic shuttling of transcription factors

    DEFF Research Database (Denmark)

    Cartwright, P; Helin, K

    2000-01-01

    To elicit the transcriptional response following intra- or extracellular stimuli, the signals need to be transmitted to their site of action within the nucleus. The nucleocytoplasmic shuttling of transcription factors is a mechanism mediating this process. The activation and inactivation...... of the transcriptional response is essential for cells to progress through the cell cycle in a normal manner. The involvement of cytoplasmic and nuclear accessory molecules, and the general nuclear membrane transport components, are essential for this process. Although nuclear import and export for different...... transcription factor families are regulated by similar mechanisms, there are several differences that allow for the specific activation of each transcription factor. This review discusses the general import and export pathways found to be common amongst many different transcription factors, and highlights...

  8. Regulation of hedgehog signaling by Myc-interacting zinc finger protein 1, Miz1.

    Directory of Open Access Journals (Sweden)

    Jiuyi Lu

    Full Text Available Smoothened (Smo mediated Hedgehog (Hh signaling plays an essential role in regulating embryonic development and postnatal tissue homeostasis. Aberrant activation of the Hh pathway contributes to the formation and progression of various cancers. In vertebrates, however, key regulatory mechanisms responsible for transducing signals from Smo to the nucleus remain to be delineated. Here, we report the identification of Myc-interacting Zinc finger protein 1 (Miz1 as a Smo and Gli2 binding protein that positively regulates Hh signaling. Overexpression of Miz1 increases Gli luciferase reporter activity, whereas knockdown of endogenous Miz1 has the opposite effect. Activation of Smo induces translocation of Miz1 to the primary cilia together with Smo and Gli2. Furthermore, Miz1 is localized to the nucleus upon Hh activation in a Smo-dependent manner, and loss of Miz1 prevents the nuclear translocation of Gli2. More importantly, silencing Miz1 expression inhibits cell proliferation in vitro and the growth of Hh-driven medulloblastoma tumors allografted in SCID mice. Taken together, these results identify Miz1 as a novel regulator in the Hh pathway that plays an important role in mediating Smo-dependent oncogenic signaling.

  9. Transcriptional regulation by competing transcription factor modules.

    Directory of Open Access Journals (Sweden)

    Rutger Hermsen

    2006-12-01

    Full Text Available Gene regulatory networks lie at the heart of cellular computation. In these networks, intracellular and extracellular signals are integrated by transcription factors, which control the expression of transcription units by binding to cis-regulatory regions on the DNA. The designs of both eukaryotic and prokaryotic cis-regulatory regions are usually highly complex. They frequently consist of both repetitive and overlapping transcription factor binding sites. To unravel the design principles of these promoter architectures, we have designed in silico prokaryotic transcriptional logic gates with predefined input-output relations using an evolutionary algorithm. The resulting cis-regulatory designs are often composed of modules that consist of tandem arrays of binding sites to which the transcription factors bind cooperatively. Moreover, these modules often overlap with each other, leading to competition between them. Our analysis thus identifies a new signal integration motif that is based upon the interplay between intramodular cooperativity and intermodular competition. We show that this signal integration mechanism drastically enhances the capacity of cis-regulatory domains to integrate signals. Our results provide a possible explanation for the complexity of promoter architectures and could be used for the rational design of synthetic gene circuits.

  10. A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease.

    Science.gov (United States)

    Sabzehei, Faezeh; Kouhpayeh, Shirin; Dastjerdeh, Mansoureh Shahbazi; Khanahmad, Hossein; Salehi, Rasoul; Naderi, Shamsi; Taghizadeh, Razieh; Rabiei, Parisa; Hejazi, Zahra; Shariati, Laleh

    2017-01-01

    Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods. At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F' that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry. The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) ( P < 0.0001). According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample.

  11. The primary structure of L37--a rat ribosomal protein with a zinc finger-like motif.

    Science.gov (United States)

    Chan, Y L; Paz, V; Olvera, J; Wool, I G

    1993-04-30

    The amino acid sequence of the rat 60S ribosomal subunit protein L37 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L37 has 96 amino acids, the NH2-terminal methionine is removed after translation of the mRNA, and has a molecular weight of 10,939. Ribosomal protein L37 has a single zinc finger-like motif of the C2-C2 type. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 13 or 14 copies of the L37 gene. The mRNA for the protein is about 500 nucleotides in length. Rat L37 is related to Saccharomyces cerevisiae ribosomal protein YL35 and to Caenorhabditis elegans L37. We have identified in the data base a DNA sequence that encodes the chicken homolog of rat L37.

  12. Separate transcriptionally regulated pathways specify distinct classes of sister dendrites in a nociceptive neuron.

    Science.gov (United States)

    O'Brien, Barbara M J; Palumbos, Sierra D; Novakovic, Michaela; Shang, Xueying; Sundararajan, Lakshmi; Miller, David M

    2017-12-15

    The dendritic processes of nociceptive neurons transduce external signals into neurochemical cues that alert the organism to potentially damaging stimuli. The receptive field for each sensory neuron is defined by its dendritic arbor, but the mechanisms that shape dendritic architecture are incompletely understood. Using the model nociceptor, the PVD neuron in C. elegans, we determined that two types of PVD lateral branches project along the dorsal/ventral axis to generate the PVD dendritic arbor: (1) Pioneer dendrites that adhere to the epidermis, and (2) Commissural dendrites that fasciculate with circumferential motor neuron processes. Previous reports have shown that the LIM homeodomain transcription factor MEC-3 is required for all higher order PVD branching and that one of its targets, the claudin-like membrane protein HPO-30, preferentially promotes outgrowth of pioneer branches. Here, we show that another MEC-3 target, the conserved TFIIA-like zinc finger transcription factor EGL-46, adopts the alternative role of specifying commissural dendrites. The known EGL-46 binding partner, the TEAD transcription factor EGL-44, is also required for PVD commissural branch outgrowth. Double mutants of hpo-30 and egl-44 show strong enhancement of the lateral branching defect with decreased numbers of both pioneer and commissural dendrites. Thus, HPO-30/Claudin and EGL-46/EGL-44 function downstream of MEC-3 and in parallel acting pathways to direct outgrowth of two distinct classes of PVD dendritic branches. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Isolation and characterization of differentially expressed genes in ...

    African Journals Online (AJOL)

    Among them, six proteins (putative fatty acid oxygenase, heat shock sks2, PriA homologue, Ap-1 like transcription factor YAP7, mung bean seed albumin, and C2H2 Zinc finger domain protein) and one protein (peroxisomal biogenesis factor 6) showed increased expression levels at the fruiting process and the mycelial ...

  14. Genome-wide identification and characterization of stress-associated protein (SAP gene family encoding A20/AN1 zinc-finger proteins in Medicago truncatula

    Directory of Open Access Journals (Sweden)

    Zhou Yong

    2018-01-01

    Full Text Available Stress associated proteins (SAPs play important roles in developmental processes, responses to various stresses and hormone stimulation in plants. However, little is known about the SAP gene family in Medicago truncatula. In this study, a total of 17 MtSAP genes encoding A20/AN1 zinc-finger proteins were characterized. Out of these 17 genes, 15 were distributed over all 8 chromosomes at different densities, and two segmental duplication events were detected. The phylogenetic analysis of these proteins and their orthologs from Arabidopsis and rice suggested that they could be classified into five out of the seven groups of SAP family genes, with genes in the same group showing similar structures and conserved domains. The cis-elements of the MtSAP promoters were studied, and many cis-elements related to stress and plant hormone responses were identified. We also investigated the stress-responsive expression patterns of the MtSAP genes under various stresses, including drought, exposure to NaCl and cold. The qRT-PCR results showed that numerous MtSAP genes exhibited transcriptional responses to multiple abiotic stresses. These results lay the foundation for further functional characterization of SAP genes. To the best of our knowledge, this is the first report of a genome-wide analysis of the SAP gene family in M. truncatula.

  15. A bio-inspired zinc finger analogue anchored in 2D hexagonal meso-porous silica for room temperature CO_2 activation via a hydrogeno-carbonate route

    International Nuclear Information System (INIS)

    Doghri, Hanene; Baranova, Elena A.; Albela, Belen; Bonneviot, Laurent; Mongia Said-Zina

    2017-01-01

    Bio-inspired diethylenetriamine-zinc(II) complexes were anchored into the nano-pores of hexagonal meso-porous MCM41-like silicas targeting a carbamate free and low temperature CO_2 recycling process. A step-by-step approach was adopted to perform an in situ synthesis in order to mimic the zinc finger of carbonic anhydrases, the fastest family of enzymes. In the presence of a surface-masking pattern of TMA"+ ions, some silanol groups were capped using grafted trimethylsilyl functions, TMSgr, (gr for grafted). After removing the masking ions, a tridentate diethylenetriamine ligand was anchored using diethylenetriamine propyl-trimethoxysilane. The so-called DETA_a_n ligands (an for anchored) were partially mono-protonated using either cyclohexane or isopropanol as a solvent. Nonetheless, up to two thirds of them were metallated by Zn(II) ions, leading to the targeted anchored zinc finger mimic [Zn(DETAan)L]+(L = Cl or OH). CO_2 is then adsorbed at room temperature and in humid ambient air by the formation of an intermediate hydrogeno-carbonate-zinc complex. Specific IR signatures at 1330 and 1400 cm"-"1 together with characteristic C 1s and Zn 2p3/2 XPS binding energies at 286.4 and 1024.6 eV advocate for a rather symmetrical bidentate [η"2-CO_3] structural unit in the anchored complex [Zn(DETA_a_n)(η"2-HCO_3"*)]"+, where the Zn(II) ion is most likely penta-coordinated. The internal pH value varied by less than 0.5 depending on the metal reacting with the DETA_a_n ligand and its ability to generate HCO_3"-, due to the buffering effect of surface silanol and amino groups according to the level of protonation of the DETA moieties measured from the N 1s XPS spectra. In contrast to nitrate ions, chloride ions were found to inhibit the formation of hydrogeno-carbonate. (authors)

  16. Characteristics of hearing loss in HDR (hypoparathyroidism, sensorineural deafness, renal dysplasia) syndrome

    NARCIS (Netherlands)

    van Looij, Marjolein A. J.; Meijers-Heijboer, Hanne; Beetz, Rolf; Thakker, Rajesh V.; Christie, Paul T.; Feenstra, Lou W.; van Zanten, Bert G. A.

    2006-01-01

    Haploinsufficiency of the zinc finger transcription factor GATA3 causes the triad of hypoparathyroidism, deafness and renal dysplasia, known by its acronym HDR syndrome. The purpose of the current study was to describe in detail the auditory phenotype in human HDR patients and compare these to

  17. Contamination by human fingers. The Midas touch

    International Nuclear Information System (INIS)

    Gwozdz, R.; Grass, F.

    2004-01-01

    Anthropogenic activity is one of the causes of contamination in the human environment: contamination of air, water, top soils, plants and food products has complex effects on human health problems. Wear and abrasion of various surfaces are constant processes in daily life, and commonly include interaction between human fingers and surfaces of every conceivable material. New methods for investigation of trace transfer processes by human fingers are described. Results of transfer for commonly used metals such as gold, silver, zinc, cadmium, tin, cobalt, nickel, chromium and iron are presented. Relationship between transfer of metals by touch and the general problem of purity in analytical activities is briefly discussed. (author)

  18. The transcription factor Th-POK negatively regulates Th17 differentiation in Vα14i NKT cells

    Science.gov (United States)

    Engel, Isaac; Zhao, Meng; Kappes, Dietmar; Taniuchi, Ichiro

    2012-01-01

    The majority of mouse Vα14 invariant natural killer T (Vα14i NKT) cells produce several cytokines, including IFNγ and IL-4, very rapidly after activation. A subset of these cells, known as NKT17 cells, however, differentiates in the thymus to preferentially produce IL-17. Here, we show that the transcription factor—known as T helper, Poxviruses, and Zinc-finger and Krüppel family, (Th-POK)—represses the formation of NKT17 cells. Vα14i NKT cells from Th-POK–mutant helper deficient (hd/hd) mice have increased transcripts of genes normally expressed by Th17 and NKT17 cells, and even heterozygosity for this mutation leads to dramatically increased numbers of Vα14i NKT cells that are poised to express IL-17, especially in the thymus and lymph nodes. In addition, using gene reporter mice, we demonstrate that NKT17 cells from wild-type mice express lower amounts of Th-POK than the majority population of Vα14i NKT cells. We also show that retroviral transduction of Th-POK represses the expression of the Th17 master regulator RORγT in Vα14i NKT-cell lines. Our data suggest that NKT17-cell differentiation is intrinsically regulated by Th-POK activity, with only low levels of Th-POK permissive for the differentiation of NKT17 cells. PMID:23034280

  19. Cyclic AMP Pathway Activation and Extracellular Zinc Induce Rapid Intracellular Zinc Mobilization in Candida albicans

    Science.gov (United States)

    Kjellerup, Lasse; Winther, Anne-Marie L.; Wilson, Duncan; Fuglsang, Anja T.

    2018-01-01

    Zinc is an essential micronutrient, required for a range of zinc-dependent enzymes and transcription factors. In mammalian cells, zinc serves as a second messenger molecule. However, a role for zinc in signaling has not yet been established in the fungal kingdom. Here, we used the intracellular zinc reporter, zinbo-5, which allowed visualization of zinc in the endoplasmic reticulum and other components of the internal membrane system in Candida albicans. We provide evidence for a link between cyclic AMP/PKA- and zinc-signaling in this major human fungal pathogen. Glucose stimulation, which triggers a cyclic AMP spike in this fungus resulted in rapid intracellular zinc mobilization and this “zinc flux” could be stimulated with phosphodiesterase inhibitors and blocked via inhibition of adenylate cyclase or PKA. A similar mobilization of intracellular zinc was generated by stimulation of cells with extracellular zinc and this effect could be reversed with the chelator EDTA. However, zinc-induced zinc flux was found to be cyclic AMP independent. In summary, we show that activation of the cyclic AMP/PKA pathway triggers intracellular zinc mobilization in a fungus. To our knowledge, this is the first described link between cyclic AMP signaling and zinc homeostasis in a human fungal pathogen. PMID:29619016

  20. Novel activation domain derived from Che-1 cofactor coupled with the artificial protein Jazz drives utrophin upregulation.

    Science.gov (United States)

    Desantis, Agata; Onori, Annalisa; Di Certo, Maria Grazia; Mattei, Elisabetta; Fanciulli, Maurizio; Passananti, Claudio; Corbi, Nicoletta

    2009-02-01

    Our aim is to upregulate the expression level of the dystrophin related gene utrophin in Duchenne muscular dystrophy, thus complementing the lack of dystrophin functions. To this end, we have engineered synthetic zinc finger based transcription factors. We have previously shown that the artificial three-zinc finger protein named Jazz fused with the Vp16 activation domain, is able to bind utrophin promoter A and to increase the endogenous level of utrophin in transgenic mice. Here, we report on an innovative artificial protein, named CJ7, that consists of Jazz DNA binding domain fused to a novel activation domain derived from the regulatory multivalent adaptor protein Che-1/AATF. This transcriptional activation domain is 100 amino acids in size and it is very powerful as compared to the Vp16 activation domain. We show that CJ7 protein efficiently promotes transcription and accumulation of the acetylated form of histone H3 on the genomic utrophin promoter locus.

  1. Overexpression of EcbHLH57 Transcription Factor from Eleusine coracana L. in Tobacco Confers Tolerance to Salt, Oxidative and Drought Stress.

    Directory of Open Access Journals (Sweden)

    K C Babitha

    Full Text Available Basic helix-loop-helix (bHLH transcription factors constitute one of the largest families in plants and are known to be involved in various developmental processes and stress tolerance. We report the characterization of a stress responsive bHLH transcription factor from stress adapted species finger millet which is homologous to OsbHLH57 and designated as EcbHLH57. The full length sequence of EcbHLH57 consisted of 256 amino acids with a conserved bHLH domain followed by leucine repeats. In finger millet, EcbHLH57 transcripts were induced by ABA, NaCl, PEG, methyl viologen (MV treatments and drought stress. Overexpression of EcbHLH57 in tobacco significantly increased the tolerance to salinity and drought stress with improved root growth. Transgenic plants showed higher photosynthetic rate and stomatal conductance under drought stress that resulted in higher biomass. Under long-term salinity stress, the transgenic plants accumulated higher seed weight/pod and pod number. The transgenic plants were also tolerant to oxidative stress and showed less accumulation of H202 and MDA levels. The overexpression of EcbHLH57 enhanced the expression of stress responsive genes such as LEA14, rd29A, rd29B, SOD, APX, ADH1, HSP70 and also PP2C and hence improved tolerance to diverse stresses.

  2. Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees.

    Science.gov (United States)

    Peer, Reut; Rivlin, Gil; Golobovitch, Sara; Lapidot, Moshe; Gal-On, Amit; Vainstein, Alexander; Tzfira, Tzvi; Flaishman, Moshe A

    2015-04-01

    Targeting a gene in apple or fig with ZFN, introduced by transient or stable transformation, should allow genome editing with high precision to advance basic science and breeding programs. Genome editing is a powerful tool for precise gene manipulation in any organism; it has recently been shown to be of great value for annual plants. Classical breeding strategies using conventional cross-breeding and induced mutations have played an important role in the development of new cultivars in fruit trees. However, fruit-tree breeding is a lengthy process with many limitations. Efficient and widely applied methods for targeted modification of fruit-tree genomes are not yet available. In this study, transgenic apple and fig lines carrying a zinc-finger nuclease (ZFNs) under the control of a heat-shock promoter were developed. Editing of a mutated uidA gene, following expression of the ZFN genes by heat shock, was confirmed by GUS staining and PCR product sequencing. Finally, whole plants with a repaired uidA gene due to deletion of a stop codon were regenerated. The ZFN-mediated gene modifications were stable and passed onto regenerants from ZFN-treated tissue cultures. This is the first demonstration of efficient and precise genome editing, using ZFN at a specific genomic locus, in two different perennial fruit trees-apple and fig. We conclude that targeting a gene in apple or fig with a ZFN introduced by transient or stable transformation should allow knockout of a gene of interest. Using this technology for genome editing allows for marker gene-independent and antibiotic selection-free genome engineering with high precision in fruit trees to advance basic science as well as nontransgenic breeding programs.

  3. The Zinc-Finger Thylakoid-Membrane Protein FIP Is Involved With Abiotic Stress Response in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Karina L. Lopes

    2018-04-01

    Full Text Available Many plant genes have their expression modulated by stress conditions. Here, we used Arabidopsis FtsH5 protease, which expression is regulated by light stress, as bait in a yeast two-hybrid screen to search for new proteins involved in the stress response. As a result, we found FIP (FtsH5 Interacting Protein, which possesses an amino proximal cleavable transit peptide, a hydrophobic membrane-anchoring region, and a carboxyl proximal C4-type zinc-finger domain. In vivo experiments using FIP fused to green fluorescent protein (GFP showed a plastid localization. This finding was corroborated by chloroplast import assays that showed FIP inserted in the thylakoid membrane. FIP expression was down-regulated in plants exposed to high light intensity, oxidative, salt, and osmotic stresses, whereas mutant plants expressing low levels of FIP were more tolerant to these abiotic stresses. Our data shows a new thylakoid-membrane protein involved with abiotic stress response in Arabidopsis thaliana.

  4. TaCHP: a wheat zinc finger protein gene down-regulated by abscisic acid and salinity stress plays a positive role in stress tolerance.

    Science.gov (United States)

    Li, Cuiling; Lv, Jian; Zhao, Xin; Ai, Xinghui; Zhu, Xinlei; Wang, Mengcheng; Zhao, Shuangyi; Xia, Guangmin

    2010-09-01

    The plant response to abiotic stresses involves both abscisic acid (ABA)-dependent and ABA-independent signaling pathways. Here we describe TaCHP, a CHP-rich (for cysteine, histidine, and proline rich) zinc finger protein family gene extracted from bread wheat (Triticum aestivum), is differentially expressed during abiotic stress between the salinity-sensitive cultivar Jinan 177 and its tolerant somatic hybrid introgression cultivar Shanrong No.3. TaCHP expressed in the roots of seedlings at the three-leaf stage, and the transcript localized within the cells of the root tip cortex and meristem. TaCHP transcript abundance was higher in Shanrong No.3 than in Jinan 177, but was reduced by the imposition of salinity or drought stress, as well as by the exogenous supply of ABA. When JN17, a salinity hypersensitive wheat cultivar, was engineered to overexpress TaCHP, its performance in the face of salinity stress was improved, and the ectopic expression of TaCHP in Arabidopsis (Arabidopsis thaliana) also improved the ability of salt tolerance. The expression level of a number of stress reporter genes (AtCBF3, AtDREB2A, AtABI2, and AtABI1) was raised in the transgenic lines in the presence of salinity stress, while that of AtMYB15, AtABA2, and AtAAO3 was reduced in its absence. The presence in the upstream region of the TaCHP open reading frame of the cis-elements ABRE, MYBRS, and MYCRS suggests that it is a component of the ABA-dependent and -independent signaling pathways involved in the plant response to abiotic stress. We suggest that TaCHP enhances stress tolerance via the promotion of CBF3 and DREB2A expression.

  5. The Journey of a Transcription Factor

    DEFF Research Database (Denmark)

    Pireyre, Marie

    Plants have developed astonishing networks regulating their metabolism to adapt to their environment. The complexity of these networks is illustrated by the expansion of families of regulators such as transcription factors in the plant kingdom. Transcription factors specifically impact...... transcriptional networks by integrating exogenous and endogenous stimuli and regulating gene expression accordingly. Regulation of transcription factors and their activation is thus highly important to modulate the transcriptional programs and increase fitness of the plant in a given environment. Plant metabolism....... The biosynthetic machinery of GLS is governed by interplay of six MYB and three bHLH transcription factors. MYB28, MYB29 and MYB76 regulate methionine-derived GLS, and MYB51, MYB34 and MYB122 regulate tryptophan-derived GLS. The three bHLH transcription factors MYC2, MYC3 and MYC4 physically interact with all six...

  6. Anti-sigma factor YlaD regulates transcriptional activity of sigma factor YlaC and sporulation via manganese-dependent redox-sensing molecular switch in Bacillus subtilis.

    Science.gov (United States)

    Kwak, Min-Kyu; Ryu, Han-Bong; Song, Sung-Hyun; Lee, Jin-Won; Kang, Sa-Ouk

    2018-05-14

    YlaD, a membrane-anchored anti-sigma factor of Bacillus subtilis , contains a HX 3 CXXC motif that functions as a redox-sensing domain and belongs to one of the zinc-coordinated anti-sigma factor families. Despite previously showing that the YlaC transcription is controlled by YlaD, experimental evidence of how the YlaC-YlaD interaction is affected by active cysteines and/or metal ions is lacking. Here, we showed that the P yla promoter is autoregulated solely by YlaC. Moreover, reduced YlaD contained zinc and iron, while oxidized YlaD did not. Cysteine substitution in YlaD led to changes in its secondary structure; Cys3 had important structural functions in YlaD, and its mutation caused dissociation from YlaC, indicating the essential requirement of a HX 3 CXXC motif for regulating interactions of YlaC with YlaD. Analyses of the far-UV CD spectrum and metal content revealed that the addition of Mn ions to Zn-YlaD changed its secondary structure and that iron was substituted for manganese. The ylaC gene expression using βGlu activity from P yla : gusA was observed at the late-exponential and early-stationary phase and the ylaC -overexpressing mutant constitutively expressed gene transcripts of clpP and sigH , an important alternative sigma factor regulated by ClpXP. Collectively, our data demonstrated that YlaD senses redox changes and elicits increase in manganese ion concentrations and that, in turn, YlaD-mediated transcriptional activity of YlaC regulates sporulation initiation under oxidative stress and manganese-substituted conditions by regulating clpP gene transcripts. This is the first report of the involvement of oxidative stress-responsive B. subtilis extracytoplasmic function sigma factors during sporulation via a manganese-dependent redox-sensing molecular switch. ©2018 The Author(s).

  7. Sphingosine-1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: Novel insights into angiogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Hui-Yan, E-mail: shy35309@sohu.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Wei, Shu-Ping, E-mail: weishuping_83@163.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Xu, Rui-Cheng, E-mail: xu_rc@sohu.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Xu, Peng-Xiao, E-mail: xupengxiao1228@sina.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Zhang, Wen-Cheng, E-mail: wenchengzhang@yahoo.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China)

    2010-05-07

    Sphingosine-1-phosphate (S1P)-induced migration and proliferation of endothelial cells are critical for angiogenesis. C2H2-zinc finger (ZNF) proteins usually play an essential role in altering gene expression and regulating the angiogenesis. The aim of this study is to investigate whether a novel human C2H2-zinc finger gene ZNF580 (Gene ID: 51157) is involved in the migration and proliferation of endothelial cells stimulated by S1P. Our study shows that EAhy926 endothelial cells express S1P1, S1P3 and S1P5 receptors. Furthermore, S1P upregulates both ZNF580 mRNA and protein levels in a concentration- and time-dependent manner. SB203580, the specific inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, blocks the S1P-induced upregulation of ZNF580. Moreover, overexpression/downexpression of ZNF580 in EAhy926 cells leads to the enhancement/decrease of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) expression as well as the migration and proliferation of EAhy926 endothelial cells. These results elucidate the important role that ZNF580 plays in the process of migration and proliferation of endothelial cells, which provides a foundation for a novel approach to regulate angiogenesis.

  8. Sphingosine-1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: Novel insights into angiogenesis

    International Nuclear Information System (INIS)

    Sun, Hui-Yan; Wei, Shu-Ping; Xu, Rui-Cheng; Xu, Peng-Xiao; Zhang, Wen-Cheng

    2010-01-01

    Sphingosine-1-phosphate (S1P)-induced migration and proliferation of endothelial cells are critical for angiogenesis. C2H2-zinc finger (ZNF) proteins usually play an essential role in altering gene expression and regulating the angiogenesis. The aim of this study is to investigate whether a novel human C2H2-zinc finger gene ZNF580 (Gene ID: 51157) is involved in the migration and proliferation of endothelial cells stimulated by S1P. Our study shows that EAhy926 endothelial cells express S1P1, S1P3 and S1P5 receptors. Furthermore, S1P upregulates both ZNF580 mRNA and protein levels in a concentration- and time-dependent manner. SB203580, the specific inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, blocks the S1P-induced upregulation of ZNF580. Moreover, overexpression/downexpression of ZNF580 in EAhy926 cells leads to the enhancement/decrease of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) expression as well as the migration and proliferation of EAhy926 endothelial cells. These results elucidate the important role that ZNF580 plays in the process of migration and proliferation of endothelial cells, which provides a foundation for a novel approach to regulate angiogenesis.

  9. Factors influencing zinc status of apparently healthy indians.

    Science.gov (United States)

    Agte, Vaishali V; Chiplonkar, Shashi A; Tarwadi, Kirtan V

    2005-10-01

    To identify dietary, environmental and socio-economic factors associated with mild zinc deficiency, three zinc status indices; erythrocyte membrane zinc (RBCMZn), plasma zinc and super oxide dismutase (SOD) were assessed in free living and apparently healthy Indian population. Dietary patterns of 232 men and 223 women (20-65 yr) from rural, industrial and urban regions of Western India were evaluated by food frequency questionnaire. RBCMZn was estimated using atomic absorption spectrometry, hemoglobin and serum ceruloplasmin by spectrophotometer. On a sub sample (48 men and 51 women) plasma zinc and SOD were also assessed. Mean RBCMZn was 0.5 +/- 0.1 micromols/g protein with 46% individuals showing zinc deficiency. Mean plasma zinc was 0.98 +/- 0.12 microg/mL with 25% men and 2.5% women having values below normal range. Mean SOD was 0.97 +/- 0.1 (u/mL cells). A significant positive correlation was observed between intakes of green leafy vegetables, other vegetables and milk products with RBCMZn status (p plasma zinc (p > 0.2). Cereal and legume intakes were negatively correlated with RBCMZn (p plasma zinc (p 0.2). Fruit and other vegetable intake were positively correlated with SOD (p Plasma zinc indicated positive association with zinc, thiamin and riboflavin intakes (p plasma zinc and SOD. Prominent determinants of zinc status were intakes of beta-carotene and zinc along with environmental conditions and family size.

  10. Genome-wide identification and characterization of cacao WRKY transcription factors and analysis of their expression in response to witches' broom disease.

    Science.gov (United States)

    Silva Monteiro de Almeida, Dayanne; Oliveira Jordão do Amaral, Daniel; Del-Bem, Luiz-Eduardo; Bronze Dos Santos, Emily; Santana Silva, Raner José; Peres Gramacho, Karina; Vincentz, Michel; Micheli, Fabienne

    2017-01-01

    Transcriptional regulation, led by transcription factors (TFs) such as those of the WRKY family, is a mechanism used by the organism to enhance or repress gene expression in response to stimuli. Here, we report on the genome-wide analysis of the Theobroma cacao WRKY TF family and also investigate the expression of WRKY genes in cacao infected by the fungus Moniliophthora perniciosa. In the cacao genome, 61 non-redundant WRKY sequences were found and classified in three groups (I to III) according to the WRKY and zinc-finger motif types. The 61 putative WRKY sequences were distributed on the 10 cacao chromosomes and 24 of them came from duplication events. The sequences were phylogenetically organized according to the general WRKY groups. The phylogenetic analysis revealed that subgroups IIa and IIb are sister groups and share a common ancestor, as well as subgroups IId and IIe. The most divergent groups according to the plant origin were IIc and III. According to the phylogenetic analysis, 7 TcWRKY genes were selected and analyzed by RT-qPCR in susceptible and resistant cacao plants infected (or not) with M. perniciosa. Some TcWRKY genes presented interesting responses to M. perniciosa such as Tc01_p014750/Tc06_p013130/AtWRKY28, Tc09_p001530/Tc06_p004420/AtWRKY40, Tc04_p016130/AtWRKY54 and Tc10_p016570/ AtWRKY70. Our results can help to select appropriate candidate genes for further characterization in cacao or in other Theobroma species.

  11. Genome-wide identification and characterization of cacao WRKY transcription factors and analysis of their expression in response to witches' broom disease

    Science.gov (United States)

    Silva Monteiro de Almeida, Dayanne; Oliveira Jordão do Amaral, Daniel; Del-Bem, Luiz-Eduardo; Bronze dos Santos, Emily; Santana Silva, Raner José; Peres Gramacho, Karina; Vincentz, Michel

    2017-01-01

    Transcriptional regulation, led by transcription factors (TFs) such as those of the WRKY family, is a mechanism used by the organism to enhance or repress gene expression in response to stimuli. Here, we report on the genome-wide analysis of the Theobroma cacao WRKY TF family and also investigate the expression of WRKY genes in cacao infected by the fungus Moniliophthora perniciosa. In the cacao genome, 61 non-redundant WRKY sequences were found and classified in three groups (I to III) according to the WRKY and zinc-finger motif types. The 61 putative WRKY sequences were distributed on the 10 cacao chromosomes and 24 of them came from duplication events. The sequences were phylogenetically organized according to the general WRKY groups. The phylogenetic analysis revealed that subgroups IIa and IIb are sister groups and share a common ancestor, as well as subgroups IId and IIe. The most divergent groups according to the plant origin were IIc and III. According to the phylogenetic analysis, 7 TcWRKY genes were selected and analyzed by RT-qPCR in susceptible and resistant cacao plants infected (or not) with M. perniciosa. Some TcWRKY genes presented interesting responses to M. perniciosa such as Tc01_p014750/Tc06_p013130/AtWRKY28, Tc09_p001530/Tc06_p004420/AtWRKY40, Tc04_p016130/AtWRKY54 and Tc10_p016570/ AtWRKY70. Our results can help to select appropriate candidate genes for further characterization in cacao or in other Theobroma species. PMID:29084273

  12. Zinc Finger Takes on a Whole New Meaning: Reducing and Monitoring Zinc Blanks in the Isotope Lab

    Science.gov (United States)

    Wilkes, E. B.; Wasylenki, L. E.; Anbar, A. D.

    2010-12-01

    In terms of avoiding contamination, zinc is one of the most difficult elements to study isotopically. The reason for this is that zinc stearate is a very common mold release agent in the production of plastics, including those most often used in isotope geochemistry clean labs. While polyethylene bottles, polypropylene centrifuge tubes, pipette tips, and Kimwipes are all potential sources of contaminant zinc, by far the largest amount of zinc is introduced to the laboratory by gloves. Most items can be effectively rid of zinc by soaking in dilute hydrochloric acid, but gloves cannot be cleaned easily, and use of gloves can quickly lead to contamination on many surfaces throughout the lab. We recently conducted several experiments in which dissolved zinc was partly adsorbed onto synthetic Mn oxyhydroxide particles. The dissolved and adsorbed pools were separated by filtration, purified with ion exchange chemistry, and analyzed for isotope composition by MC-ICP-MS. We used a commercially purchased ICP standard solution both as our standard (delta66/64Zn = 0) and as the source of the zinc in the experiments. Whenever gloves were worn during purification, process blanks contained as much as 150 ng Zn, and both the dissolved and adsorbed pools of zinc came out enriched in heavy isotopes relative to the starting pool, contrary to our expectation of mass balance. When gloves were not worn, blanks were brands of vinyl gloves, including one brand recommended to us for being “low” in zinc, measured +10‰ relative to our standard. We therefore concluded that glove zinc contaminated most of our experimental samples. We were only able to see such clear evidence of contamination because (1) we were doing an experiment in which we expected one light and one heavy pool of zinc compared to our standard, and (2) we happened to use an ICP standard solution for delta = 0 that is strongly enriched in light isotopes relative to both brands of gloves. We caution others who measure

  13. Global analysis of WRKY transcription factor superfamily in Setaria identifies potential candidates involved in abiotic stress signaling.

    Science.gov (United States)

    Muthamilarasan, Mehanathan; Bonthala, Venkata S; Khandelwal, Rohit; Jaishankar, Jananee; Shweta, Shweta; Nawaz, Kashif; Prasad, Manoj

    2015-01-01

    Transcription factors (TFs) are major players in stress signaling and constitute an integral part of signaling networks. Among the major TFs, WRKY proteins play pivotal roles in regulation of transcriptional reprogramming associated with stress responses. In view of this, genome- and transcriptome-wide identification of WRKY TF family was performed in the C4model plants, Setaria italica (SiWRKY) and S. viridis (SvWRKY), respectively. The study identified 105 SiWRKY and 44 SvWRKY proteins that were computationally analyzed for their physicochemical properties. Sequence alignment and phylogenetic analysis classified these proteins into three major groups, namely I, II, and III with majority of WRKY proteins belonging to group II (53 SiWRKY and 23 SvWRKY), followed by group III (39 SiWRKY and 11 SvWRKY) and group I (10 SiWRKY and 6 SvWRKY). Group II proteins were further classified into 5 subgroups (IIa to IIe) based on their phylogeny. Domain analysis showed the presence of WRKY motif and zinc finger-like structures in these proteins along with additional domains in a few proteins. All SiWRKY genes were physically mapped on the S. italica genome and their duplication analysis revealed that 10 and 8 gene pairs underwent tandem and segmental duplications, respectively. Comparative mapping of SiWRKY and SvWRKY genes in related C4 panicoid genomes demonstrated the orthologous relationships between these genomes. In silico expression analysis of SiWRKY and SvWRKY genes showed their differential expression patterns in different tissues and stress conditions. Expression profiling of candidate SiWRKY genes in response to stress (dehydration and salinity) and hormone treatments (abscisic acid, salicylic acid, and methyl jasmonate) suggested the putative involvement of SiWRKY066 and SiWRKY082 in stress and hormone signaling. These genes could be potential candidates for further characterization to delineate their functional roles in abiotic stress signaling.

  14. Transcriptome analysis of salinity responsiveness in contrasting genotypes of finger millet (Eleusine coracana L.) through RNA-sequencing.

    Science.gov (United States)

    Rahman, Hifzur; Jagadeeshselvam, N; Valarmathi, R; Sachin, B; Sasikala, R; Senthil, N; Sudhakar, D; Robin, S; Muthurajan, Raveendran

    2014-07-01

    Finger millet (Eleusine coracana L.) is a hardy cereal known for its superior level of tolerance against drought, salinity, diseases and its nutritional properties. In this study, attempts were made to unravel the physiological and molecular basis of salinity tolerance in two contrasting finger millet genotypes viz., CO 12 and Trichy 1. Physiological studies revealed that the tolerant genotype Trichy 1 had lower Na(+) to K(+) ratio in leaves and shoots, higher growth rate (osmotic tolerance) and ability to accumulate higher amount of total soluble sugar in leaves under salinity stress. We sequenced the salinity responsive leaf transcriptome of contrasting finger millet genotypes using IonProton platform and generated 27.91 million reads. Mapping and annotation of finger millet transcripts against rice gene models led to the identification of salinity responsive genes and genotype specific responses. Several functional groups of genes like transporters, transcription factors, genes involved in cell signaling, osmotic homeostasis and biosynthesis of compatible solutes were found to be highly up-regulated in the tolerant Trichy 1. Salinity stress inhibited photosynthetic capacity and photosynthesis related genes in the susceptible genotype CO 12. Several genes involved in cell growth and differentiation were found to be up-regulated in both the genotypes but more specifically in tolerant genotype. Genes involved in flavonoid biosynthesis were found to be down-regulated specifically in the salinity tolerant Trichy 1. This study provides a genome-wide transcriptional analysis of two finger millet genotypes differing in their level of salinity tolerance during a gradually progressing salinity stress under greenhouse conditions.

  15. The ciliogenic transcription factor RFX3 regulates early midline distribution of guidepost neurons required for corpus callosum development.

    Directory of Open Access Journals (Sweden)

    Carine Benadiba

    Full Text Available The corpus callosum (CC is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3 is a transcription factor involved in the control of ciliogenesis, and Rfx3-deficient mice show several hallmarks of ciliopathies including left-right asymmetry defects and hydrocephalus. Here we show that Rfx3-deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8 at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3 repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies.

  16. Pak2 Controls Acquisition of NKT Cell Fate by Regulating Expression of the Transcription Factors PLZF and Egr2

    Science.gov (United States)

    O’Hagan, Kyle L.; Zhao, Jie; Pryshchep, Olga; Wang, Chyung-Ru

    2015-01-01

    NKT cells constitute a small population of T cells developed in the thymus that produce large amounts of cytokines and chemokines in response to lipid Ags. Signaling through the Vα14-Jα18 TCR instructs commitment to the NKT cell lineage, but the precise signaling mechanisms that instruct their lineage choice are unclear. In this article, we report that the cytoskeletal remodeling protein, p21-activated kinase 2 (Pak2), was essential for NKT cell development. Loss of Pak2 in T cells reduced stage III NKT cells in the thymus and periphery. Among different NKT cell subsets, Pak2 was necessary for the generation and function of NKT1 and NKT2 cells, but not NKT17 cells. Mechanistically, expression of Egr2 and promyelocytic leukemia zinc finger (PLZF), two key transcription factors for acquiring the NKT cell fate, were markedly diminished in the absence of Pak2. Diminished expression of Egr2 and PLZF were not caused by aberrant TCR signaling, as determined using a Nur77-GFP reporter, but were likely due to impaired induction and maintenance of signaling lymphocyte activation molecule 6 expression, a TCR costimulatory receptor required for NKT cell development. These data suggest that Pak2 controls thymic NKT cell development by providing a signal that links Egr2 to induce PLZF, in part by regulating signaling lymphocyte activation molecule 6 expression. PMID:26519537

  17. Genome-wide identification and characterization of WRKY transcriptional factor family in apple and analysis of their responses to waterlogging and drought stress.

    Science.gov (United States)

    Meng, Dong; Li, Yuanyuan; Bai, Yang; Li, Mingjun; Cheng, Lailiang

    2016-06-01

    As one of the largest transcriptional factor families in plants, WRKY genes play significant roles in various biotic and abiotic stress responses. Although the WRKY gene family has been characterized in a few plant species, the details remain largely unknown in the apple (Malus domestica Borkh.). In this study, we identified a total of 127 MdWRKYs from the apple genome, which were divided into four subgroups according to the WRKY domains and zinc finger motif. Most of them were mapped onto the apple's 17 chromosomes and were expressed in more than one tissue, including shoot tips, mature leaves, fruit and apple calli. We then contrasted WRKY expression patterns between calli grown in solid medium (control) and liquid medium (representing waterlogging stress) and found that 34 WRKY genes were differentially expressed between the two growing conditions. Finally, we determined the expression patterns of 10 selected WRKY genes in an apple rootstock, G41, in response to waterlogging and drought stress, which identified candidate genes involved in responses to water stress for functional analysis. Our data provide interesting candidate MdWRKYs for future functional analysis and demonstrate that apple callus is a useful system for characterizing gene expression and function in apple. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  18. Zinc-induced Self-association of Complement C3b and Factor H

    Science.gov (United States)

    Nan, Ruodan; Tetchner, Stuart; Rodriguez, Elizabeth; Pao, Po-Jung; Gor, Jayesh; Lengyel, Imre; Perkins, Stephen J.

    2013-01-01

    The sub-retinal pigment epithelial deposits that are a hallmark of age-related macular degeneration contain both C3b and millimolar levels of zinc. C3 is the central protein of complement, whereas C3u is formed by the spontaneous hydrolysis of the thioester bridge in C3. During activation, C3 is cleaved to form active C3b, then C3b is inactivated by Factor I and Factor H to form the C3c and C3d fragments. The interaction of zinc with C3 was quantified using analytical ultracentrifugation and x-ray scattering. C3, C3u, and C3b associated strongly in >100 μm zinc, whereas C3c and C3d showed weak association. With zinc, C3 forms soluble oligomers, whereas C3u and C3b precipitate. We conclude that the C3, C3u, and C3b association with zinc depended on the relative positions of C3d and C3c in each protein. Computational predictions showed that putative weak zinc binding sites with different capacities exist in all five proteins, in agreement with experiments. Factor H forms large oligomers in >10 μm zinc. In contrast to C3b or Factor H alone, the solubility of the central C3b-Factor H complex was much reduced at 60 μm zinc and even more so at >100 μm zinc. The removal of the C3b-Factor H complex by zinc explains the reduced C3u/C3b inactivation rates by zinc. Zinc-induced precipitation may contribute to the initial development of sub-retinal pigment epithelial deposits in the retina as well as reducing the progression to advanced age-related macular degeneration in higher risk patients. PMID:23661701

  19. Prevalence of self-reported finger deformations and occupational risk factors among professional cooks: a cross-sectional study.

    Science.gov (United States)

    Nagasu, Miwako; Sakai, Kazuhiro; Kogi, Kazutaka; Ito, Akiyoshi; Feskens, Edith J M; Tomita, Shigeru; Temmyo, Yoshiomi; Ueno, Mitsuo; Miyagi, Shigeji

    2011-05-26

    Previous studies have pointed out that the school lunch workers in Japan are suffering from work-related disorders including finger deformations. The purpose of this study was to investigate the prevalence of self-reported finger deformations and the association with job-related risk factors. A cross-sectional questionnaire study of 5,719 subjects (response rate: 81%, 982 men and 4,737 women) was undertaken during September 2003 to February 2004. Finger deformations were found among 11.7% of the men and 35.6% of the women studied, with significant differences among sex, age and sex-age groups. For both men and women the pattern of finger deformations across the hand was similar for the right and the left hand. For women, the deformations were found in about 10% of the distal interphalangeal joints of all fingers. Based on multiple logistic regression analyses, the factors female sex, age, the number of cooked lunches per cook and cooking activities were independently associated with the prevalence of finger deformations. High prevalence odds ratios were found for those frequently carrying or using tools by hands such as delivering containers, distributing meals, preparing dishes, washing equipment, cutting and stirring foods. Among the school lunch workers studied, women had a higher prevalence of finger deformations on all joints of both hands. Various cooking tasks were associated with the prevalence of finger deformations. The results suggest that improvements in working conditions are important for preventing work-related disorders such as finger deformations.

  20. Tzfp represses the androgen receptor in mouse testis.

    Science.gov (United States)

    Furu, Kari; Klungland, Arne

    2013-01-01

    The testis zinc finger protein (Tzfp), also known as Repressor of GATA, belongs to the BTB/POZ zinc finger family of transcription factors and is thought to play a role in spermatogenesis due to its remarkably high expression in testis. Despite many attempts to find the in vivo role of the protein, the molecular function is still largely unknown. Here, we address this issue using a novel mouse model with a disrupted Tzfp gene. Homozygous Tzfp null mice are born at reduced frequency but appear viable and fertile. Sertoli cells in testes lacking Tzfp display an increase in Androgen Receptor (AR) signaling, and several genes in the testis, including Gata1, Aie1 and Fanc, show increased expression. Our results indicate that Tzfp function as a transcriptional regulator and that loss of the protein leads to alterations in AR signaling and reduced number of apoptotic cells in the testicular tubules.

  1. Directing traffic on DNA-How transcription factors relieve or induce transcriptional interference.

    Science.gov (United States)

    Hao, Nan; Palmer, Adam C; Dodd, Ian B; Shearwin, Keith E

    2017-03-15

    Transcriptional interference (TI) is increasingly recognized as a widespread mechanism of gene control, particularly given the pervasive nature of transcription, both sense and antisense, across all kingdoms of life. Here, we discuss how transcription factor binding kinetics strongly influence the ability of a transcription factor to relieve or induce TI.

  2. Selection for a Zinc-Finger Protein Contributes to Seed Oil Increase during Soybean Domestication.

    Science.gov (United States)

    Li, Qing-Tian; Lu, Xiang; Song, Qing-Xin; Chen, Hao-Wei; Wei, Wei; Tao, Jian-Jun; Bian, Xiao-Hua; Shen, Ming; Ma, Biao; Zhang, Wan-Ke; Bi, Ying-Dong; Li, Wei; Lai, Yong-Cai; Lam, Sin-Man; Shui, Guang-Hou; Chen, Shou-Yi; Zhang, Jin-Song

    2017-04-01

    Seed oil is a momentous agronomical trait of soybean ( Glycine max ) targeted by domestication in breeding. Although multiple oil-related genes have been uncovered, knowledge of the regulatory mechanism of seed oil biosynthesis is currently limited. We demonstrate that the seed-preferred gene GmZF351 , encoding a tandem CCCH zinc finger protein, is selected during domestication. Further analysis shows that GmZF351 facilitates oil accumulation by directly activating WRINKLED1 , BIOTIN CARBOXYL CARRIER PROTEIN2 , 3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE III , DIACYLGLYCEROL O-ACYLTRANSFERASE1 , and OLEOSIN2 in transgenic Arabidopsis ( Arabidopsis thaliana ) seeds. Overexpression of GmZF351 in transgenic soybean also activates lipid biosynthesis genes, thereby accelerating seed oil accumulation. The ZF351 haplotype from the cultivated soybean group and the wild soybean ( Glycine soja ) subgroup III correlates well with high gene expression level, seed oil contents and promoter activity, suggesting that selection of GmZF351 expression leads to increased seed oil content in cultivated soybean. Our study provides novel insights into the regulatory mechanism for seed oil accumulation, and the manipulation of GmZF351 may have great potential in the improvement of oil production in soybean and other related crops. © 2017 American Society of Plant Biologists. All Rights Reserved.

  3. Prevalence of self-reported finger deformations and occupational risk factors among professional cooks: a cross-sectional study

    Directory of Open Access Journals (Sweden)

    Tomita Shigeru

    2011-05-01

    Full Text Available Abstract Background Previous studies have pointed out that the school lunch workers in Japan are suffering from work-related disorders including finger deformations. The purpose of this study was to investigate the prevalence of self-reported finger deformations and the association with job-related risk factors. Methods A cross-sectional questionnaire study of 5,719 subjects (response rate: 81%, 982 men and 4,737 women was undertaken during September 2003 to February 2004. Results Finger deformations were found among 11.7% of the men and 35.6% of the women studied, with significant differences among sex, age and sex-age groups. For both men and women the pattern of finger deformations across the hand was similar for the right and the left hand. For women, the deformations were found in about 10% of the distal interphalangeal joints of all fingers. Based on multiple logistic regression analyses, the factors female sex, age, the number of cooked lunches per cook and cooking activities were independently associated with the prevalence of finger deformations. High prevalence odds ratios were found for those frequently carrying or using tools by hands such as delivering containers, distributing meals, preparing dishes, washing equipment, cutting and stirring foods. Conclusions Among the school lunch workers studied, women had a higher prevalence of finger deformations on all joints of both hands. Various cooking tasks were associated with the prevalence of finger deformations. The results suggest that improvements in working conditions are important for preventing work-related disorders such as finger deformations.

  4. Generation of SNCA Cell Models Using Zinc Finger Nuclease (ZFN) Technology for Efficient High-Throughput Drug Screening.

    Science.gov (United States)

    Dansithong, Warunee; Paul, Sharan; Scoles, Daniel R; Pulst, Stefan M; Huynh, Duong P

    2015-01-01

    Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by loss of dopaminergic neurons of the substantia nigra. The hallmark of PD is the appearance of neuronal protein aggregations known as Lewy bodies and Lewy neurites, of which α-synuclein forms a major component. Familial PD is rare and is associated with missense mutations of the SNCA gene or increases in gene copy number resulting in SNCA overexpression. This suggests that lowering SNCA expression could be therapeutic for PD. Supporting this hypothesis, SNCA reduction was neuroprotective in cell line and rodent PD models. We developed novel cell lines expressing SNCA fused to the reporter genes luciferase (luc) or GFP with the objective to enable high-throughput compound screening (HTS) for small molecules that can lower SNCA expression. Because SNCA expression is likely regulated by far-upstream elements (including the NACP-REP1 located at 8852 bp upstream of the transcription site), we employed zinc finger nuclease (ZFN) genome editing to insert reporter genes in-frame downstream of the SNCA gene in order to retain native SNCA expression control. This ensured full retention of known and unknown up- and downstream genetic elements controlling SNCA expression. Treatment of cells with the histone deacetylase inhibitor valproic acid (VPA) resulted in significantly increased SNCA-luc and SNCA-GFP expression supporting the use of our cell lines for identifying small molecules altering complex modes of expression control. Cells expressing SNCA-luc treated with a luciferase inhibitor or SNCA siRNA resulted in Z'-scores ≥ 0.75, suggesting the suitability of these cell lines for use in HTS. This study presents a novel use of genome editing for the creation of cell lines expressing α-synuclein fusion constructs entirely under native expression control. These cell lines are well suited for HTS for compounds that lower SNCA expression directly or by acting at long-range sites to the SNCA

  5. A Family of Zinc Finger Proteins Is Required forChromosome-specific Pairing and Synapsis during Meiosis in C.elegans

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, Carolyn M.; Dernburg, Abby F.

    2006-06-07

    Homologous chromosome pairing and synapsis are prerequisitefor accurate chromosome segregation during meiosis. Here, we show that afamily of four related C2H2 zinc-finger proteins plays a central role inthese events in C. elegans. These proteins are encoded within a tandemgene cluster. In addition to the X-specific HIM-8 protein, threeadditional paralogs collectively mediate the behavior of the fiveautosomes. Each chromosome relies on a specific member of the family topair and synapse with its homolog. These "ZIM" proteins concentrate atspecial regions called meiotic pairing centers on the correspondingchromosomes. These sites are dispersed along the nuclear envelope duringearly meiotic prophase, suggesting a role analogous to thetelomere-mediated meiotic bouquet in other organisms. To gain insightinto the evolution of these components, wecharacterized homologs in C.briggsae and C. remanei, which revealed changes in copy number of thisgene family within the nematode lineage.

  6. Determination of the characteristics of a Schottky barrier formed by latent finger mark corrosion of brass

    International Nuclear Information System (INIS)

    Bond, J W

    2009-01-01

    The ideality factor (η) and barrier height (φ B ) for a metal-copper(I) oxide rectifying contact formed by the latent finger mark corrosion of α phase brass have been determined from forward bias I/V characteristics in the range 0.4 V ≤ V ≤ 0.55 V. Rectifying contacts formed from the finger mark deposits of different people gave η = 1.5-1.6 ± 0.1 and φ B = 0.49-0.52 ± 0.04 V. A Mott-Schottky plot of capacitance-voltage measurements in reverse bias gave the built in potential ψ bi = 0.4 ± 0.1 V, the gradient of the plot confirming the conductivity of the finger mark corrosion as p type. X-ray photoelectron spectroscopy spectra of the corrosion showed that Cu(I), Cu(II) and Zn(II) can co-exist on the surface, the Cu(I) : Cu(II) and Zn : Cu ratios determining whether a rectifying contact is formed. Initial findings suggest that when the concentration of Cu(I) dominates the Cu(I) : Cu(II) ratio (approximately 6 : 1), or when Cu(II) is absent, a rectifying contact can be formed subject to the Zn : Cu ratio being approximately 1 : 3. As the surface concentration of zinc increases, the rectifying contact is degraded until the concentration of zinc approaches that of copper when no evidence of a Schottky barrier is observed and the contact appears ohmic.

  7. Structural analysis and dimerization profile of the SCAN domain of the pluripotency factor Zfp206

    KAUST Repository

    Liang, Yu

    2012-06-26

    Zfp206 (also named as Zscan10) belongs to the subfamily of C2H2 zinc finger transcription factors, which is characterized by the N-terminal SCAN domain. The SCAN domain mediates self-association and association between the members of SCAN family transcription factors, but the structural basis and selectivity determinants for complex formation is unknown. Zfp206 is important for maintaining the pluripotency of embryonic stem cells presumably by combinatorial assembly of itself or other SCAN family members on enhancer regions. To gain insights into the folding topology and selectivity determinants for SCAN dimerization, we solved the 1.85 crystal structure of the SCAN domain of Zfp206. In vitro binding studies using a panel of 20 SCAN proteins indicate that the SCAN domain Zfp206 can selectively associate with other members of SCAN family transcription factors. Deletion mutations showed that the N-terminal helix 1 is critical for heterodimerization. Double mutations and multiple mutations based on the Zfp206SCAN-Zfp110SCAN model suggested that domain swapped topology is a possible preference for Zfp206SCAN-Zfp110SCAN heterodimer. Together, we demonstrate that the Zfp206SCAN constitutes a protein module that enables C2H2 transcription factor dimerization in a highly selective manner using a domain-swapped interface architecture and identify novel partners for Zfp206 during embryonal development. 2012 The Author(s).

  8. Structural analysis and dimerization profile of the SCAN domain of the pluripotency factor Zfp206

    KAUST Repository

    Liang, Yu; Huimei Hong, Felicia; Ganesan, Pugalenthi; Jiang, Sizun; Jauch, Ralf; Stanton, Lawrence W.; Kolatkar, Prasanna R.

    2012-01-01

    Zfp206 (also named as Zscan10) belongs to the subfamily of C2H2 zinc finger transcription factors, which is characterized by the N-terminal SCAN domain. The SCAN domain mediates self-association and association between the members of SCAN family transcription factors, but the structural basis and selectivity determinants for complex formation is unknown. Zfp206 is important for maintaining the pluripotency of embryonic stem cells presumably by combinatorial assembly of itself or other SCAN family members on enhancer regions. To gain insights into the folding topology and selectivity determinants for SCAN dimerization, we solved the 1.85 crystal structure of the SCAN domain of Zfp206. In vitro binding studies using a panel of 20 SCAN proteins indicate that the SCAN domain Zfp206 can selectively associate with other members of SCAN family transcription factors. Deletion mutations showed that the N-terminal helix 1 is critical for heterodimerization. Double mutations and multiple mutations based on the Zfp206SCAN-Zfp110SCAN model suggested that domain swapped topology is a possible preference for Zfp206SCAN-Zfp110SCAN heterodimer. Together, we demonstrate that the Zfp206SCAN constitutes a protein module that enables C2H2 transcription factor dimerization in a highly selective manner using a domain-swapped interface architecture and identify novel partners for Zfp206 during embryonal development. 2012 The Author(s).

  9. Model of how plants sense zinc deficiency

    DEFF Research Database (Denmark)

    Assuncao, Ana G.L.; Persson, Daniel Olof; Husted, Søren

    2013-01-01

    to develop plant-based solutions addressing nutrient-use-efficiency and adaptation to nutrient-limited or -toxic soils. Recently two transcription factors of the bZIP family (basic-region leucine zipper) have been identified in Arabidopsis and shown to be pivotal in the adaptation response to zinc deficiency...

  10. A Novel Gli3 Enhancer Controls the Gli3 Spatiotemporal Expression Pattern through a TALE Homeodomain Protein Binding Site ▿‡

    OpenAIRE

    Coy, Sarah; Caamaño, Jorge H.; Carvajal, Jaime; Cleary, Michael L.; Borycki, Anne-Gaëlle

    2011-01-01

    The zinc finger transcription factor Gli3 is an essential mediator of hedgehog signaling. Gli3 has a dynamic expression pattern during embryonic development. In the neural tube, Gli3 transcripts are patterned along the anteroposterior and dorsoventral axes such that the initial broad expression in the posterior neural tube becomes dorsally restricted as neurogenesis takes place. Little is known about the molecular mechanisms that regulate this dynamic expression. Here, we report on a phylogen...

  11. TcoF-DB: dragon database for human transcription co-factors and transcription factor interacting proteins

    KAUST Repository

    Schaefer, Ulf; Schmeier, Sebastian; Bajic, Vladimir B.

    2010-01-01

    The initiation and regulation of transcription in eukaryotes is complex and involves a large number of transcription factors (TFs), which are known to bind to the regulatory regions of eukaryotic DNA. Apart from TF-DNA binding, protein-protein interaction involving TFs is an essential component of the machinery facilitating transcriptional regulation. Proteins that interact with TFs in the context of transcription regulation but do not bind to the DNA themselves, we consider transcription co-factors (TcoFs). The influence of TcoFs on transcriptional regulation and initiation, although indirect, has been shown to be significant with the functionality of TFs strongly influenced by the presence of TcoFs. While the role of TFs and their interaction with regulatory DNA regions has been well-studied, the association between TFs and TcoFs has so far been given less attention. Here, we present a resource that is comprised of a collection of human TFs and the TcoFs with which they interact. Other proteins that have a proven interaction with a TF, but are not considered TcoFs are also included. Our database contains 157 high-confidence TcoFs and additionally 379 hypothetical TcoFs. These have been identified and classified according to the type of available evidence for their involvement in transcriptional regulation and their presence in the cell nucleus. We have divided TcoFs into four groups, one of which contains high-confidence TcoFs and three others contain TcoFs which are hypothetical to different extents. We have developed the Dragon Database for Human Transcription Co-Factors and Transcription Factor Interacting Proteins (TcoF-DB). A web-based interface for this resource can be freely accessed at http://cbrc.kaust.edu.sa/tcof/ and http://apps.sanbi.ac.za/tcof/. © The Author(s) 2010.

  12. TcoF-DB: dragon database for human transcription co-factors and transcription factor interacting proteins

    KAUST Repository

    Schaefer, Ulf

    2010-10-21

    The initiation and regulation of transcription in eukaryotes is complex and involves a large number of transcription factors (TFs), which are known to bind to the regulatory regions of eukaryotic DNA. Apart from TF-DNA binding, protein-protein interaction involving TFs is an essential component of the machinery facilitating transcriptional regulation. Proteins that interact with TFs in the context of transcription regulation but do not bind to the DNA themselves, we consider transcription co-factors (TcoFs). The influence of TcoFs on transcriptional regulation and initiation, although indirect, has been shown to be significant with the functionality of TFs strongly influenced by the presence of TcoFs. While the role of TFs and their interaction with regulatory DNA regions has been well-studied, the association between TFs and TcoFs has so far been given less attention. Here, we present a resource that is comprised of a collection of human TFs and the TcoFs with which they interact. Other proteins that have a proven interaction with a TF, but are not considered TcoFs are also included. Our database contains 157 high-confidence TcoFs and additionally 379 hypothetical TcoFs. These have been identified and classified according to the type of available evidence for their involvement in transcriptional regulation and their presence in the cell nucleus. We have divided TcoFs into four groups, one of which contains high-confidence TcoFs and three others contain TcoFs which are hypothetical to different extents. We have developed the Dragon Database for Human Transcription Co-Factors and Transcription Factor Interacting Proteins (TcoF-DB). A web-based interface for this resource can be freely accessed at http://cbrc.kaust.edu.sa/tcof/ and http://apps.sanbi.ac.za/tcof/. © The Author(s) 2010.

  13. Genome-wide analysis of WRKY transcription factors in white pear (Pyrus bretschneideri) reveals evolution and patterns under drought stress.

    Science.gov (United States)

    Huang, Xiaosan; Li, Kongqing; Xu, Xiaoyong; Yao, Zhenghong; Jin, Cong; Zhang, Shaoling

    2015-12-24

    WRKY transcription factors (TFs) constitute one of the largest protein families in higher plants, and its members contain one or two conserved WRKY domains, about 60 amino acid residues with the WRKYGQK sequence followed by a C2H2 or C2HC zinc finger motif. WRKY proteins play significant roles in plant development, and in responses to biotic and abiotic stresses. Pear (Pyrus bretschneideri) is one of the most important fruit crops in the world and is frequently threatened by abiotic stress, such as drought, affecting growth, development and productivity. Although the pear genome sequence has been released, little is known about the WRKY TFs in pear, especially in respond to drought stress at the genome-wide level. We identified a total of 103 WRKY TFs in the pear genome. Based on the structural features of WRKY proteins and topology of the phylogenetic tree, the pear WRKY (PbWRKY) family was classified into seven groups (Groups 1, 2a-e, and 3). The microsyteny analysis indicated that 33 (32%) PbWRKY genes were tandemly duplicated and 57 genes (55.3%) were segmentally duplicated. RNA-seq experiment data and quantitative real-time reverse transcription PCR revealed that PbWRKY genes in different groups were induced by drought stress, and Group 2a and 3 were mainly involved in the biological pathways in response to drought stress. Furthermore, adaptive evolution analysis detected a significant positive selection for Pbr001425 in Group 3, and its expression pattern differed from that of other members in this group. The present study provides a solid foundation for further functional dissection and molecular evolution of WRKY TFs in pear, especially for improving the water-deficient resistance of pear through manipulation of the PbWRKYs.

  14. The WRKY transcription factor family in Brachypodium distachyon.

    Science.gov (United States)

    Tripathi, Prateek; Rabara, Roel C; Langum, Tanner J; Boken, Ashley K; Rushton, Deena L; Boomsma, Darius D; Rinerson, Charles I; Rabara, Jennifer; Reese, R Neil; Chen, Xianfeng; Rohila, Jai S; Rushton, Paul J

    2012-06-22

    A complete assembled genome sequence of wheat is not yet available. Therefore, model plant systems for wheat are very valuable. Brachypodium distachyon (Brachypodium) is such a system. The WRKY family of transcription factors is one of the most important families of plant transcriptional regulators with members regulating important agronomic traits. Studies of WRKY transcription factors in Brachypodium and wheat therefore promise to lead to new strategies for wheat improvement. We have identified and manually curated the WRKY transcription factor family from Brachypodium using a pipeline designed to identify all potential WRKY genes. 86 WRKY transcription factors were found, a total higher than all other current databases. We therefore propose that our numbering system (BdWRKY1-BdWRKY86) becomes the standard nomenclature. In the JGI v1.0 assembly of Brachypodium with the MIPS/JGI v1.0 annotation, nine of the transcription factors have no gene model and eleven gene models are probably incorrectly predicted. In total, twenty WRKY transcription factors (23.3%) do not appear to have accurate gene models. To facilitate use of our data, we have produced The Database of Brachypodium distachyon WRKY Transcription Factors. Each WRKY transcription factor has a gene page that includes predicted protein domains from MEME analyses. These conserved protein domains reflect possible input and output domains in signaling. The database also contains a BLAST search function where a large dataset of WRKY transcription factors, published genes, and an extensive set of wheat ESTs can be searched. We also produced a phylogram containing the WRKY transcription factor families from Brachypodium, rice, Arabidopsis, soybean, and Physcomitrella patens, together with published WRKY transcription factors from wheat. This phylogenetic tree provides evidence for orthologues, co-orthologues, and paralogues of Brachypodium WRKY transcription factors. The description of the WRKY transcription factor

  15. Tamarix hispida zinc finger protein ThZFP1 participates in salt and osmotic stress tolerance by increasing proline content and SOD and POD activities.

    Science.gov (United States)

    Zang, Dandan; Wang, Chao; Ji, Xiaoyu; Wang, Yucheng

    2015-06-01

    Zinc finger proteins (ZFPs) are a large family that play important roles in various biological processes, such as signal transduction, RNA binding, morphogenesis, transcriptional regulation, abiotic or biotic stress response. However, the functions of ZFPs involved in abiotic stress are largely not known. In the present study, we cloned and functionally characterized a ZFP gene, ThZFP1, from Tamarix hispida. The expression of ThZFP1 is highly induced by NaCl, mannitol or ABA treatment. To study the function of ThZFP1 involved in abiotic stress response, transgenic T. hispida plants with overexpression or knockdown of ThZFP1 were generated using a transient transformation system. Gain- and loss-of-function studies of ThZFP1 suggested that ThZFP1 can induce the expression of a series of genes, including delta-pyrroline-5-carboxylate synthetase (P5CS), peroxidase (POD) and superoxide dismutase (SOD), leading to accumulation of proline and enhanced activities of SOD and POD. These physiological changes enhanced proline content and reactive oxygen species (ROS) scavenging capability when exposed to salt or osmotic stress. All the results obtained from T. hispida plants were further confirmed by analyses of the transgenic Arabidopsis plants overexpressing ThZFP1. These data together suggested that ThZFP1 positively regulates proline accumulation and activities of SOD and POD under salt and osmotic stress conditions. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  16. The evolutionary duplication and probable demise of an endodermal GATA factor in Caenorhabditis elegans.

    Science.gov (United States)

    Fukushige, Tetsunari; Goszczynski, Barbara; Tian, Helen; McGhee, James D

    2003-10-01

    We describe the elt-4 gene from the nematode Caenorhabditis elegans. elt-4 is predicted to encode a very small (72 residues, 8.1 kD) GATA-type zinc finger transcription factor. The elt-4 gene is located approximately 5 kb upstream of the C. elegans elt-2 gene, which also encodes a GATA-type transcription factor; the zinc finger DNA-binding domains are highly conserved (24/25 residues) between the two proteins. The elt-2 gene is expressed only in the intestine and is essential for normal intestinal development. This article explores whether elt-4 also has a role in intestinal development. Reporter fusions to the elt-4 promoter or reporter insertions into the elt-4 coding regions show that elt-4 is indeed expressed in the intestine, beginning at the 1.5-fold stage of embryogenesis and continuing into adulthood. elt-4 reporter fusions are also expressed in nine cells of the posterior pharynx. Ectopic expression of elt-4 cDNA within the embryo does not cause detectable ectopic expression of biochemical markers of gut differentiation; furthermore, ectopic elt-4 expression neither inhibits nor enhances the ectopic marker expression caused by ectopic elt-2 expression. A deletion allele of elt-4 was isolated but no obvious phenotype could be detected, either in the gut or elsewhere; brood sizes, hatching efficiencies, and growth rates were indistinguishable from wild type. We found no evidence that elt-4 provided backup functions for elt-2. We used microarray analysis to search for genes that might be differentially expressed between L1 larvae of the elt-4 deletion strain and wild-type worms. Paired hybridizations were repeated seven times, allowing us to conclude, with some confidence, that no candidate target transcript could be identified as significantly up- or downregulated by loss of elt-4 function. In vitro binding experiments could not detect specific binding of ELT-4 protein to candidate binding sites (double-stranded oligonucleotides containing single or multiple

  17. Lead-zinc interactions in the production of osteocalcin by ROS 17/2.8 osteoblastic bone cells

    International Nuclear Information System (INIS)

    Pounds, J.G.

    1991-01-01

    The serum level of osteocalcin, a bone specific protein produced by osteoblasts and used clinically as a marker of osteoblast acceptive, is decreased in lead intoxicated children. Previous studies suggest that the reduced osteocalcin production appears to be the result of impaired transcriptional regulation of this 1,25-dihydroxyvitamin D 3 gene product, and not translation. As part of a study to investigate the potential interaction of Pb 2+ with Zn 2+ , and with the zinc fingers of the vitamin D receptor, ROS cells were treated with 0, 5, 10, or 25 μM lead acetate for 24 hr, in the presence of 10, 30, or 50 μM Zn followed by an additional 24 hr treatment with lead with 1,25-dihydroxyvitamin D 3 (100 pg/ml media). At the end of this period a radioimmunoassay was conducted to determine the amount of osteocalcin in the cells and secreted in the media. 1,25-dihydroxyvitamin D 3 caused an increase in osteocalcin secreted into the media in cultures containing 0 μM lead, but this increase was inhibited by lead in a concentration dependent manner, so that osteocalcin secretion in 10 or 25 μM lead treated groups was less than cultures without 1,25-dihydroxyvitamin D 3 treatment. This inhibitory effect of lead was blocked by increasing the medium zinc concentration of 50 μM. Increasing medium Pb 2+ concentrations decreased the amount of 65 Zn taken up by cells by ∼30%, which was nullified by increasing medium Zn. These results suggest that lead produces a localized and specific Zn deficiency in the vitamin D receptor zinc finger, and perhaps other zinc metalloproteins, and that these effects of lead are not mediated through general effects on RNA or protein synthesis

  18. Modeling, molecular dynamics, and docking assessment of transcription factor rho: a potential drug target in Brucella melitensis 16M

    Directory of Open Access Journals (Sweden)

    Pradeepkiran JA

    2015-03-01

    Full Text Available Jangampalli Adi Pradeepkiran,1 Konidala Kranthi Kumar,1 Yellapu Nanda Kumar,2 Matcha Bhaskar11Division of Animal Biotechnology, Department of Zoology, Sri Venkateswara University, Tirupati, 2Biomedical Informatics Centre, Vector Control Research Centre, Indian Council of Medical Research, Pondicherry, India Abstract: The zoonotic disease brucellosis, a chronic condition in humans affecting renal and cardiac systems and causing osteoarthritis, is caused by Brucella, a genus of Gram-negative, facultative, intracellular pathogens. The mode of transmission and the virulence of the pathogens are still enigmatic. Transcription regulatory elements, such as rho proteins, play an important role in the termination of transcription and/or the selection of genes in Brucella. Adverse effects of the transcription inhibitors play a key role in the non-successive transcription challenges faced by the pathogens. In the investigation presented here, we computationally predicted the transcription termination factor rho (TtFRho inhibitors against Brucella melitensis 16M via a structure-based method. In view the unknown nature of its crystal structure, we constructed a robust three-dimensional homology model of TtFRho’s structure by comparative modeling with the crystal structure of the Escherichia coli TtFRho (Protein Data Bank ID: 1PVO as a template in MODELLER (v 9.10. The modeled structure was optimized by applying a molecular dynamics simulation for 2 ns with the CHARMM (Chemistry at HARvard Macromolecular Mechanics 27 force field in NAMD (NAnoscale Molecular Dynamics program; v 2.9 and then evaluated by calculating the stereochemical quality of the protein. The flexible docking for the interaction phenomenon of the template consists of ligand-related inhibitor molecules from the ZINC (ZINC Is Not Commercial database using a structure-based virtual screening strategy against minimized TtFRho. Docking simulations revealed two inhibitors compounds – ZINC

  19. Highly transparent front electrodes with metal fingers for p-i-n thin-film silicon solar cells

    Directory of Open Access Journals (Sweden)

    Moulin Etienne

    2015-01-01

    Full Text Available The optical and electrical properties of transparent conductive oxides (TCOs, traditionally used in thin-film silicon (TF-Si solar cells as front-electrode materials, are interlinked, such that an increase in TCO transparency is generally achieved at the cost of reduced lateral conductance. Combining a highly transparent TCO front electrode of moderate conductance with metal fingers to support charge collection is a well-established technique in wafer-based technologies or for TF-Si solar cells in the substrate (n-i-p configuration. Here, we extend this concept to TF-Si solar cells in the superstrate (p-i-n configuration. The metal fingers are used in conjunction with a millimeter-scale textured foil, attached to the glass superstrate, which provides an antireflective and retroreflective effect; the latter effect mitigates the shadowing losses induced by the metal fingers. As a result, a substantial increase in power conversion efficiency, from 8.7% to 9.1%, is achieved for 1-μm-thick microcrystalline silicon solar cells deposited on a highly transparent thermally treated aluminum-doped zinc oxide layer combined with silver fingers, compared to cells deposited on a state-of-the-art zinc oxide layer.

  20. Fatty Acid–Regulated Transcription Factors in the Liver

    Science.gov (United States)

    Jump, Donald B.; Tripathy, Sasmita; Depner, Christopher M.

    2014-01-01

    Fatty acid regulation of hepatic gene transcription was first reported in the early 1990s. Several transcription factors have been identified as targets of fatty acid regulation. This regulation is achieved by direct fatty acid binding to the transcription factor or by indirect mechanisms where fatty acids regulate signaling pathways controlling the expression of transcription factors or the phosphorylation, ubiquitination, or proteolytic cleavage of the transcription factor. Although dietary fatty acids are well-established regulators of hepatic transcription factors, emerging evidence indicates that endogenously generated fatty acids are equally important in controlling transcription factors in the context of glucose and lipid homeostasis. Our first goal in this review is to provide an up-to-date examination of the molecular and metabolic bases of fatty acid regulation of key transcription factors controlling hepatic metabolism. Our second goal is to link these mechanisms to nonalcoholic fatty liver disease (NAFLD), a growing health concern in the obese population. PMID:23528177

  1. Mipu1, a novel direct target gene, is involved in hypoxia inducible factor 1-mediated cytoprotection.

    Directory of Open Access Journals (Sweden)

    Kangkai Wang

    Full Text Available Mipu1 (myocardial ischemic preconditioning up-regulated protein 1, recently identified in our lab, is a novel zinc-finger transcription factor which is up-regulated during ischemic preconditioning. However, it is not clear what transcription factor contributes to its inducible expression. In the present study, we reported that HIF-1 regulates the inducible expression of Mipu1 which is involved in the cytoprotection of HIF-1α against oxidative stress by inhibiting Bax expression. Our results showed that the inducible expression of Mipu1 was associated with the expression and activation of transcription factor HIF-1 as indicated by cobalt chloride (CoCl2 treatment, HIF-1α overexpression and knockdown assays. EMSA and luciferase reporter gene assays showed that HIF-1α bound to the hypoxia response element (HRE within Mipu1 promoter region and promoted its transcription. Moreover, our results revealed that Mipu1 inhibited the expression of Bax, an important pro-apoptosis protein associated with the intrinsic pathway of apoptosis, elevating the cytoprotection of HIF-1 against hydrogen peroxide (H2O2-mediated injury in H9C2 cells. Our findings implied that Bax may be a potential target gene of transcription factor Mipu1, and provided a novel insight for understanding the cytoprotection of HIF-1 and new clues for further elucidating the mechanisms by which Mipu1 protects cell against pathological stress.

  2. Zinc-finger protein 418 overexpression protects against cardiac hypertrophy and fibrosis.

    Directory of Open Access Journals (Sweden)

    Liming Pan

    Full Text Available This study aimed to investigated the effect and mechanism of zinc-finger protein 418 (ZNF418 on cardiac hypertrophy caused by aortic banding (AB, phenylephrine (PE or angiotensin II (Ang II in vivo and in vitro.The expression of ZNF418 in hearts of patients with dilated cardiomyopathy (DCM or hypertrophic cardiomyopathy (HCM and AB-induced cardiac hypertrophy mice, as well as in Ang II- or PE-induced hypertrophic primary cardiomyocytes was detected by western blotting. Then, the expression of ZNF418 was up-regulated or down-regulated in AB-induced cardiac hypertrophy mice and Ang II -induced hypertrophic primary cardiomyocytes. The hypertrophic responses and fibrosis were evaluated by echocardiography and histological analysis. The mRNA levels of hypertrophy markers and fibrotic markers were detected by RT-qPCR. Furthermore, the phosphorylation and total levels of c-Jun were measured by western blotting.ZNF418 was markedly down-regulated in hearts of cardiac hypertrophy and hypertrophic primary cardiomyocytes. Down-regulated ZNF418 exacerbated the myocyte size and fibrosis, moreover increased the mRNA levels of ANP, BNP, β-MHC, MCIP1.4, collagen 1a, collagen III, MMP-2 and fibronection in hearts of AB-treated ZNF418 knockout mice or Ang II-treated cardiomyocytes with AdshZNF418. Conversely, these hypertrophic responses were reduced in the ZNF418 transgenic (TG mice treated by AB and the AdZNF418-transfected primary cardiomyocytes treated by Ang II. Additionally, the deficiency of ZNF418 enhanced the phosphorylation level of c-jun, and overexpression of ZNF418 suppressed the phosphorylation level of c-jun in vivo and in vitro.ZNF418 maybe attenuate hypertrophic responses by inhibiting the activity of c-jun/AP-1.

  3. Tzfp represses the androgen receptor in mouse testis.

    Directory of Open Access Journals (Sweden)

    Kari Furu

    Full Text Available The testis zinc finger protein (Tzfp, also known as Repressor of GATA, belongs to the BTB/POZ zinc finger family of transcription factors and is thought to play a role in spermatogenesis due to its remarkably high expression in testis. Despite many attempts to find the in vivo role of the protein, the molecular function is still largely unknown. Here, we address this issue using a novel mouse model with a disrupted Tzfp gene. Homozygous Tzfp null mice are born at reduced frequency but appear viable and fertile. Sertoli cells in testes lacking Tzfp display an increase in Androgen Receptor (AR signaling, and several genes in the testis, including Gata1, Aie1 and Fanc, show increased expression. Our results indicate that Tzfp function as a transcriptional regulator and that loss of the protein leads to alterations in AR signaling and reduced number of apoptotic cells in the testicular tubules.

  4. Pak2 Controls Acquisition of NKT Cell Fate by Regulating Expression of the Transcription Factors PLZF and Egr2.

    Science.gov (United States)

    O'Hagan, Kyle L; Zhao, Jie; Pryshchep, Olga; Wang, Chyung-Ru; Phee, Hyewon

    2015-12-01

    NKT cells constitute a small population of T cells developed in the thymus that produce large amounts of cytokines and chemokines in response to lipid Ags. Signaling through the Vα14-Jα18 TCR instructs commitment to the NKT cell lineage, but the precise signaling mechanisms that instruct their lineage choice are unclear. In this article, we report that the cytoskeletal remodeling protein, p21-activated kinase 2 (Pak2), was essential for NKT cell development. Loss of Pak2 in T cells reduced stage III NKT cells in the thymus and periphery. Among different NKT cell subsets, Pak2 was necessary for the generation and function of NKT1 and NKT2 cells, but not NKT17 cells. Mechanistically, expression of Egr2 and promyelocytic leukemia zinc finger (PLZF), two key transcription factors for acquiring the NKT cell fate, were markedly diminished in the absence of Pak2. Diminished expression of Egr2 and PLZF were not caused by aberrant TCR signaling, as determined using a Nur77-GFP reporter, but were likely due to impaired induction and maintenance of signaling lymphocyte activation molecule 6 expression, a TCR costimulatory receptor required for NKT cell development. These data suggest that Pak2 controls thymic NKT cell development by providing a signal that links Egr2 to induce PLZF, in part by regulating signaling lymphocyte activation molecule 6 expression. Copyright © 2015 by The American Association of Immunologists, Inc.

  5. Improved ethanol production at high temperature by consolidated bioprocessing using Saccharomyces cerevisiae strain engineered with artificial zinc finger protein.

    Science.gov (United States)

    Khatun, M Mahfuza; Yu, Xinshui; Kondo, Akihiko; Bai, Fengwu; Zhao, Xinqing

    2017-12-01

    In this work, the consolidated bioprocessing (CBP) yeast Saccharomyces cerevisiae MNII/cocδBEC3 was transformed by an artificial zinc finger protein (AZFP) library to improve its thermal tolerance, and the strain MNII-AZFP with superior growth at 42°C was selected. Improved degradation of acid swollen cellulose by 45.9% led to an increase in ethanol production, when compared to the control strain. Moreover, the fermentation of Jerusalem artichoke stalk (JAS) by MNII-AZFP was shortened by 12h at 42°C with a concomitant improvement in ethanol production. Comparative transcriptomics analysis suggested that the AZFP in the mutant exerted beneficial effect by modulating the expression of multiple functional genes. These results provide a feasible strategy for efficient ethanol production from JAS and other cellulosic biomass through CBP based-fermentation at elevated temperatures. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. In Vivo Zinc Finger Nuclease-mediated Targeted Integration of a Glucose-6-phosphatase Transgene Promotes Survival in Mice With Glycogen Storage Disease Type IA

    Science.gov (United States)

    Landau, Dustin J; Brooks, Elizabeth Drake; Perez-Pinera, Pablo; Amarasekara, Hiruni; Mefferd, Adam; Li, Songtao; Bird, Andrew; Gersbach, Charles A; Koeberl, Dwight D

    2016-01-01

    Glycogen storage disease type Ia (GSD Ia) is caused by glucose-6-phosphatase (G6Pase) deficiency in association with severe, life-threatening hypoglycemia that necessitates lifelong dietary therapy. Here we show that use of a zinc-finger nuclease (ZFN) targeted to the ROSA26 safe harbor locus and a ROSA26-targeting vector containing a G6PC donor transgene, both delivered with adeno-associated virus (AAV) vectors, markedly improved survival of G6Pase knockout (G6Pase-KO) mice compared with mice receiving the donor vector alone (P Ia, as compared with normal littermates, at 8 months following vector administration (P Ia. PMID:26865405

  7. Regulation of hippocampus-dependent memory by the zinc finger protein Zbtb20 in mature CA1 neurons.

    Science.gov (United States)

    Ren, Anjing; Zhang, Huan; Xie, Zhifang; Ma, Xianhua; Ji, Wenli; He, David Z Z; Yuan, Wenjun; Ding, Yu-Qiang; Zhang, Xiao-Hui; Zhang, Weiping J

    2012-10-01

    The mammalian hippocampus harbours neural circuitry that is crucial for associative learning and memory. The mechanisms that underlie the development and regulation of this complex circuitry are not fully understood. Our previous study established an essential role for the zinc finger protein Zbtb20 in the specification of CA1 field identity in the developing hippocampus. Here, we show that conditionally deleting Zbtb20 specifically in mature CA1 pyramidal neurons impaired hippocampus-dependent memory formation, without affecting hippocampal architecture or the survival, identity and basal excitatory synaptic activity of CA1 pyramidal neurons. We demonstrate that mature CA1-specific Zbtb20 knockout mice exhibited reductions in long-term potentiation (LTP) and NMDA receptor (NMDAR)-mediated excitatory post-synaptic currents. Furthermore, we show that activity-induced phosphorylation of ERK and CREB is impaired in the hippocampal CA1 of Zbtb20 mutant mice. Collectively, these results indicate that Zbtb20 in mature CA1 plays an important role in LTP and memory by regulating NMDAR activity, and activation of ERK and CREB.

  8. The artificial gene Jazz, a transcriptional regulator of utrophin, corrects the dystrophic pathology in mdx mice.

    Science.gov (United States)

    Di Certo, Maria Grazia; Corbi, Nicoletta; Strimpakos, Georgios; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Guglielmotti, Angelo; Batassa, Enrico Maria; Pisani, Cinzia; Floridi, Aristide; Benassi, Barbara; Fanciulli, Maurizio; Magrelli, Armando; Mattei, Elisabetta; Passananti, Claudio

    2010-03-01

    The absence of the cytoskeletal protein dystrophin results in Duchenne muscular dystrophy (DMD). The utrophin protein is the best candidate for dystrophin replacement in DMD patients. To obtain therapeutic levels of utrophin expression in dystrophic muscle, we developed an alternative strategy based on the use of artificial zinc finger transcription factors (ZF ATFs). The ZF ATF 'Jazz' was recently engineered and tested in vivo by generating a transgenic mouse specifically expressing Jazz at the muscular level. To validate the ZF ATF technology for DMD treatment we generated a second mouse model by crossing Jazz-transgenic mice with dystrophin-deficient mdx mice. Here, we show that the artificial Jazz protein restores sarcolemmal integrity and prevents the development of the dystrophic disease in mdx mice. This exclusive animal model establishes the notion that utrophin-based therapy for DMD can be efficiently developed using ZF ATF technology and candidates Jazz as a novel therapeutic molecule for DMD therapy.

  9. Comparative analysis of function and interaction of transcription factors in nematodes: Extensive conservation of orthology coupled to rapid sequence evolution

    Directory of Open Access Journals (Sweden)

    Singh Rama S

    2008-08-01

    Full Text Available Abstract Background Much of the morphological diversity in eukaryotes results from differential regulation of gene expression in which transcription factors (TFs play a central role. The nematode Caenorhabditis elegans is an established model organism for the study of the roles of TFs in controlling the spatiotemporal pattern of gene expression. Using the fully sequenced genomes of three Caenorhabditid nematode species as well as genome information from additional more distantly related organisms (fruit fly, mouse, and human we sought to identify orthologous TFs and characterized their patterns of evolution. Results We identified 988 TF genes in C. elegans, and inferred corresponding sets in C. briggsae and C. remanei, containing 995 and 1093 TF genes, respectively. Analysis of the three gene sets revealed 652 3-way reciprocal 'best hit' orthologs (nematode TF set, approximately half of which are zinc finger (ZF-C2H2 and ZF-C4/NHR types and HOX family members. Examination of the TF genes in C. elegans and C. briggsae identified the presence of significant tandem clustering on chromosome V, the majority of which belong to ZF-C4/NHR family. We also found evidence for lineage-specific duplications and rapid evolution of many of the TF genes in the two species. A search of the TFs conserved among nematodes in Drosophila melanogaster, Mus musculus and Homo sapiens revealed 150 reciprocal orthologs, many of which are associated with important biological processes and human diseases. Finally, a comparison of the sequence, gene interactions and function indicates that nematode TFs conserved across phyla exhibit significantly more interactions and are enriched in genes with annotated mutant phenotypes compared to those that lack orthologs in other species. Conclusion Our study represents the first comprehensive genome-wide analysis of TFs across three nematode species and other organisms. The findings indicate substantial conservation of transcription

  10. The Transcription Factor Encyclopedia

    DEFF Research Database (Denmark)

    Yusuf, Dimas; Butland, Stefanie L; Swanson, Magdalena I

    2012-01-01

    mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written......ABSTRACT: Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130...

  11. Nonintrusive Finger-Vein Recognition System Using NIR Image Sensor and Accuracy Analyses According to Various Factors

    Directory of Open Access Journals (Sweden)

    Tuyen Danh Pham

    2015-07-01

    Full Text Available Biometrics is a technology that enables an individual person to be identified based on human physiological and behavioral characteristics. Among biometrics technologies, face recognition has been widely used because of its advantages in terms of convenience and non-contact operation. However, its performance is affected by factors such as variation in the illumination, facial expression, and head pose. Therefore, fingerprint and iris recognitions are preferred alternatives. However, the performance of the former can be adversely affected by the skin condition, including scarring and dryness. In addition, the latter has the disadvantages of high cost, large system size, and inconvenience to the user, who has to align their eyes with the iris camera. In an attempt to overcome these problems, finger-vein recognition has been vigorously researched, but an analysis of its accuracies according to various factors has not received much attention. Therefore, we propose a nonintrusive finger-vein recognition system using a near infrared (NIR image sensor and analyze its accuracies considering various factors. The experimental results obtained with three databases showed that our system can be operated in real applications with high accuracy; and the dissimilarity of the finger-veins of different people is larger than that of the finger types and hands.

  12. Nonintrusive Finger-Vein Recognition System Using NIR Image Sensor and Accuracy Analyses According to Various Factors.

    Science.gov (United States)

    Pham, Tuyen Danh; Park, Young Ho; Nguyen, Dat Tien; Kwon, Seung Yong; Park, Kang Ryoung

    2015-07-13

    Biometrics is a technology that enables an individual person to be identified based on human physiological and behavioral characteristics. Among biometrics technologies, face recognition has been widely used because of its advantages in terms of convenience and non-contact operation. However, its performance is affected by factors such as variation in the illumination, facial expression, and head pose. Therefore, fingerprint and iris recognitions are preferred alternatives. However, the performance of the former can be adversely affected by the skin condition, including scarring and dryness. In addition, the latter has the disadvantages of high cost, large system size, and inconvenience to the user, who has to align their eyes with the iris camera. In an attempt to overcome these problems, finger-vein recognition has been vigorously researched, but an analysis of its accuracies according to various factors has not received much attention. Therefore, we propose a nonintrusive finger-vein recognition system using a near infrared (NIR) image sensor and analyze its accuracies considering various factors. The experimental results obtained with three databases showed that our system can be operated in real applications with high accuracy; and the dissimilarity of the finger-veins of different people is larger than that of the finger types and hands.

  13. Global analysis of WRKY transcription factor superfamily in Setaria identifies potential candidates involved in abiotic stress signalling

    Directory of Open Access Journals (Sweden)

    Mehanathan eMuthamilarasan

    2015-10-01

    Full Text Available Transcription factors (TFs are major players in stress signalling and constitute an integral part of signalling networks. Among the major TFs, WRKY proteins play pivotal roles in regulation of transcriptional reprogramming associated with stress responses. In view of this, genome- and transcriptome-wide identification of WRKY TF family was performed in the C4 model plants, Setaria italica (SiWRKY and S. viridis (SvWRKY, respectively. The study identified 105 SiWRKY and 44 SvWRKY proteins that were computationally analysed for their physicochemical properties. Sequence alignment and phylogenetic analysis classified these proteins into three major groups, namely I, II and III with majority of WRKY proteins belonging to group II (53 SiWRKY and 23 SvWRKY, followed by group III (39 SiWRKY and 11 SvWRKY and group I (10 SiWRKY and 6 SvWRKY. Group II proteins were further classified into 5 subgroups (IIa to IIe based on their phylogeny. Domain analysis showed the presence of WRKY motif and zinc finger-like structures in these proteins along with additional domains in a few proteins. All SiWRKY genes were physically mapped on the S. italica genome and their duplication analysis revealed that 10 and 8 gene pairs underwent tandem and segmental duplications, respectively. Comparative mapping of SiWRKY and SvWRKY genes in related C4 panicoid genomes demonstrated the orthologous relationships between these genomes. In silico expression analysis of SiWRKY and SvWRKY genes showed their differential expression patterns in different tissues and stress conditions. Expression profiling of candidate SiWRKY genes in response to stress (dehydration and salinity and hormone treatments (abscisic acid, salicylic acid and methyl jasmonate suggested the putative involvement of SiWRKY066 and SiWRKY082 in stress and hormone signalling. These genes could be potential candidates for further characterization to delineate their functional roles in abiotic stress signalling.

  14. Global analysis of WRKY transcription factor superfamily in Setaria identifies potential candidates involved in abiotic stress signaling

    Science.gov (United States)

    Muthamilarasan, Mehanathan; Bonthala, Venkata S.; Khandelwal, Rohit; Jaishankar, Jananee; Shweta, Shweta; Nawaz, Kashif; Prasad, Manoj

    2015-01-01

    Transcription factors (TFs) are major players in stress signaling and constitute an integral part of signaling networks. Among the major TFs, WRKY proteins play pivotal roles in regulation of transcriptional reprogramming associated with stress responses. In view of this, genome- and transcriptome-wide identification of WRKY TF family was performed in the C4model plants, Setaria italica (SiWRKY) and S. viridis (SvWRKY), respectively. The study identified 105 SiWRKY and 44 SvWRKY proteins that were computationally analyzed for their physicochemical properties. Sequence alignment and phylogenetic analysis classified these proteins into three major groups, namely I, II, and III with majority of WRKY proteins belonging to group II (53 SiWRKY and 23 SvWRKY), followed by group III (39 SiWRKY and 11 SvWRKY) and group I (10 SiWRKY and 6 SvWRKY). Group II proteins were further classified into 5 subgroups (IIa to IIe) based on their phylogeny. Domain analysis showed the presence of WRKY motif and zinc finger-like structures in these proteins along with additional domains in a few proteins. All SiWRKY genes were physically mapped on the S. italica genome and their duplication analysis revealed that 10 and 8 gene pairs underwent tandem and segmental duplications, respectively. Comparative mapping of SiWRKY and SvWRKY genes in related C4 panicoid genomes demonstrated the orthologous relationships between these genomes. In silico expression analysis of SiWRKY and SvWRKY genes showed their differential expression patterns in different tissues and stress conditions. Expression profiling of candidate SiWRKY genes in response to stress (dehydration and salinity) and hormone treatments (abscisic acid, salicylic acid, and methyl jasmonate) suggested the putative involvement of SiWRKY066 and SiWRKY082 in stress and hormone signaling. These genes could be potential candidates for further characterization to delineate their functional roles in abiotic stress signaling. PMID:26635818

  15. The correlation of prenatal zinc concentration and deficiency with anthropometric factors.

    Directory of Open Access Journals (Sweden)

    Parichehr Hanachi

    2014-03-01

    Full Text Available To determine the status of serum zinc in pregnant women in different gestational ages and correlation with socio-demographic and anthropometric factors in Iranian women referring to prenatal care public health clinics.We analyzed the zinc concentrations in plasma samples obtained at different gestational ages from 961 women and recorded BMI at the first trimester in pregnant women who were screened for a trial designed to evaluate the zinc concentration. Subjects were from different socio economical backgrounds and attended public health clinics for their prenatal care. All analyses were performed by SPSS (version 16. P values < 0.05 were considered significant.The results showed that after plasma zinc concentrations were adjusted with Parity, weight (early pregnancy, BMI (at early pregnancy, age and educational statues. Plasma zinc deficiency declined as gestational age progressed, however it was not significant. There was no significant correlation between zinc concentration, anthropometric, method of contraception and socio factors. However, there were significant relation between parity (p = 0.007 and weight at early pregnancy (p= 0.039 with serum zinc levels.We conclude that plasma zinc concentrations decreased during the late first trimester to the early third trimester and with parity. These findings may indicate that the deficient levels of zinc in the latter third of pregnancy suggest a tendency for insufficient maternal nutrition. However larger studies are required to support this finding.

  16. Differing Dynamics of Intrapersonal and Interpersonal Coordination: Two-finger and Four-Finger Tapping Experiments.

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    Kentaro Kodama

    Full Text Available Finger-tapping experiments were conducted to examine whether the dynamics of intrapersonal and interpersonal coordination systems can be described equally by the Haken-Kelso-Bunz model, which describes inter-limb coordination dynamics. This article reports the results of finger-tapping experiments conducted in both systems. Two within-subject factors were investigated: the phase mode and the number of fingers. In the intrapersonal experiment (Experiment 1, the participants were asked to tap, paced by a gradually hastening auditory metronome, looking at their fingers moving, using the index finger in the two finger condition, or the index and middle finger in the four-finger condition. In the interpersonal experiment (Experiment 2, pairs of participants performed the task while each participant used the outside hand, tapping with the index finger in the two finger condition, or the index and middle finger in the four-finger condition. Some results did not agree with the HKB model predictions. First, from Experiment 1, no significant difference was observed in the movement stability between the in-phase and anti-phase modes in the two finger condition. Second, from Experiment 2, no significant difference was found in the movement stability between the in-phase and anti-phase mode in the four-finger condition. From these findings, different coordination dynamics were inferred between intrapersonal and interpersonal coordination systems against prediction from the previous studies. Results were discussed according to differences between intrapersonal and interpersonal coordination systems in the availability of perceptual information and the complexity in the interaction between limbs derived from a nested structure.

  17. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 Synergistically Activate Transcription of Fatty-acid Synthase Gene (FASN)*S⃞

    Science.gov (United States)

    Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F.; Hur, Man-Wook

    2008-01-01

    FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation. PMID:18682402

  18. Influence of heat processing on the bioaccessibility of zinc and iron from cereals and pulses consumed in India.

    Science.gov (United States)

    Hemalatha, Sreeramaiah; Platel, Kalpana; Srinivasan, Krishnapura

    2007-01-01

    Influence of heat processing on the bioaccessibility of zinc and iron from food grains consumed in India was evaluated. Cereals - rice (Oryza sativa), finger millet (Eleusine coracana), sorghum (Sorghum vulgare), wheat (Triticum aestivum), and maize (Zea mays), and pulses - chickpea (Cicer arietinum) - whole and decorticated, green gram (Phaseolus aureus) - whole and decorticated, decorticated black gram (Phaseolus mungo), decorticated red gram (Cajanus cajan), cowpea (Vigna catjang), and French bean (Phaseolus vulgaris) were examined for zinc and iron bioaccessibility by employing an in vitro dialysability procedure. Both pressure-cooking and microwave heating were tested for their influence on mineral bioaccessibility. Zinc bioaccessibility from food grains was considerably reduced upon pressure-cooking, especially in pulses. Among cereals, pressure-cooking decreased zinc bioaccessibility by 63% and 57% in finger millet and rice, respectively. All the pressure-cooked cereals showed similar percent zinc bioaccessibility with the exception of finger millet. Bioaccessibility of zinc from pulses was generally lower as a result of pressure-cooking or microwave heating. The decrease in bioaccessibility of zinc caused by microwave heating ranged from 11.4% in chickpea (whole) to 63% in cowpea. Decrease in zinc bioaccessibility was 48% in pressure-cooked whole chickpea, 45% and 55% in pressure-cooked or microwave-heated whole green gram, 32% and 22% in pressure-cooked or microwave-heated decorticated green gram, and 45% in microwave-heated black gram. Iron bioaccessibility, on the other hand, was significantly enhanced generally from all the food grains studied upon heat treatment. Thus, heat treatment of grains produced contrasting effect on zinc and iron bioaccessibility.

  19. The KRAB Zinc Finger Protein Roma/Zfp157 Is a Critical Regulator of Cell-Cycle Progression and Genomic Stability

    Directory of Open Access Journals (Sweden)

    Teresa L.F. Ho

    2016-04-01

    Full Text Available Regulation of DNA replication and cell division is essential for tissue growth and maintenance of genomic integrity and is particularly important in tissues that undergo continuous regeneration such as mammary glands. We have previously shown that disruption of the KRAB-domain zinc finger protein Roma/Zfp157 results in hyperproliferation of mammary epithelial cells (MECs during pregnancy. Here, we delineate the mechanism by which Roma engenders this phenotype. Ablation of Roma in MECs leads to unscheduled proliferation, replication stress, DNA damage, and genomic instability. Furthermore, mouse embryonic fibroblasts (MEFs depleted for Roma exhibit downregulation of p21Cip1 and geminin and have accelerated replication fork velocities, which is accompanied by a high rate of mitotic errors and polyploidy. In contrast, overexpression of Roma in MECs halts cell-cycle progression, whereas siRNA-mediated p21Cip1 knockdown ameliorates, in part, this phenotype. Thus, Roma is an essential regulator of the cell cycle and is required to maintain genomic stability.

  20. Glycogen synthase kinase 3 alpha phosphorylates and regulates the osteogenic activity of Osterix.

    Science.gov (United States)

    Li, Hongyan; Jeong, Hyung Min; Choi, You Hee; Lee, Sung Ho; Jeong, Hye Gwang; Jeong, Tae Cheon; Lee, Kwang Youl

    2013-05-10

    Osteoblast-specific transcription factor Osterix is a zinc-finger transcription factor that required for osteoblast differentiation and new bone formation. The function of Osterix can be modulated by post-translational modification. Glycogen synthase kinase 3 alpha (GSK3α) is a multifunctional serine/threonine protein kinase that plays a role in the Wnt signaling pathways and is implicated in the control of several regulatory proteins and transcription factors. In the present study, we investigated how GSK3α regulates Osterix during osteoblast differentiation. Wide type GSK3α up-regulated the protein level, protein stability and transcriptional activity of Osterix. These results suggest that GSK3α regulates osteogenic activity of Osterix. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

    Science.gov (United States)

    Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

    2017-10-01

    Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

  2. Hsf1p and Msn2/4p cooperate in the expression of Saccharomyces cerevisiae genes HSP26 and HSP104 in a gene- and stress type-dependent manner.

    Science.gov (United States)

    Amorós, M; Estruch, F

    2001-03-01

    Saccharomyces cerevisiae possesses several transcription factors involved in the transcriptional activation of stress-induced genes. Among them, the heat shock factor (Hsf1p) and the zinc finger proteins of the general stress response (Msn2p and Msn4p) have been shown to play a major role in stress protection. Some heat shock protein (HSP) genes contain both heat shock elements (HSEs) and stress response elements (STREs), suggesting the involvement of both transcription factors in their regulation. Analysis of the stress-induced expression of two of these genes, HSP26 and HSP104, reveals that the contribution of Hsf1p and Msn2/4p is different depending on the gene and the stress condition.

  3. Transcriptomic profiling of Arabidopsis gene expression in response to varying micronutrient zinc supply

    Directory of Open Access Journals (Sweden)

    Herlânder Azevedo

    2016-03-01

    Full Text Available Deficiency of the micronutrient zinc is a widespread condition in agricultural soils, causing a negative impact on crop quality and yield. Nevertheless, there is an insufficient knowledge on the regulatory and molecular mechanisms underlying the plant response to inadequate zinc nutrition [1]. This information should contribute to the development of plant-based solutions with improved nutrient-use-efficiency traits in crops. Previously, the transcription factors bZIP19 and bZIP23 were identified as essential regulators of the response to zinc deficiency in Arabidopsis thaliana [2]. A microarray experiment comparing gene expression between roots of wild-type and the mutant bzip19 bzip23, exposed to zinc deficiency, led to the identification of differentially expressed genes related with zinc homeostasis, namely its transport and plant internal translocation [2]. Here, we provide the detailed methodology, bioinformatics analysis and quality controls related to the microarray gene expression profiling published by Assunção and co-workers [2]. Most significantly, the present dataset comprises new experimental variables, including analysis of shoot tissue, and zinc sufficiency and excess supply. Thus, it expands from 8 to 42 microarrays hybridizations, which have been deposited at the Gene Expression Omnibus (GEO under the accession number GSE77286. Overall, it provides a resource for research on the molecular basis and regulatory events of the plant response to zinc supply, emphasizing the importance of Arabidopsis bZIP19 and bZIP23 transcription factors. Keywords: Microarray, Micronutrient, Zinc deficiency, Arabidopsis, bZIP

  4. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    International Nuclear Information System (INIS)

    Watanabe, Masahito; Umeyama, Kazuhiro; Matsunari, Hitomi; Takayanagi, Shuko; Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka; Nakauchi, Hiromitsu

    2010-01-01

    Research highlights: → EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. → ZFNs induced targeted mutations in porcine primary cultured cells. → Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  5. Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

    Directory of Open Access Journals (Sweden)

    Tatiana Flisikowska

    Full Text Available Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+ and IgG(+ B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

  6. High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs

    DEFF Research Database (Denmark)

    Duda, Katarzyna; Lonowski, Lindsey A; Kofoed-Nielsen, Michael

    2014-01-01

    Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing...... higher genome editing rates. For ZFNs, this approach, combined with delivery of donors as single-stranded oligodeoxynucleotides and nucleases as messenger ribonucleic acid, enabled high knockin efficiencies in demanding applications, including biallelic codon conversion frequencies reaching 30......-70% at high transfection efficiencies and ∼2% at low transfection efficiencies, simultaneous homozygous knockin mutation of two genes with ∼1.5% efficiency as well as generation of cell pools with almost complete codon conversion via three consecutive targeting and FACS events. Observed off-target effects...

  7. Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase

    Science.gov (United States)

    Morellet, Nelly; Li, Xianghong; Wieninger, Silke A; Taylor, Jennifer L; Bischerour, Julien; Moriau, Séverine; Lescop, Ewen; Bardiaux, Benjamin; Mathy, Nathalie; Assrir, Nadine; Bétermier, Mireille; Nilges, Michael; Hickman, Alison B; Dyda, Fred; Craig, Nancy L; Guittet, Eric

    2018-01-01

    Abstract The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure–function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5′-TGCGT-3′/3′-ACGCA-5′ motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity. PMID:29385532

  8. In-house zinc SAD phasing at Cu Kα edge.

    Science.gov (United States)

    Kim, Min-Kyu; Lee, Sangmin; An, Young Jun; Jeong, Chang-Sook; Ji, Chang-Jun; Lee, Jin-Won; Cha, Sun-Shin

    2013-07-01

    De novo zinc single-wavelength anomalous dispersion (Zn-SAD) phasing has been demonstrated with the 1.9 Å resolution data of glucose isomerase and 2.6 Å resolution data of Staphylococcus aureus Fur (SaFur) collected using in-house Cu Kα X-ray source. The successful in-house Zn-SAD phasing of glucose isomerase, based on the anomalous signals of both zinc ions introduced to crystals by soaking and native sulfur atoms, drove us to determine the structure of SaFur, a zinc-containing transcription factor, by Zn-SAD phasing using in-house X-ray source. The abundance of zinc-containing proteins in nature, the easy zinc derivatization of the protein surface, no need of synchrotron access, and the successful experimental phasing with the modest 2.6 Å resolution SAD data indicate that inhouse Zn-SAD phasing can be widely applicable to structure determination.

  9. Selection for a Zinc-Finger Protein Contributes to Seed Oil Increase during Soybean Domestication1[OPEN

    Science.gov (United States)

    Li, Qing-Tian; Lu, Xiang; Song, Qing-Xin; Chen, Hao-Wei; Wei, Wei; Tao, Jian-Jun; Ma, Biao; Bi, Ying-Dong; Li, Wei; Lai, Yong-Cai; Shui, Guang-Hou; Chen, Shou-Yi

    2017-01-01

    Seed oil is a momentous agronomical trait of soybean (Glycine max) targeted by domestication in breeding. Although multiple oil-related genes have been uncovered, knowledge of the regulatory mechanism of seed oil biosynthesis is currently limited. We demonstrate that the seed-preferred gene GmZF351, encoding a tandem CCCH zinc finger protein, is selected during domestication. Further analysis shows that GmZF351 facilitates oil accumulation by directly activating WRINKLED1, BIOTIN CARBOXYL CARRIER PROTEIN2, 3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE III, DIACYLGLYCEROL O-ACYLTRANSFERASE1, and OLEOSIN2 in transgenic Arabidopsis (Arabidopsis thaliana) seeds. Overexpression of GmZF351 in transgenic soybean also activates lipid biosynthesis genes, thereby accelerating seed oil accumulation. The ZF351 haplotype from the cultivated soybean group and the wild soybean (Glycine soja) subgroup III correlates well with high gene expression level, seed oil contents and promoter activity, suggesting that selection of GmZF351 expression leads to increased seed oil content in cultivated soybean. Our study provides novel insights into the regulatory mechanism for seed oil accumulation, and the manipulation of GmZF351 may have great potential in the improvement of oil production in soybean and other related crops. PMID:28184009

  10. Eukaryotic translation initiator protein 1A isoform, CCS-3, enhances the transcriptional repression of p21CIP1 by proto-oncogene FBI-1 (Pokemon/ZBTB7A).

    Science.gov (United States)

    Choi, Won-Il; Kim, Youngsoo; Kim, Yuri; Yu, Mi-young; Park, Jungeun; Lee, Choong-Eun; Jeon, Bu-Nam; Koh, Dong-In; Hur, Man-Wook

    2009-01-01

    FBI-1, a member of the POK (POZ and Kruppel) family of transcription factors, plays a role in differentiation, oncogenesis, and adipogenesis. eEF1A is a eukaryotic translation elongation factor involved in several cellular processes including embryogenesis, oncogenic transformation, cell proliferation, and cytoskeletal organization. CCS-3, a potential cervical cancer suppressor, is an isoform of eEF1A. We found that eEF1A forms a complex with FBI-1 by co-immunoprecipitation, SDS-PAGE, and MALDI-TOF Mass analysis of the immunoprecipitate. GST fusion protein pull-downs showed that FBI-1 directly interacts with eEF1A and CCS-3 via the zinc finger and POZ-domain of FBI-1. FBI-1 co-localizes with either eEF1A or CCS-3 at the nuclear periplasm. CCS-3 enhances transcriptional repression of the p21CIP1 gene (hereafter referred to as p21) by FBI-1. The POZ-domain of FBI-1 interacts with the co-repressors, SMRT and BCoR. We found that CCS-3 also interacts with the co-repressors independently. The molecular interaction between the co-repressors and CCS-3 at the POZ-domain of FBI-1 appears to enhance FBI-1 mediated transcriptional repression. Our data suggest that CCS-3 may be important in cell differentiation, tumorigenesis, and oncogenesis by interacting with the proto-oncogene FBI-1 and transcriptional co-repressors. Copyright 2009 S. Karger AG, Basel.

  11. NAC transcription factors: structurally distinct, functionally diverse

    DEFF Research Database (Denmark)

    Olsen, Addie Nina; Ernst, Heidi A; Leggio, Leila Lo

    2005-01-01

    level and localization, and to the first indications of NAC participation in transcription factor networks. The recent determination of the DNA and protein binding NAC domain structure offers insight into the molecular functions of the protein family. Research into NAC transcription factors has......NAC proteins constitute one of the largest families of plant-specific transcription factors, and the family is present in a wide range of land plants. Here, we summarize the biological and molecular functions of the NAC family, paying particular attention to the intricate regulation of NAC protein...

  12. Structural basis for ubiquitin recognition by ubiquitin-binding zinc finger of FAAP20.

    Directory of Open Access Journals (Sweden)

    Aya Toma

    Full Text Available Several ubiquitin-binding zinc fingers (UBZs have been reported to preferentially bind K63-linked ubiquitin chains. In particular, the UBZ domain of FAAP20 (FAAP20-UBZ, a member of the Fanconi anemia core complex, seems to recognize K63-linked ubiquitin chains, in order to recruit the complex to DNA interstrand crosslinks and mediate DNA repair. By contrast, it is reported that the attachment of a single ubiquitin to Rev1, a translesion DNA polymerase, increases binding of Rev1 to FAAP20. To clarify the specificity of FAAP20-UBZ, we determined the crystal structure of FAAP20-UBZ in complex with K63-linked diubiquitin at 1.9 Å resolution. In this structure, FAAP20-UBZ interacts only with one of the two ubiquitin moieties. Consistently, binding assays using surface plasmon resonance spectrometry showed that FAAP20-UBZ binds ubiquitin and M1-, K48- and K63-linked diubiquitin chains with similar affinities. Residues in the vicinity of Ala168 within the α-helix and the C-terminal Trp180 interact with the canonical Ile44-centered hydrophobic patch of ubiquitin. Asp164 within the α-helix and the C-terminal loop mediate a hydrogen bond network, which reinforces ubiquitin-binding of FAAP20-UBZ. Mutations of the ubiquitin-interacting residues disrupted binding to ubiquitin in vitro and abolished the accumulation of FAAP20 to DNA damage sites in vivo. Finally, structural comparison among FAAP20-UBZ, WRNIP1-UBZ and RAD18-UBZ revealed distinct modes of ubiquitin binding. UBZ family proteins could be divided into at least three classes, according to their ubiquitin-binding modes.

  13. A transcription factor active on the epidermal growth factor receptor gene

    International Nuclear Information System (INIS)

    Kageyama, R.; Merlino, G.T.; Pastan, I.

    1988-01-01

    The authors have developed an in vitro transcription system for the epidermal growth factor receptor (EGFR) oncogene by using nuclear extracts of A431 human epidermoid carcinoma cells, which overproduce EGFR. They found that a nuclear factor, termed EGFR-specific transcription factor (ETF), specifically stimulated EGFR transcription by 5- to 10-fold. In this report, ETF, purified by using sequence-specific oligonucleotide affinity chromatography, is shown by renaturing material eluted from a NaDodSO 4 /polyacrylamide gel to be a protein with a molecular mass of 120 kDa. ETF binds to the promoter region, as measured by DNase I footprinting and gel-mobility-shift assays, and specifically stimulates the transcription of the EGFR gene in a reconstituted in vitro transcription system. These results suggest that ETF could play a role in the overexpression of the cellular oncogene EGFR

  14. Study on differential transcriptional profile in human hepatocyte exposed to different doses γ ray

    International Nuclear Information System (INIS)

    Li Jianguo; Wen Jianhua; Duan Zhikai; Tian Yu; Wang Fang; Zuo Yahui

    2009-01-01

    The study analyzed the differential transcriptional profile of normal human hepatic cell and human hepatic cell radiated with three different doses (0.5 Gy, 2 Gy, 4 Gy γ ray) by gene chip technique. The results showed that the whole differentially expressed genes of three different doses have 284 in 14112 human genes analyzed, in which 261 genes were up-regulated and 23 genes were down-regulated. These genes are mainly associated with interferon receptor, mitochondrial regulation, homo sapiens hepatitis A virus cellular receptor, cell cycle regulation, kinase and zinc finger protein etc. RT-PCR results indicated that up-regulated expression of gene HAVcr-1, HAVcr-2, MFTC, MOAP1 and down-regulated expression of gene TRIP12, DCN are consistent with gene chip data. (authors)

  15. Dynamic transcriptomic profiles of zebrafish gills in response to zinc supplementation

    Directory of Open Access Journals (Sweden)

    Cunningham Phil

    2010-10-01

    Full Text Available Abstract Background Dietary zinc supplementation may help to promote growth, boost the immune system, protect against diabetes, and aid recovery from diarrhoea. We exploited the zebrafish (Danio rerio gill as a unique vertebrate ion transporting epithelium model to study the time-dependent regulatory networks of gene-expression leading to homeostatic control during zinc supplementation. This organ forms a conduit for zinc uptake whilst exhibiting conservation of zinc trafficking components. Results Fish were maintained with either zinc supplemented water (4.0 μM and diet (2023 mg zinc kg-1 or water and diet containing Zn2+ at 0.25 μM and 233 mg zinc kg-1, respectively. Gill tissues were harvested at five time points (8 hours to 14 days and transcriptome changes analysed in quintuplicate using a 16 K microarray with results anchored to gill Zn2+ influx and whole body nutrient composition (protein, carbohydrate, lipid, elements. The number of regulated genes increased up to day 7 but declined as the fish acclimated. In total 525 genes were regulated (having a fold-change more than 1.8 fold change and an adjusted P-value less than 0.1 which is controlling a 10% False discovery rate, FDR by zinc supplementation, but little overlap was observed between genes regulated at successive time-points. Many genes displayed cyclic expression, typical for homeostatic control mechanisms. Annotation enrichment analysis revealed strong overrepresentation of "transcription factors", with specific association evident with "steroid hormone receptors". A suite of genes linked to "development" were also statistically overrepresented. More specifically, early regulation of genes was linked to a few key transcription factors (e.g. Mtf1, Jun, Stat1, Ppara, Gata3 and was followed by hedgehog and bone morphogenic protein signalling. Conclusions The results suggest that zinc supplementation reactivated developmental pathways in the gill and stimulated stem cell

  16. Efficient methods for targeted mutagenesis in zebrafish using zinc-finger nucleases: data from targeting of nine genes using CompoZr or CoDA ZFNs.

    Directory of Open Access Journals (Sweden)

    Raman Sood

    Full Text Available Recently, it has been shown that targeted mutagenesis using zinc-finger nucleases (ZFNs and transcription activator-like effector nucleases (TALENs can be used to generate knockout zebrafish lines for analysis of their function and/or developing disease models. A number of different methods have been developed for the design and assembly of gene-specific ZFNs and TALENs, making them easily available to most zebrafish researchers. Regardless of the choice of targeting nuclease, the process of generating mutant fish is similar. It is a time-consuming and multi-step process that can benefit significantly from development of efficient high throughput methods. In this study, we used ZFNs assembled through either the CompoZr (Sigma-Aldrich or the CoDA (context-dependent assembly platforms to generate mutant zebrafish for nine genes. We report our improved high throughput methods for 1 evaluation of ZFNs activity by somatic lesion analysis using colony PCR, eliminating the need for plasmid DNA extractions from a large number of clones, and 2 a sensitive founder screening strategy using fluorescent PCR with PIG-tailed primers that eliminates the stutter bands and accurately identifies even single nucleotide insertions and deletions. Using these protocols, we have generated multiple mutant alleles for seven genes, five of which were targeted with CompoZr ZFNs and two with CoDA ZFNs. Our data also revealed that at least five-fold higher mRNA dose was required to achieve mutagenesis with CoDA ZFNs than with CompoZr ZFNs, and their somatic lesion frequency was lower (<5% when compared to CopmoZr ZFNs (9-98%. This work provides high throughput protocols for efficient generation of zebrafish mutants using ZFNs and TALENs.

  17. Two C3H Type Zinc Finger Protein Genes, CpCZF1 and CpCZF2, from Chimonanthus praecox Affect Stamen Development in Arabidopsis

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    Huamin Liu

    2017-08-01

    Full Text Available Wintersweet (Chimonanthus praecox is a popular garden plant because of its flowering time, sweet fragrance, and ornamental value. However, research into the molecular mechanism that regulates flower development in wintersweet is still limited. In this study, we sought to investigate the molecular characteristics, expression patterns, and potential functions of two C3H-type zinc finger (CZF protein genes, CpCZF1 and CpCZF2, which were isolated from the wintersweet flowers based on the flower developmental transcriptome database. CpCZF1 and CpCZF2 were more highly expressed in flower organs than in vegetative tissues, and during the flower development, their expression profiles were associated with flower primordial differentiation, especially that of petal and stamen primordial differentiation. Overexpression of either CpCZF1 or CpCZF2 caused alterations on stamens in transgenic Arabidopsis. The expression levels of the stamen identity-related genes, such as AGAMOUS (AG, PISTILLATA (PI, SEPALLATA1 (SEP1, SEPALLATA2 (SEP2, SEPALLATA3 (SEP3, APETALA1 (AP1, APETALA2 (AP2, and boundary gene RABBIT EAR (RBE were significantly up-regulated in CpCZF1 overexpression lines. Additionally, the transcripts of AG, PI, APETALA3 SEP1-3, AP1, and RBE were markedly increased in CpCZF2 overexpressed plant inflorescences. Moreover, CpCZF1 and CpCZF2 could interact with each other by using yeast two-hybrid and bimolecular fluorescence complementation assays. Our results suggest that CpCZF1 and CpCZF2 may be involved in the regulation of stamen development and cause the formation of abnormal flowers in transgenic Arabidopsis plants.

  18. Alterations in protein kinase C activity and processing during zinc-deficiency-induced cell death.

    Science.gov (United States)

    Chou, Susan S; Clegg, Michael S; Momma, Tony Y; Niles, Brad J; Duffy, Jodie Y; Daston, George P; Keen, Carl L

    2004-10-01

    Protein kinases C (PKCs) are a family of serine/threonine kinases that are critical for signal transduction pathways involved in growth, differentiation and cell death. All PKC isoforms have four conserved domains, C1-C4. The C1 domain contains cysteine-rich finger-like motifs, which bind two zinc atoms. The zinc-finger motifs modulate diacylglycerol binding; thus, intracellular zinc concentrations could influence the activity and localization of PKC family members. 3T3 cells were cultured in zinc-deficient or zinc-supplemented medium for up to 32 h. Cells cultured in zinc-deficient medium had decreased zinc content, lowered cytosolic classical PKC activity, increased caspase-3 processing and activity, and reduced cell number. Zinc-deficient cytosols had decreased activity and expression levels of PKC-alpha, whereas PKC-alpha phosphorylation was not altered. Inhibition of PKC-alpha with Gö6976 had no effect on cell number in the zinc-deficient group. Proteolysis of the novel PKC family member, PKC-delta, to its 40-kDa catalytic fragment occurred in cells cultured in the zinc-deficient medium. Occurrence of the PKC-delta fragment in mitochondria was co-incident with caspase-3 activation. Addition of the PKC-delta inhibitor, rottlerin, or zinc to deficient medium reduced or eliminated proteolysis of PKC-delta, activated caspase-3 and restored cell number. Inhibition of caspase-3 processing by Z-DQMD-FMK (Z-Asp-Gln-Met-Asp-fluoromethylketone) did not restore cell number in the zinc-deficient group, but resulted in processing of full-length PKC-delta to a 56-kDa fragment. These results support the concept that intracellular zinc concentrations influence PKC activity and processing, and that zinc-deficiency-induced apoptosis occurs in part through PKC-dependent pathways.

  19. Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage.

    Science.gov (United States)

    Takagi, Yuichiro; Masuda, Claudio A; Chang, Wei-Hau; Komori, Hirofumi; Wang, Dong; Hunter, Tony; Joazeiro, Claudio A P; Kornberg, Roger D

    2005-04-15

    Core transcription factor (TF) IIH purified from yeast possesses an E3 ubiquitin (Ub) ligase activity, which resides, at least in part, in a RING finger (RNF) domain of the Ssl1 subunit. Yeast strains mutated in the Ssl1 RNF domain are sensitive to ultraviolet (UV) light and to methyl methanesulfonate (MMS). This increased sensitivity to DNA-damaging agents does not reflect a deficiency in nucleotide excision repair. Rather, it correlates with reduced transcriptional induction of genes involved in DNA repair, suggesting that the E3 Ub ligase activity of TFIIH mediates the transcriptional response to DNA damage.

  20. The conserved basic residues and the charged amino acid residues at the α-helix of the zinc finger motif regulate the nuclear transport activity of triple C2H2 zinc finger proteins

    Science.gov (United States)

    Lin, Chih-Ying

    2018-01-01

    Zinc finger (ZF) motifs on proteins are frequently recognized as a structure for DNA binding. Accumulated reports indicate that ZF motifs contain nuclear localization signal (NLS) to facilitate the transport of ZF proteins into nucleus. We investigated the critical factors that facilitate the nuclear transport of triple C2H2 ZF proteins. Three conserved basic residues (hot spots) were identified among the ZF sequences of triple C2H2 ZF proteins that reportedly have NLS function. Additional basic residues can be found on the α-helix of the ZFs. Using the ZF domain (ZFD) of Egr-1 as a template, various mutants were constructed and expressed in cells. The nuclear transport activity of various mutants was estimated by analyzing the proportion of protein localized in the nucleus. Mutation at any hot spot of the Egr-1 ZFs reduced the nuclear transport activity. Changes of the basic residues at the α-helical region of the second ZF (ZF2) of the Egr-1 ZFD abolished the NLS activity. However, this activity can be restored by substituting the acidic residues at the homologous positions of ZF1 or ZF3 with basic residues. The restored activity dropped again when the hot spots at ZF1 or the basic residues in the α-helix of ZF3 were mutated. The variations in nuclear transport activity are linked directly to the binding activity of the ZF proteins with importins. This study was extended to other triple C2H2 ZF proteins. SP1 and KLF families, similar to Egr-1, have charged amino acid residues at the second (α2) and the third (α3) positions of the α-helix. Replacing the amino acids at α2 and α3 with acidic residues reduced the NLS activity of the SP1 and KLF6 ZFD. The reduced activity can be restored by substituting the α3 with histidine at any SP1 and KLF6 ZFD. The results show again the interchangeable role of ZFs and charge residues in the α-helix in regulating the NLS activity of triple C2H2 ZF proteins. PMID:29381770

  1. Zinc Deficiency Induces Apoptosis via Mitochondrial p53- and Caspase-Dependent Pathways in Human Neuronal Precursor Cells

    Science.gov (United States)

    Seth, Rohit; Corniola, Rikki S.; Gower-Winter, Shannon D.; Morgan, Thomas J., Jr.; Bishop, Brian; Levenson, Cathy W.

    2015-01-01

    Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent…

  2. Differential sensitivities of cellular XPA and PARP-1 to arsenite inhibition and zinc rescue.

    Science.gov (United States)

    Ding, Xiaofeng; Zhou, Xixi; Cooper, Karen L; Huestis, Juliana; Hudson, Laurie G; Liu, Ke Jian

    2017-09-15

    Arsenite directly binds to the zinc finger domains of the DNA repair protein poly (ADP ribose) polymerase (PARP)-1, and inhibits PARP-1 activity in the base excision repair (BER) pathway. PARP inhibition by arsenite enhances ultraviolet radiation (UVR)-induced DNA damage in keratinocytes, and the increase in DNA damage is reduced by zinc supplementation. However, little is known about the effects of arsenite and zinc on the zinc finger nucleotide excision repair (NER) protein xeroderma pigmentosum group A (XPA). In this study, we investigated the difference in response to arsenite exposure between XPA and PARP-1, and the differential effectiveness of zinc supplementation in restoring protein DNA binding and DNA damage repair. Arsenite targeted both XPA and PARP-1 in human keratinocytes, resulting in zinc loss from each protein and a pronounced decrease in XPA and PARP-1 binding to chromatin as demonstrated by Chip-on-Western assays. Zinc effectively restored DNA binding of PARP-1 and XPA to chromatin when zinc concentrations were equal to those of arsenite. In contrast, zinc was more effective in rescuing arsenite-augmented direct UVR-induced DNA damage than oxidative DNA damage. Taken together, our findings indicate that arsenite interferes with PARP-1 and XPA binding to chromatin, and that zinc supplementation fully restores DNA binding activity to both proteins in the cellular context. Interestingly, rescue of arsenite-inhibited DNA damage repair by supplemental zinc was more sensitive for DNA damage repaired by the XPA-associated NER pathway than for the PARP-1-dependent BER pathway. This study expands our understanding of arsenite's role in DNA repair inhibition and co-carcinogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Specific recognition of linear polyubiquitin by A20 zinc finger 7 is involved in NF-κB regulation

    Science.gov (United States)

    Tokunaga, Fuminori; Nishimasu, Hiroshi; Ishitani, Ryuichiro; Goto, Eiji; Noguchi, Takuya; Mio, Kazuhiro; Kamei, Kiyoko; Ma, Averil; Iwai, Kazuhiro; Nureki, Osamu

    2012-01-01

    LUBAC (linear ubiquitin chain assembly complex) activates the canonical NF-κB pathway through linear polyubiquitination of NEMO (NF-κB essential modulator, also known as IKKγ) and RIP1. However, the regulatory mechanism of LUBAC-mediated NF-κB activation remains elusive. Here, we show that A20 suppresses LUBAC-mediated NF-κB activation by binding linear polyubiquitin via the C-terminal seventh zinc finger (ZF7), whereas CYLD suppresses it through deubiquitinase (DUB) activity. We determined the crystal structures of A20 ZF7 in complex with linear diubiquitin at 1.70–1.98 Å resolutions. The crystal structures revealed that A20 ZF7 simultaneously recognizes the Met1-linked proximal and distal ubiquitins, and that genetic mutations associated with B cell lymphomas map to the ubiquitin-binding sites. Our functional analysis indicated that the binding of A20 ZF7 to linear polyubiquitin contributes to the recruitment of A20 into a TNF receptor (TNFR) signalling complex containing LUBAC and IκB kinase (IKK), which results in NF-κB suppression. These findings provide new insight into the regulation of immune and inflammatory responses. PMID:23032187

  4. Neuronal migration is regulated by endogenous RNAi and chromatin-binding factor ZFP-1/AF10 in Caenorhabditis elegans.

    Science.gov (United States)

    Kennedy, Lisa M; Grishok, Alla

    2014-05-01

    Endogenous short RNAs and the conserved plant homeodomain (PHD) zinc-finger protein ZFP-1/AF10 regulate overlapping sets of genes in Caenorhabditis elegans, which suggests that they control common biological pathways. We have shown recently that the RNAi factor RDE-4 and ZFP-1 negatively modulate transcription of the insulin/PI3 signaling-dependent kinase PDK-1 to promote C. elegans fitness. Moreover, we have demonstrated that the insulin/IGF-1-PI3K-signaling pathway regulates the activity of the DAF-16/FOXO transcription factor in the hypodermis to nonautonomously promote the anterior migrations of the hermaphrodite-specific neurons (HSNs) during embryogenesis of C. elegans. In this study, we implicate the PHD-containing isoform of ZFP-1 and endogenous RNAi in the regulation of HSN migration. ZFP-1 affects HSN migration in part through its negative effect on pdk-1 transcription and modulation of downstream DAF-16 activity. We also identify a novel role for ZFP-1 and RNAi pathway components, including RDE-4, in the regulation of HSN migration in parallel with DAF-16. Therefore, the coordinated activities of DAF-16, ZFP-1, and endogenous RNAi contribute to gene regulation during development to ensure proper neuronal positioning.

  5. Crystal optimization and preliminary diffraction data analysis of the SCAN domain of Zfp206

    International Nuclear Information System (INIS)

    Liang, Yu; Choo, Siew Hua; Rossbach, Michael; Baburajendran, Nithya; Palasingam, Paaventhan; Kolatkar, Prasanna R

    2012-01-01

    Crystals of the SCAN domain of Zfp206 are tetragonal, belonging to space group I422 with unit-cell parameters a = 67.57, c = 87.54 Å and one molecule in the asymmetric unit, and diffract to 1.85 Å resolution. Zfp206 (also named Zscan10) is a transcription factor that plays an important role in maintaining the pluripotent state of embryonic stem cells. Zfp206 is a member of the SCAN-domain family of C 2 H 2 zinc-finger transcription factors. The SCAN domain is a highly conserved motif of 84 residues which mediates the self-association of and heterodimerization between SCAN-domain family transcription factors. The SCAN domain may therefore be the key to the selective oligomerization of and may combinatorially enhance the regulatory versatility of C 2 H 2 zinc fingers. This paper describes crystallization attempts with the SCAN domain of Zfp206 (Zfp206SCAN) and optimization strategies to obtain diffraction-quality crystals. The best diffracting crystal was grown in a solution consisting of 0.3 M ammonium sulfate, 0.1 M Tris–HCl pH 8.6, 25% PEG 3350, 0.1 M ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) using the hanging-drop vapour-diffusion technique. Optimized crystals diffracted to 1.85 Å resolution and belonged to space group I422, with unit-cell parameters a = 67.57, c = 87.54 Å. A Matthews analysis indicated the presence of one Zfp206SCAN molecule per asymmetric unit

  6. Transcriptional Activation Domains of the Candida albicans Gcn4p and Gal4p Homologs▿ †

    OpenAIRE

    Martchenko, Mikhail; Levitin, Anastasia; Whiteway, Malcolm

    2006-01-01

    Many putative transcription factors in the pathogenic fungus Candida albicans contain sequence similarity to well-defined transcriptional regulators in the budding yeast Saccharomyces cerevisiae, but this sequence similarity is often limited to the DNA binding domains of the molecules. The Gcn4p and Gal4p proteins of Saccharomyces cerevisiae are highly studied and well-understood eukaryotic transcription factors of the basic leucine zipper (Gcn4p) and C6 zinc cluster (Gal4p) families; C. albi...

  7. DNA residence time is a regulatory factor of transcription repression

    Science.gov (United States)

    Clauß, Karen; Popp, Achim P.; Schulze, Lena; Hettich, Johannes; Reisser, Matthias; Escoter Torres, Laura; Uhlenhaut, N. Henriette

    2017-01-01

    Abstract Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation. PMID:28977492

  8. Identification and characterization of finger millet OPAQUE2 transcription factor gene under different nitrogen inputs for understanding their role during accumulation of prolamin seed storage protein.

    Science.gov (United States)

    Gaur, Vikram Singh; Kumar, Lallan; Gupta, Supriya; Jaiswal, J P; Pandey, Dinesh; Kumar, Anil

    2018-03-01

    In this study, we report the isolation and characterization of the mRNA encoding OPAQUE2 (O2) like TF of finger millet (FM) ( Eleusine coracana) ( EcO2 ). Full-length EcO2 mRNA was isolated using conserved primers designed by aligning O2 mRNAs of different cereals followed by 3' and 5' RACE (Rapid Amplification of cDNA Ends). The assembled full-length EcO2 mRNA was found to contain an ORF of 1248-nt coding the 416 amino acids O2 protein. Domain analysis revealed the presence of the BLZ and bZIP-C domains which is a characteristic feature of O2 proteins. Phylogenetic analysis of EcO2 protein with other bZIP proteins identified using finger millet transcriptome data and O2 proteins of other cereals showed that EcO2 shared high sequence similarity with barley BLZ1 protein. Transcripts of EcO2 were detected in root, stem, leaves, and seed development stages. Furthermore, to investigate nitrogen responsiveness and the role of EcO2 in regulating seed storage protein gene expression, the expression profiles of EcO2 along with an α-prolamin gene were studied during the seed development stages of two FM genotypes (GE-3885 and GE-1437) differing in grain protein content (13.8 and 6.2%, respectively) grown under increasing nitrogen inputs. Compared to GE-1437, the EcO2 was relatively highly expressed during the S2 stage of seed development which further increased as nitrogen input was increased. The Ecα - prolamin gene was strongly induced in the high protein genotype (GE-3885) at all nitrogen inputs. These results indicate the presence of nitrogen responsiveness regulatory elements which might play an important role in accumulating protein in FM genotypes through modulating EcO2 expression by sensing plant nitrogen status.

  9. C3HC4-type RING finger protein NbZFP1 is involved in growth and fruit development in Nicotiana benthamiana.

    Directory of Open Access Journals (Sweden)

    Wenxian Wu

    Full Text Available C3HC4-type RING finger proteins constitute a large family in the plant kingdom and play important roles in various physiological processes of plant life. In this study, a C3HC4-type zinc finger gene was isolated from Nicotiana benthamiana. Sequence analysis indicated that the gene encodes a 24-kDa protein with 191 amino acids containing one typical C3HC4-type zinc finger domain; this gene was named NbZFP1. Transient expression of pGDG-NbZFP1 demonstrated that NbZFP1 was localized to the chloroplast, especially in the chloroplasts of cells surrounding leaf stomata. Virus-induced gene silencing (VIGS analysis indicated that silencing of NbZFP1 hampered fruit development, although the height of the plants was normal. An overexpression construct was then designed and transferred into Nicotiana benthamiana, and PCR and Southern blot showed that the NbZFP1 gene was successfully integrated into the Nicotiana benthamiana genome. The transgenic lines showed typical compactness, with a short internode length and sturdy stems. This is the first report describing the function of a C3HC4-type RING finger protein in tobacco.

  10. A novel human AP endonuclease with conserved zinc-finger-like motifs involved in DNA strand break responses

    Science.gov (United States)

    Kanno, Shin-ichiro; Kuzuoka, Hiroyuki; Sasao, Shigeru; Hong, Zehui; Lan, Li; Nakajima, Satoshi; Yasui, Akira

    2007-01-01

    DNA damage causes genome instability and cell death, but many of the cellular responses to DNA damage still remain elusive. We here report a human protein, PALF (PNK and APTX-like FHA protein), with an FHA (forkhead-associated) domain and novel zinc-finger-like CYR (cysteine–tyrosine–arginine) motifs that are involved in responses to DNA damage. We found that the CYR motif is widely distributed among DNA repair proteins of higher eukaryotes, and that PALF, as well as a Drosophila protein with tandem CYR motifs, has endo- and exonuclease activities against abasic site and other types of base damage. PALF accumulates rapidly at single-strand breaks in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner in human cells. Indeed, PALF interacts directly with PARP1 and is required for its activation and for cellular resistance to methyl-methane sulfonate. PALF also interacts directly with KU86, LIGASEIV and phosphorylated XRCC4 proteins and possesses endo/exonuclease activity at protruding DNA ends. Various treatments that produce double-strand breaks induce formation of PALF foci, which fully coincide with γH2AX foci. Thus, PALF and the CYR motif may play important roles in DNA repair of higher eukaryotes. PMID:17396150

  11. Zinc finger nuclease: a new approach for excising HIV-1 proviral DNA from infected human T cells.

    Science.gov (United States)

    Qu, Xiying; Wang, Pengfei; Ding, Donglin; Wang, Xiaohui; Zhang, Gongmin; Zhou, Xin; Liu, Lin; Zhu, Xiaoli; Zeng, Hanxian; Zhu, Huanzhang

    2014-09-01

    A major reason that Acquired Immune Deficiency Syndrome (AIDS) cannot be completely cured is the human immunodeficiency virus 1 (HIV-1) provirus integrated into the human genome. Though existing therapies can inhibit replication of HIV-1, they cannot eradicate it. A molecular therapy gains popularity due to its specifically targeting to HIV-1 infected cells and effectively removing the HIV-1, regardless of viral genes being active or dormant. Now, we propose a new method which can excellently delete the HIV provirus from the infected human T cell genome. First, we designed zinc-finger nucleases (ZFNs) that target a sequence within the long terminal repeat (LTR) U3 region that is highly conserved in whole clade. Then, we screened out one pair of ZFN and named it as ZFN-U3. We discovered that ZFN-U3 can exactly target and eliminate the full-length HIV-1 proviral DNA after the infected human cell lines treated with it, and the frequency of its excision was about 30 % without cytotoxicity. These results prove that ZFN-U3 can efficiently excise integrated HIV-1 from the human genome in infected cells. This method to delete full length HIV-1 in human genome can therefore provide a novel approach to cure HIV-infected individuals in the future.

  12. Runx transcription factors in neuronal development

    Directory of Open Access Journals (Sweden)

    Shiga Takashi

    2008-08-01

    Full Text Available Abstract Runt-related (Runx transcription factors control diverse aspects of embryonic development and are responsible for the pathogenesis of many human diseases. In recent years, the functions of this transcription factor family in the nervous system have just begun to be understood. In dorsal root ganglion neurons, Runx1 and Runx3 play pivotal roles in the development of nociceptive and proprioceptive sensory neurons, respectively. Runx appears to control the transcriptional regulation of neurotrophin receptors, numerous ion channels and neuropeptides. As a consequence, Runx contributes to diverse aspects of the sensory system in higher vertebrates. In this review, we summarize recent progress in determining the role of Runx in neuronal development.

  13. Different Binding Properties and Function of CXXC Zinc Finger Domains in Dnmt1 and Tet1

    Science.gov (United States)

    Meilinger, Daniela; Bultmann, Sebastian; Fellinger, Karin; Hasenöder, Stefan; Wang, Mengxi; Qin, Weihua; Söding, Johannes; Spada, Fabio; Leonhardt, Heinrich

    2011-01-01

    Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1−/− embryonic stem cells (ESCs) just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1−/− ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites. PMID:21311766

  14. Different binding properties and function of CXXC zinc finger domains in Dnmt1 and Tet1.

    Directory of Open Access Journals (Sweden)

    Carina Frauer

    2011-02-01

    Full Text Available Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1⁻/⁻ embryonic stem cells (ESCs just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1⁻/⁻ ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites.

  15. Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID using zinc-finger nucleases.

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    Tomoji Mashimo

    Full Text Available BACKGROUND: Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc. METHODOLOGY/PRINCIPAL FINDINGS: We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID. Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype. CONCLUSIONS AND SIGNIFICANCE: The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.

  16. A Finger Exoskeleton Robot for Finger Movement Rehabilitation

    Directory of Open Access Journals (Sweden)

    Tzu-Heng Hsu

    2017-07-01

    Full Text Available In this study, a finger exoskeleton robot has been designed and presented. The prototype device was designed to be worn on the dorsal side of the hand to assist in the movement and rehabilitation of the fingers. The finger exoskeleton is 3D-printed to be low-cost and has a transmission mechanism consisting of rigid serial links which is actuated by a stepper motor. The actuation of the robotic finger is by a sliding motion and mimics the movement of the human finger. To make it possible for the patient to use the rehabilitation device anywhere and anytime, an Arduino™ control board and a speech recognition board were used to allow voice control. As the robotic finger follows the patients voice commands the actual motion is analyzed by Tracker image analysis software. The finger exoskeleton is designed to flex and extend the fingers, and has a rotation range of motion (ROM of 44.2°.

  17. Potential Role of Activating Transcription Factor 5 during Osteogenesis

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    Luisa Vicari

    2016-01-01

    Full Text Available Human adipose-derived stem cells are an abundant population of stem cells readily isolated from human adipose tissue that can differentiate into connective tissue lineages including bone, cartilage, fat, and muscle. Activating transcription factor 5 is a transcription factor of the ATF/cAMP response element-binding protein (CREB family. It is transcribed in two types of mRNAs (activating transcription factor 5 isoform 1 and activating transcription factor 5 isoform 2, encoding the same single 30-kDa protein. Although it is well demonstrated that it regulates the proliferation, differentiation, and apoptosis, little is known about its potential role in osteogenic differentiation. The aim of this study was to evaluate the expression levels of the two isoforms and protein during osteogenic differentiation of human adipose-derived stem cells. Our data indicate that activating transcription factor 5 is differentially expressed reaching a peak of expression at the stage of bone mineralization. These findings suggest that activating transcription factor 5 could play an interesting regulatory role during osteogenesis, which would provide a powerful tool to study bone physiology.

  18. Potential Role of Activating Transcription Factor 5 during Osteogenesis.

    Science.gov (United States)

    Vicari, Luisa; Calabrese, Giovanna; Forte, Stefano; Giuffrida, Raffaella; Colarossi, Cristina; Parrinello, Nunziatina Laura; Memeo, Lorenzo

    2016-01-01

    Human adipose-derived stem cells are an abundant population of stem cells readily isolated from human adipose tissue that can differentiate into connective tissue lineages including bone, cartilage, fat, and muscle. Activating transcription factor 5 is a transcription factor of the ATF/cAMP response element-binding protein (CREB) family. It is transcribed in two types of mRNAs (activating transcription factor 5 isoform 1 and activating transcription factor 5 isoform 2), encoding the same single 30-kDa protein. Although it is well demonstrated that it regulates the proliferation, differentiation, and apoptosis, little is known about its potential role in osteogenic differentiation. The aim of this study was to evaluate the expression levels of the two isoforms and protein during osteogenic differentiation of human adipose-derived stem cells. Our data indicate that activating transcription factor 5 is differentially expressed reaching a peak of expression at the stage of bone mineralization. These findings suggest that activating transcription factor 5 could play an interesting regulatory role during osteogenesis, which would provide a powerful tool to study bone physiology.

  19. Polyphenol Compound as a Transcription Factor Inhibitor.

    Science.gov (United States)

    Park, Seyeon

    2015-10-30

    A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor-DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein-protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1), c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and β-catenin/T cell factor (Tcf)).

  20. SoyDB: a knowledge database of soybean transcription factors

    Directory of Open Access Journals (Sweden)

    Valliyodan Babu

    2010-01-01

    Full Text Available Abstract Background Transcription factors play the crucial rule of regulating gene expression and influence almost all biological processes. Systematically identifying and annotating transcription factors can greatly aid further understanding their functions and mechanisms. In this article, we present SoyDB, a user friendly database containing comprehensive knowledge of soybean transcription factors. Description The soybean genome was recently sequenced by the Department of Energy-Joint Genome Institute (DOE-JGI and is publicly available. Mining of this sequence identified 5,671 soybean genes as putative transcription factors. These genes were comprehensively annotated as an aid to the soybean research community. We developed SoyDB - a knowledge database for all the transcription factors in the soybean genome. The database contains protein sequences, predicted tertiary structures, putative DNA binding sites, domains, homologous templates in the Protein Data Bank (PDB, protein family classifications, multiple sequence alignments, consensus protein sequence motifs, web logo of each family, and web links to the soybean transcription factor database PlantTFDB, known EST sequences, and other general protein databases including Swiss-Prot, Gene Ontology, KEGG, EMBL, TAIR, InterPro, SMART, PROSITE, NCBI, and Pfam. The database can be accessed via an interactive and convenient web server, which supports full-text search, PSI-BLAST sequence search, database browsing by protein family, and automatic classification of a new protein sequence into one of 64 annotated transcription factor families by hidden Markov models. Conclusions A comprehensive soybean transcription factor database was constructed and made publicly accessible at http://casp.rnet.missouri.edu/soydb/.

  1. Interdependence of free zinc changes and protein complex assembly - insights into zinc signal regulation.

    Science.gov (United States)

    Kocyła, Anna; Adamczyk, Justyna; Krężel, Artur

    2018-01-24

    Cellular zinc (Zn(ii)) is bound with proteins that are part of the proteomes of all domains of life. It is mostly utilized as a catalytic or structural protein cofactor, which results in a vast number of binding architectures. The Zn(ii) ion is also important for the formation of transient protein complexes with a Zn(ii)-dependent quaternary structure that is formed upon cellular zinc signals. The mechanisms by which proteins associate with and dissociate from Zn(ii) and the connection with cellular Zn(ii) changes remain incompletely understood. In this study, we aimed to examine how zinc protein domains with various Zn(ii)-binding architectures are formed under free Zn(ii) concentration changes and how formation of the Zn(ii)-dependent assemblies is related to the protein concentration and reactivity. To accomplish these goals we chose four zinc domains with different Zn(ii)-to-protein binding stoichiometries: classical zinc finger (ZnP), LIM domain (Zn 2 P), zinc hook (ZnP 2 ) and zinc clasp (ZnP 1 P 2 ) folds. Our research demonstrated a lack of changes in the saturation level of intraprotein zinc binding sites, despite various peptide concentrations, while homo- and heterodimers indicated a concentration-dependent tendency. In other words, at a certain free Zn(ii) concentration, the fraction of a formed dimeric complex increases or decreases with subunit concentration changes. Secondly, even small or local changes in free Zn(ii) may significantly affect protein saturation depending on its architecture, function and subcellular concentration. In our paper, we indicate the importance of interdependence of free Zn(ii) availability and protein subunit concentrations for cellular zinc signal regulation.

  2. Transcriptomic profiling of Arabidopsis gene expression in response to varying micronutrient zinc supply

    DEFF Research Database (Denmark)

    Azevedo, Herlânder; Azinheiro, Sarah Gaspar; Muñoz-Mérida, Antonio

    2016-01-01

    Deficiency of the micronutrient zinc is a widespread condition in agricultural soils, causing a negative impact on crop quality and yield. Nevertheless, there is an insufficient knowledge on the regulatory and molecular mechanisms underlying the plant response to inadequate zinc nutrition [1......]. This information should contribute to the development of plant-based solutions with improved nutrient-use-efficiency traits in crops. Previously, the transcription factors bZIP19 and bZIP23 were identified as essential regulators of the response to zinc deficiency in Arabidopsis thaliana [2]. A microarray...... experiment comparing gene expression between roots of wild-type and the mutant bzip19 bzip23, exposed to zinc deficiency, led to the identification of differentially expressed genes related with zinc homeostasis, namely its transport and plant internal translocation [2]. Here, we provide the detailed...

  3. Radiation activation of transcription factors in mammalian cells

    International Nuclear Information System (INIS)

    Kraemer, M.; Stein, B.; Mai, S.; Kunz, E.; Koenig, H.; Ponta, H.; Herrlich, P.; Rahmsdorf, H.J.; Loferer, H.; Grunicke, H.H.

    1990-01-01

    In mammalian cells radiation induces the enhanced transcription of several genes. The cis acting elements in the control region of inducible genes have been delimited by site directed mutagenesis. Several different elements have been found in different genes. They do not only activate gene transcription in response to radiation but also in response to growth factors and to tumor promoter phorbol esters. The transcription factors binding to these elements are present also in non-irradiated cells, but their DNA binding activity and their transactivating capability is increased upon irradiation. The signal chain linking the primary radiation induced signal (damaged DNA) to the activation of transcription factors involves the action of (a) protein kinase(s). (orig.)

  4. Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance

    Science.gov (United States)

    Shahbazi Dastjerdeh, Mansoureh; Kouhpayeh, Shirin; Sabzehei, Faezeh; Khanahmad, Hossein; Salehi, Mansour; Mohammadi, Zahra; Shariati, Laleh; Hejazi, Zahra; Rabiei, Parisa; Manian, Mostafa

    2016-01-01

    Background: The evolution of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) has been accelerated recently by the indiscriminate application of antibiotics. Antibiotic resistance has challenged the success of medical interventions and therefore is considered a hazardous threat to human health. Objectives: The present study aimed to describe the use of zinc finger nuclease (ZFN) technology to target and disrupt a plasmid-encoded β-lactamase, which prevents horizontal gene transfer-mediated evolution of ARBs. Materials and Methods: An engineered ZFN was designed to target a specific sequence in the ampicillin resistance gene (ampR) of the pTZ57R plasmid. The Escherichia coli bacteria already contained the pZFN kanamycin-resistant (kanaR) plasmid as the case or the pP15A, kanaR empty vector as the control, were transformed with the pTZ57R; the ability of the designed ZFN to disrupt the β-lactamase gene was evaluated with the subsequent disturbed ability of the bacteria to grow on ampicillin (amp) and ampicillin-kanamycin (amp-kana)-containing media. The effect of mild hypothermia on the ZFN gene targeting efficiency was also evaluated. Results: The growth of bacteria in the case group on the amp and amp-kana-containing media was significantly lower compared with the control group at 37°C (P < 0.001). Despite being more efficient in hypothermic conditions at 30°C (P < 0.001), there were no significant associations between the incubation temperature and the ZFN gene targeting efficiency. Conclusions: Our findings revealed that the ZFN technology could be employed to overcome ampicillin resistance by the targeted disruption of the ampicillin resistance gene, which leads to inactivation of β-lactam synthesis. Therefore, ZFN technology could be engaged to decrease the antibiotic resistance issue with the construction of a ZFN archive against different ARGs. To tackle the resistance issue at the environmental level, recombinant phages

  5. OsDOG, a gibberellin-induced A20/AN1 zinc-finger protein, negatively regulates gibberellin-mediated cell elongation in rice.

    Science.gov (United States)

    Liu, Yaju; Xu, Yunyuan; Xiao, Jun; Ma, Qibin; Li, Dan; Xue, Zhen; Chong, Kang

    2011-07-01

    The A20/AN1 zinc-finger proteins (ZFPs) play pivotal roles in animal immune responses and plant stress responses. From previous gibberellin (GA) microarray data and A20/AN1 ZFP family member association, we chose Oryza sativa dwarf rice with overexpression of gibberellin-induced gene (OsDOG) to examine its function in the GA pathway. OsDOG was induced by gibberellic acid (GA(3)) and repressed by the GA-synthesis inhibitor paclobutrazol. Different transgenic lines with constitutive expression of OsDOG showed dwarf phenotypes due to deficiency of cell elongation. Additional GA(1) and real-time PCR quantitative assay analyses confirmed that the decrease of GA(1) in the overexpression lines resulted from reduced expression of GA3ox2 and enhanced expression of GA2ox1 and GA2ox3. Adding exogenous GA rescued the constitutive expression phenotypes of the transgenic lines. OsDOG has a novel function in regulating GA homeostasis and in negative maintenance of plant cell elongation in rice. Copyright © 2011 Elsevier GmbH. All rights reserved.

  6. Comparison of Transcription Factor Binding Site Models

    KAUST Repository

    Bhuyan, Sharifulislam

    2012-05-01

    Modeling of transcription factor binding sites (TFBSs) and TFBS prediction on genomic sequences are important steps to elucidate transcription regulatory mechanism. Dependency of transcription regulation on a great number of factors such as chemical specificity, molecular structure, genomic and epigenetic characteristics, long distance interaction, makes this a challenging problem. Different experimental procedures generate evidence that DNA-binding domains of transcription factors show considerable DNA sequence specificity. Probabilistic modeling of TFBSs has been moderately successful in identifying patterns from a family of sequences. In this study, we compare performances of different probabilistic models and try to estimate their efficacy over experimental TFBSs data. We build a pipeline to calculate sensitivity and specificity from aligned TFBS sequences for several probabilistic models, such as Markov chains, hidden Markov models, Bayesian networks. Our work, containing relevant statistics and evaluation for the models, can help researchers to choose the most appropriate model for the problem at hand.

  7. Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases.

    Directory of Open Access Journals (Sweden)

    Anna Osiak

    Full Text Available Gene knockout in murine embryonic stem cells (ESCs has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs. Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.

  8. Robust finger vein ROI localization based on flexible segmentation.

    Science.gov (United States)

    Lu, Yu; Xie, Shan Juan; Yoon, Sook; Yang, Jucheng; Park, Dong Sun

    2013-10-24

    Finger veins have been proved to be an effective biometric for personal identification in the recent years. However, finger vein images are easily affected by influences such as image translation, orientation, scale, scattering, finger structure, complicated background, uneven illumination, and collection posture. All these factors may contribute to inaccurate region of interest (ROI) definition, and so degrade the performance of finger vein identification system. To improve this problem, in this paper, we propose a finger vein ROI localization method that has high effectiveness and robustness against the above factors. The proposed method consists of a set of steps to localize ROIs accurately, namely segmentation, orientation correction, and ROI detection. Accurate finger region segmentation and correct calculated orientation can support each other to produce higher accuracy in localizing ROIs. Extensive experiments have been performed on the finger vein image database, MMCBNU_6000, to verify the robustness of the proposed method. The proposed method shows the segmentation accuracy of 100%. Furthermore, the average processing time of the proposed method is 22 ms for an acquired image, which satisfies the criterion of a real-time finger vein identification system.

  9. Robust Finger Vein ROI Localization Based on Flexible Segmentation

    Directory of Open Access Journals (Sweden)

    Dong Sun Park

    2013-10-01

    Full Text Available Finger veins have been proved to be an effective biometric for personal identification in the recent years. However, finger vein images are easily affected by influences such as image translation, orientation, scale, scattering, finger structure, complicated background, uneven illumination, and collection posture. All these factors may contribute to inaccurate region of interest (ROI definition, and so degrade the performance of finger vein identification system. To improve this problem, in this paper, we propose a finger vein ROI localization method that has high effectiveness and robustness against the above factors. The proposed method consists of a set of steps to localize ROIs accurately, namely segmentation, orientation correction, and ROI detection. Accurate finger region segmentation and correct calculated orientation can support each other to produce higher accuracy in localizing ROIs. Extensive experiments have been performed on the finger vein image database, MMCBNU_6000, to verify the robustness of the proposed method. The proposed method shows the segmentation accuracy of 100%. Furthermore, the average processing time of the proposed method is 22 ms for an acquired image, which satisfies the criterion of a real-time finger vein identification system.

  10. Robust Finger Vein ROI Localization Based on Flexible Segmentation

    Science.gov (United States)

    Lu, Yu; Xie, Shan Juan; Yoon, Sook; Yang, Jucheng; Park, Dong Sun

    2013-01-01

    Finger veins have been proved to be an effective biometric for personal identification in the recent years. However, finger vein images are easily affected by influences such as image translation, orientation, scale, scattering, finger structure, complicated background, uneven illumination, and collection posture. All these factors may contribute to inaccurate region of interest (ROI) definition, and so degrade the performance of finger vein identification system. To improve this problem, in this paper, we propose a finger vein ROI localization method that has high effectiveness and robustness against the above factors. The proposed method consists of a set of steps to localize ROIs accurately, namely segmentation, orientation correction, and ROI detection. Accurate finger region segmentation and correct calculated orientation can support each other to produce higher accuracy in localizing ROIs. Extensive experiments have been performed on the finger vein image database, MMCBNU_6000, to verify the robustness of the proposed method. The proposed method shows the segmentation accuracy of 100%. Furthermore, the average processing time of the proposed method is 22 ms for an acquired image, which satisfies the criterion of a real-time finger vein identification system. PMID:24284769

  11. METALLOTHIONEIN: CLASSIFICATION, BIOCHEMICAL FEATURES AND CLINICAL APPLICATIONS

    Directory of Open Access Journals (Sweden)

    Tooba Naz Shamsi

    2014-03-01

    Full Text Available Metallothionein (MT is a ubiquitous low molecular weight protein with high cysteine content and has strong affinity for heavy metals. MT provides protection against heavy metal toxicity, oxidative stress, and participates in the regulation of physiological metals like zinc (Zn2+ and copper (Cu. Abnormal MT expression and function presumably leads to various diseases like diabetes, cancer and neuro-degenerative diseases. MT gene expression is induced by a high variety of stimuli like metal exposure, oxidative stress, glucocorticoids, hydric stress etc. The level of the response to these inducers depends on the MT gene. These activities are regulated through intracellular metal ion modulation and free radical scavenging. MT participates in the uptake, transport, and regulation of zinc in biological system. It regulates zinc homeostasis by binding and releasing zinc ions which are a key element for the activation and binding of certain transcription factors through its participation in the zinc finger region of the protein. It also seems to be important for the regulation of tumor suppressor protein, p 53. Because MT plays an important role in transcription factor regulation, problems with MT function or expression may lead to malignant transformation of cells and ultimately cancer. There are variou

  12. Polyphenol Compound as a Transcription Factor Inhibitor

    Directory of Open Access Journals (Sweden)

    Seyeon Park

    2015-10-01

    Full Text Available A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor–DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein–protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1, c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB and β-catenin/T cell factor (Tcf.

  13. Proto-oncogene FBI-1 (Pokemon/ZBTB7A) Represses Transcription of the Tumor Suppressor Rb Gene via Binding Competition with Sp1 and Recruitment of Co-repressors*S⃞

    Science.gov (United States)

    Jeon, Bu-Nam; Yoo, Jung-Yoon; Choi, Won-Il; Lee, Choong-Eun; Yoon, Ho-Geun; Hur, Man-Wook

    2008-01-01

    FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at bp –308 to –188 of the Rb promoter region. The Rb promoter also contains two Sp1 binding sites: GC-box 1 (bp –65 to –56) and GC-box 2 (bp –18 to –9), the latter of which is also bound by FBI-1. We found that FRE3 (bp –244 to –236) is also a Sp1 binding element. FBI-1 represses transcription of the Rb gene not only by binding to the FREs, but also by competing with Sp1 at the GC-box 2 and the FRE3. By binding to the FREs and/or the GC-box, FBI-1 represses transcription of the Rb gene through its POZ-domain, which recruits a co-repressor-histone deacetylase complex and deacetylates histones H3 and H4 at the Rb gene promoter. FBI-1 inhibits C2C12 myoblast cell differentiation by repressing Rb gene expression. PMID:18801742

  14. NAC Transcription Factors in Senescence: From Molecular Structure to Function in Crops

    Directory of Open Access Journals (Sweden)

    Dagmara Podzimska-Sroka

    2015-07-01

    Full Text Available Within the last decade, NAC transcription factors have been shown to play essential roles in senescence, which is the focus of this review. Transcriptome analyses associate approximately one third of Arabidopsis NAC genes and many crop NAC genes with senescence, thereby implicating NAC genes as important regulators of the senescence process. The consensus DNA binding site of the NAC domain is used to predict NAC target genes, and protein interaction sites can be predicted for the intrinsically disordered transcription regulatory domains of NAC proteins. The molecular characteristics of these domains determine the interactions in gene regulatory networks. Emerging local NAC-centered gene regulatory networks reveal complex molecular mechanisms of stress- and hormone-regulated senescence and basic physiological steps of the senescence process. For example, through molecular interactions involving the hormone abscisic acid, Arabidopsis NAP promotes chlorophyll degradation, a hallmark of senescence. Furthermore, studies of the functional rice ortholog, OsNAP, suggest that NAC genes can be targeted to obtain specific changes in lifespan control and nutrient remobilization in crop plants. This is also exemplified by the wheat NAM1 genes which promote senescence and increase grain zinc, iron, and protein content. Thus, NAC genes are promising targets for fine-tuning senescence for increased yield and quality.

  15. Gene repair of an Usher syndrome causing mutation by zinc-finger nuclease mediated homologous recombination.

    Science.gov (United States)

    Overlack, Nora; Goldmann, Tobias; Wolfrum, Uwe; Nagel-Wolfrum, Kerstin

    2012-06-26

    Human Usher syndrome (USH) is the most frequent cause of inherited deaf-blindness. It is clinically and genetically heterogeneous, assigned to three clinical types of which the most severe type is USH1. No effective treatment for the ophthalmic component of USH exists. Gene augmentation is an attractive strategy for hereditary retinal diseases. However, several USH genes, like USH1C, are expressed in various isoforms, hampering gene augmentation. As an alternative treatment strategy, we applied the zinc-finger nuclease (ZFN) technology for targeted gene repair of an USH1C, causing mutation by homologous recombination. We designed ZFNs customized for the p.R31X nonsense mutation in Ush1c. We evaluated ZFNs for DNA cleavage capability and analyzed ZFNs biocompatibilities by XTT assays. We demonstrated ZFNs mediated gene repair on genomic level by digestion assays and DNA sequencing, and on protein level by indirect immunofluorescence and Western blot analyses. The specifically designed ZFNs did not show cytotoxic effects in a p.R31X cell line. We demonstrated that ZFN induced cleavage of their target sequence. We showed that simultaneous application of ZFN and rescue DNA induced gene repair of the disease-causing mutation on the genomic level, resulting in recovery of protein expression. In our present study, we analyzed for the first time ZFN-activated gene repair of an USH gene. The data highlight the ability of ZFNs to induce targeted homologous recombination and mediate gene repair in USH. We provide further evidence that the ZFN technology holds great potential to recover disease-causing mutations in inherited retinal disorders.

  16. Analysis and optimization of acoustic wave micro-resonators integrating piezoelectric zinc oxide layers

    Science.gov (United States)

    Mortada, O.; Zahr, A. H.; Orlianges, J.-C.; Crunteanu, A.; Chatras, M.; Blondy, P.

    2017-02-01

    This paper reports on the design, simulation, fabrication, and test results of ZnO-based contour-mode micro-resonators integrating piezoelectric zinc oxide (ZnO) layers. The inter-digitated (IDT) type micro-resonators are fabricated on ZnO films and suspended top of 2 μm thick silicon membranes using silicon-on insulator technology. We analyze several possibilities of increasing the quality factor (Q) and the electromechanical coupling coefficient (kt2) of the devices by varying the numbers and lengths of the IDT electrodes and using different thicknesses of the ZnO layer. We designed and fabricated IDTs of different finger numbers (n = 25, 40, 50, and 80) and lengths (L = 100/130/170/200 μm) for three different thicknesses of ZnO films (200, 600, and 800 nm). The measured Q factor confirms that reducing the length and the number of IDT fingers enables us to reach better electrical performances at resonant frequencies around 700 MHz. The extracted results for an optimized micro-resonator device having an IDT length of 100 μm and 40 finger electrodes show a Q of 1180 and a kt2 of 7.4%. We demonstrate also that the reduction of the ZnO thickness from 800 nm to 200 nm increases the quality factor from 430 to 1600, respectively, around 700 MHz. Experimental data are in very good agreement with theoretical simulations of the fabricated devices

  17. Protein-protein interactions in the regulation of WRKY transcription factors.

    Science.gov (United States)

    Chi, Yingjun; Yang, Yan; Zhou, Yuan; Zhou, Jie; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2013-03-01

    It has been almost 20 years since the first report of a WRKY transcription factor, SPF1, from sweet potato. Great progress has been made since then in establishing the diverse biological roles of WRKY transcription factors in plant growth, development, and responses to biotic and abiotic stress. Despite the functional diversity, almost all analyzed WRKY proteins recognize the TTGACC/T W-box sequences and, therefore, mechanisms other than mere recognition of the core W-box promoter elements are necessary to achieve the regulatory specificity of WRKY transcription factors. Research over the past several years has revealed that WRKY transcription factors physically interact with a wide range of proteins with roles in signaling, transcription, and chromatin remodeling. Studies of WRKY-interacting proteins have provided important insights into the regulation and mode of action of members of the important family of transcription factors. It has also emerged that the slightly varied WRKY domains and other protein motifs conserved within each of the seven WRKY subfamilies participate in protein-protein interactions and mediate complex functional interactions between WRKY proteins and between WRKY and other regulatory proteins in the modulation of important biological processes. In this review, we summarize studies of protein-protein interactions for WRKY transcription factors and discuss how the interacting partners contribute, at different levels, to the establishment of the complex regulatory and functional network of WRKY transcription factors.

  18. Ret Finger Protein: An E3 Ubiquitin Ligase Juxtaposed to the XY Body in Meiosis

    Directory of Open Access Journals (Sweden)

    Isabelle Gillot

    2009-01-01

    Full Text Available During prophase I of male meiosis, the sex chromosomes form a compact structure called XY body that associates with the nuclear membrane of pachytene spermatocytes. Ret Finger Protein is a transcriptional repressor, able to interact with both nuclear matrix-associated proteins and double-stranded DNA. We report the precise and unique localization of Ret Finger Protein in pachytene spermatocytes, in which Ret Finger Protein takes place of lamin B1, between the XY body and the inner nuclear membrane. This localization of Ret Finger Protein does not seem to be associated with O-glycosylation or sumoylation. In addition, we demonstrate that Ret Finger Protein contains an E3 ubiquitin ligase activity. These observations lead to an attractive hypothesis in which Ret Finger Protein would be involved in the positioning and the attachment of XY body to the nuclear lamina of pachytene spermatocytes.

  19. Emerging Functions of Transcription Factors in Malaria Parasite

    Directory of Open Access Journals (Sweden)

    Renu Tuteja

    2011-01-01

    Full Text Available Transcription is a process by which the genetic information stored in DNA is converted into mRNA by enzymes known as RNA polymerase. Bacteria use only one RNA polymerase to transcribe all of its genes while eukaryotes contain three RNA polymerases to transcribe the variety of eukaryotic genes. RNA polymerase also requires other factors/proteins to produce the transcript. These factors generally termed as transcription factors (TFs are either associated directly with RNA polymerase or add in building the actual transcription apparatus. TFs are the most common tools that our cells use to control gene expression. Plasmodium falciparum is responsible for causing the most lethal form of malaria in humans. It shows most of its characteristics common to eukaryotic transcription but it is assumed that mechanisms of transcriptional control in P. falciparum somehow differ from those of other eukaryotes. In this article we describe the studies on the main TFs such as myb protein, high mobility group protein and ApiA2 family proteins from malaria parasite. These studies show that these TFs are slowly emerging to have defined roles in the regulation of gene expression in the parasite.

  20. Genome and Transcriptome sequence of Finger millet (Eleusine coracana (L.) Gaertn.) provides insights into drought tolerance and nutraceutical properties.

    Science.gov (United States)

    Hittalmani, Shailaja; Mahesh, H B; Shirke, Meghana Deepak; Biradar, Hanamareddy; Uday, Govindareddy; Aruna, Y R; Lohithaswa, H C; Mohanrao, A

    2017-06-15

    Finger millet (Eleusine coracana (L.) Gaertn.) is an important staple food crop widely grown in Africa and South Asia. Among the millets, finger millet has high amount of calcium, methionine, tryptophan, fiber, and sulphur containing amino acids. In addition, it has C4 photosynthetic carbon assimilation mechanism, which helps to utilize water and nitrogen efficiently under hot and arid conditions without severely affecting yield. Therefore, development and utilization of genomic resources for genetic improvement of this crop is immensely useful. Experimental results from whole genome sequencing and assembling process of ML-365 finger millet cultivar yielded 1196 Mb covering approximately 82% of total estimated genome size. Genome analysis showed the presence of 85,243 genes and one half of the genome is repetitive in nature. The finger millet genome was found to have higher colinearity with foxtail millet and rice as compared to other Poaceae species. Mining of simple sequence repeats (SSRs) yielded abundance of SSRs within the finger millet genome. Functional annotation and mining of transcription factors revealed finger millet genome harbors large number of drought tolerance related genes. Transcriptome analysis of low moisture stress and non-stress samples revealed the identification of several drought-induced candidate genes, which could be used in drought tolerance breeding. This genome sequencing effort will strengthen plant breeders for allele discovery, genetic mapping, and identification of candidate genes for agronomically important traits. Availability of genomic resources of finger millet will enhance the novel breeding possibilities to address potential challenges of finger millet improvement.

  1. Zfp296 is a novel, pluripotent-specific reprogramming factor.

    Directory of Open Access Journals (Sweden)

    Gerrit Fischedick

    Full Text Available Expression of the four transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM is sufficient to reprogram somatic cells into induced pluripotent stem (iPSCs. However, this process is slow and inefficient compared with the fusion of somatic cells with embryonic stem cells (ESCs, indicating that ESCs express additional factors that can enhance the efficiency of reprogramming. We had previously developed a method to detect and isolate early neural induction intermediates during the differentiation of mouse ESCs. Using the gene expression profiles of these intermediates, we identified 23 ESC-specific transcripts and tested each for the ability to enhance iPSC formation. Of the tested factors, zinc finger protein 296 (Zfp296 led to the largest increase in mouse iPSC formation. We confirmed that Zfp296 was specifically expressed in pluripotent stem cells and germ cells. Zfp296 in combination with OSKM induced iPSC formation earlier and more efficiently than OSKM alone. Through mouse chimera and teratoma formation, we demonstrated that the resultant iPSCs were pluripotent. We showed that Zfp296 activates transcription of the Oct4 gene via the germ cell-specific conserved region 4 (CR4, and when overexpressed in mouse ESCs leads to upregulation of Nanog expression and downregulation of the expression of differentiation markers, including Sox17, Eomes, and T, which is consistent with the observation that Zfp296 enhances the efficiency of reprogramming. In contrast, knockdown of Zfp296 in ESCs leads to the expression of differentiation markers. Finally, we demonstrated that expression of Zfp296 in ESCs inhibits, but does not block, differentiation into neural cells.

  2. Transcription Factor Functional Protein-Protein Interactions in Plant Defense Responses

    Directory of Open Access Journals (Sweden)

    Murilo S. Alves

    2014-03-01

    Full Text Available Responses to biotic stress in plants lead to dramatic reprogramming of gene expression, favoring stress responses at the expense of normal cellular functions. Transcription factors are master regulators of gene expression at the transcriptional level, and controlling the activity of these factors alters the transcriptome of the plant, leading to metabolic and phenotypic changes in response to stress. The functional analysis of interactions between transcription factors and other proteins is very important for elucidating the role of these transcriptional regulators in different signaling cascades. In this review, we present an overview of protein-protein interactions for the six major families of transcription factors involved in plant defense: basic leucine zipper containing domain proteins (bZIP, amino-acid sequence WRKYGQK (WRKY, myelocytomatosis related proteins (MYC, myeloblastosis related proteins (MYB, APETALA2/ ETHYLENE-RESPONSIVE ELEMENT BINDING FACTORS (AP2/EREBP and no apical meristem (NAM, Arabidopsis transcription activation factor (ATAF, and cup-shaped cotyledon (CUC (NAC. We describe the interaction partners of these transcription factors as molecular responses during pathogen attack and the key components of signal transduction pathways that take place during plant defense responses. These interactions determine the activation or repression of response pathways and are crucial to understanding the regulatory networks that modulate plant defense responses.

  3. NUR TRANSCRIPTION FACTORS IN STRESS AND ADDICTION

    Directory of Open Access Journals (Sweden)

    Danae eCampos-Melo

    2013-12-01

    Full Text Available The Nur transcription factors Nur77 (NGFI-B, NR4A1, Nurr1 (NR4A2 and Nor-1 (NR4A3 are a sub-family of orphan members of the nuclear receptor superfamily. These transcription factors are products of immediate early genes, whose expression is rapidly and transiently induced in the central nervous system by several types of stimuli. Nur factors are present throughout the hypothalamus-pituitary-adrenal axis where are prominently induced in response to stress. Drugs of abuse and stress also induce the expression of Nur factors in nuclei of the motivation/reward circuit of the brain, indicating their participation in the process of drug addiction and in non-hypothalamic responses to stress. Repeated use of addictive drugs and chronic stress induce long-lasting dysregulation of the brain motivation/reward circuit, due to reprogramming of gene expression and enduring alterations in neuronal function. Here, we review the data supporting that Nur transcription factors are key players in the molecular basis of the dysregulation of neuronal circuits involved in chronic stress and addiction.

  4. Zinc Serum Level Can Be a Risk Factor In Babol Stroke Patients?

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    Alijan AhmadiAhangar

    2017-02-01

    Full Text Available Background and Purpose: Stroke is the second leading cause of death worldwide. The role of zinc as a contributing factor in the pathogenesis of stroke was considered. Results: This cross-sectional study on 100 stroke patients in Ayatollah Rouhani Hospital and 100 control group from cohort master plan "Ageing and health projects Amirkola was conducted. Zinc levels Serum simultaneously with other blood tests in the early hours of hospitalization. Zinc serum level was defined 70 to 120 micrograms per deciliter. Findings: The difference in mean of zinc level in patients and control group was not significant (102.6±47.7 in control group vs 100.9±35.8 in patient, p=0.7. Difference in zinc Serum level had statically significant with IHD (under70 0 cases (0, 70 to120 8 cases (24, 120 and upper24 cases (75, p=0.003 and with type of stroke (under70 (3(3.3 hemorrhagic vs 0(0 ischemic, 70 to 120(19(21 vs6 (60, 120 and upper68 (75.6 vs4 (40, p=0.025 and also with patient and control group (under70 (3(3 in patient's vs 20(20 control group, 70 to 120(25(25 vs54 (54, 120 and upper72 (72 vs26 (26, p<0001. In patients group 72(73.5 of cases had zinc serum level above 120. HLP difference was significant in patient and control group (50(50 in control group vs 35(35 in patients, p=0.04. Regression logistic show that IHD (p<0001, OR=30, CI=6-152, HLP (p<0001, OR=4, CI=9.09-1.85, zinc serum level (p<0001, OR=15.5, CI=4-59.8 had significant role. Conclusions: Zinc serum levels, Ischemic Heart Disease, Hyperlipidemia were most risk factor that play role in Babol stroke patients.

  5. The Prdm13 histone methyltransferase encoding gene is a Ptf1a-Rbpj downstream target that suppresses glutamatergic and promotes GABAergic neuronal fate in the dorsal neural tube

    DEFF Research Database (Denmark)

    Hanotel, Julie; Bessodes, Nathalie; Thélie, Aurore

    2014-01-01

    The basic helix-loop-helix (bHLH) transcriptional activator Ptf1a determines inhibitory GABAergic over excitatory glutamatergic neuronal cell fate in progenitors of the vertebrate dorsal spinal cord, cerebellum and retina. In an in situ hybridization expression survey of PR domain containing genes...... encoding putative chromatin-remodeling zinc finger transcription factors in Xenopus embryos, we identified Prdm13 as a histone methyltransferase belonging to the Ptf1a synexpression group. Gain and loss of Ptf1a function analyses in both frog and mice indicates that Prdm13 is positively regulated by Ptf1a...

  6. HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3 Transcriptional Activity.

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    Gary T ZeRuth

    Full Text Available The transcription factor Gli-similar 3 (Glis3 plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.

  7. Hepatic ZIP14-mediated zinc transport is required for adaptation to endoplasmic reticulum stress.

    Science.gov (United States)

    Kim, Min-Hyun; Aydemir, Tolunay B; Kim, Jinhee; Cousins, Robert J

    2017-07-18

    Extensive endoplasmic reticulum (ER) stress damages the liver, causing apoptosis and steatosis despite the activation of the unfolded protein response (UPR). Restriction of zinc from cells can induce ER stress, indicating that zinc is essential to maintain normal ER function. However, a role for zinc during hepatic ER stress is largely unknown despite important roles in metabolic disorders, including obesity and nonalcoholic liver disease. We have explored a role for the metal transporter ZIP14 during pharmacologically and high-fat diet-induced ER stress using Zip14 -/- (KO) mice, which exhibit impaired hepatic zinc uptake. Here, we report that ZIP14-mediated hepatic zinc uptake is critical for adaptation to ER stress, preventing sustained apoptosis and steatosis. Impaired hepatic zinc uptake in Zip14 KO mice during ER stress coincides with greater expression of proapoptotic proteins. ER stress-induced Zip14 KO mice show greater levels of hepatic steatosis due to higher expression of genes involved in de novo fatty acid synthesis, which are suppressed in ER stress-induced WT mice. During ER stress, the UPR-activated transcription factors ATF4 and ATF6α transcriptionally up-regulate Zip14 expression. We propose ZIP14 mediates zinc transport into hepatocytes to inhibit protein-tyrosine phosphatase 1B (PTP1B) activity, which acts to suppress apoptosis and steatosis associated with hepatic ER stress. Zip14 KO mice showed greater hepatic PTP1B activity during ER stress. These results show the importance of zinc trafficking and functional ZIP14 transporter activity for adaptation to ER stress associated with chronic metabolic disorders.

  8. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

    Directory of Open Access Journals (Sweden)

    Nicholas D Weber

    Full Text Available Despite an existing effective vaccine, hepatitis B virus (HBV remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB, imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.

  9. Trigger finger

    Science.gov (United States)

    ... digit; Trigger finger release; Locked finger; Digital flexor tenosynovitis ... cut or hand Yellow or green drainage from the cut Hand pain or discomfort Fever If your trigger finger returns, call your surgeon. You may need another surgery.

  10. Zinc finger protein 219-like (ZNF219L) and Sox9a regulate synuclein-γ2 (sncgb) expression in the developing notochord of zebrafish.

    Science.gov (United States)

    Lien, Huang-Wei; Yang, Chung-Hsiang; Cheng, Chia-Hsiung; Liao, Yung-Feng; Han, Yu-San; Huang, Chang-Jen

    2013-12-13

    Zebrafish synuclein-γ2 (sncgb) has been reported to be expressed specifically in the notochord. However, the mechanism by which the sncgb gene promoter is regulated has not been described. In this paper, we demonstrate that Zinc finger protein 219-like (ZNF219L) and sox9a are involved in the regulation of sncgb gene expression. Furthermore, we observed that over-expression of both ZNF219L and Sox9a resulted in increased sncgb expression. In addition, ZNF219L is physically associated with Sox9a, and simultaneous morpholino knockdown of znf219L and sox9a caused a synergistic decrease of sncgb expression in the notochord. Taken together, our results reveal that coordination of ZNF219L with Sox9a is involved in the regulation of notochord-specific expression of sncgb. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  11. A deeper look into transcription regulatory code by preferred pair distance templates for transcription factor binding sites

    KAUST Repository

    Kulakovskiy, Ivan V.; Belostotsky, A. A.; Kasianov, Artem S.; Esipova, Natalia G.; Medvedeva, Yulia; Eliseeva, Irina A.; Makeev, Vsevolod J.

    2011-01-01

    Motivation: Modern experimental methods provide substantial information on protein-DNA recognition. Studying arrangements of transcription factor binding sites (TFBSs) of interacting transcription factors (TFs) advances understanding

  12. Characterization and metal-induced gene transcription of two new copper zinc superoxide dismutases in the solitary ascidian Ciona intestinalis

    International Nuclear Information System (INIS)

    Ferro, Diana; Franchi, Nicola; Mangano, Valentina; Bakiu, Rigers; Cammarata, Matteo; Parrinello, Nicolò; Santovito, Gianfranco; Ballarin, Loriano

    2013-01-01

    Highlights: •Ciona intestinalis express two copper-zinc superoxide dismutases (Cu,Zn SODs), one extracellular (Ci-SODa) and one intracellular isoform (Ci-SODb). •Promoters contain consensus sequences similar to mammalian MRE. •Metal exposure results in a significant increase of gene transcription: ci-soda is induced especially by copper and zinc, the increase of ci-sodb transcription is more evident after cadmium exposure. •Genes are mostly transcribed in circulating hemocytes and in ovarian follicular cells. -- Abstract: Antioxidant enzymes are known to protect living organisms against the oxidative stress risk, also induced by metals. In the present study, we describe the purification and molecular characterization of two Cu,Zn superoxide dismutases (SODs), referred to as Ci-SODa and Ci-SODb, from Ciona intestinalis, a basal chordate widely distributed in temperate shallow seawater. The putative amino acid sequences were compared with Cu,Zn SODs from other metazoans and phylogenetic analyses indicate that the two putative Ci-SODs are more related to invertebrate SODs than vertebrate ones. Both phylogenetic and preliminary homology modeling analyses suggest that Ci-SODa and Ci-SODb are extracellular and intracellular isoform, respectively. The mRNA of the two Cu,Zn SODs was localized in hemocytes and in ovarian follicular cells, as revealed by in situ hybridization. The time course of SOD mRNA levels in the presence of three different metals showed upregulation of ci-soda and inhibition of ci-sodb. Spectrophotometric analysis confirms the presence of SOD activity in Ciona tissues. Our in silico analyses of the ci-soda promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE), suggesting that the transcription of these genes directly depends on metals. These data emphasize the importance of complex metal regulation of ci-soda and ci-sodb transcription, as components of an efficient detoxification pathway

  13. Characterization and metal-induced gene transcription of two new copper zinc superoxide dismutases in the solitary ascidian Ciona intestinalis

    Energy Technology Data Exchange (ETDEWEB)

    Ferro, Diana [Department of Biology, University of Padova, Padova (Italy); Institute for Evolution and Biodiversity, Westfälische Wilhelms-Universität, Münster (Germany); Franchi, Nicola [Department of Biology, University of Padova, Padova (Italy); Department of Biological, Chemical, Pharmaceutical Science and Technology, University of Palermo, Palermo (Italy); Mangano, Valentina [Department of Biological, Chemical, Pharmaceutical Science and Technology, University of Palermo, Palermo (Italy); Bakiu, Rigers [Department of Crop Production, Agricultural University of Tirana, Tirana (Albania); Cammarata, Matteo; Parrinello, Nicolò [Department of Biological, Chemical, Pharmaceutical Science and Technology, University of Palermo, Palermo (Italy); Santovito, Gianfranco, E-mail: gianfranco.santovito@unipd.it [Department of Biology, University of Padova, Padova (Italy); Ballarin, Loriano [Department of Biology, University of Padova, Padova (Italy)

    2013-09-15

    Highlights: •Ciona intestinalis express two copper-zinc superoxide dismutases (Cu,Zn SODs), one extracellular (Ci-SODa) and one intracellular isoform (Ci-SODb). •Promoters contain consensus sequences similar to mammalian MRE. •Metal exposure results in a significant increase of gene transcription: ci-soda is induced especially by copper and zinc, the increase of ci-sodb transcription is more evident after cadmium exposure. •Genes are mostly transcribed in circulating hemocytes and in ovarian follicular cells. -- Abstract: Antioxidant enzymes are known to protect living organisms against the oxidative stress risk, also induced by metals. In the present study, we describe the purification and molecular characterization of two Cu,Zn superoxide dismutases (SODs), referred to as Ci-SODa and Ci-SODb, from Ciona intestinalis, a basal chordate widely distributed in temperate shallow seawater. The putative amino acid sequences were compared with Cu,Zn SODs from other metazoans and phylogenetic analyses indicate that the two putative Ci-SODs are more related to invertebrate SODs than vertebrate ones. Both phylogenetic and preliminary homology modeling analyses suggest that Ci-SODa and Ci-SODb are extracellular and intracellular isoform, respectively. The mRNA of the two Cu,Zn SODs was localized in hemocytes and in ovarian follicular cells, as revealed by in situ hybridization. The time course of SOD mRNA levels in the presence of three different metals showed upregulation of ci-soda and inhibition of ci-sodb. Spectrophotometric analysis confirms the presence of SOD activity in Ciona tissues. Our in silico analyses of the ci-soda promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE), suggesting that the transcription of these genes directly depends on metals. These data emphasize the importance of complex metal regulation of ci-soda and ci-sodb transcription, as components of an efficient detoxification pathway

  14. Integration of tactile input across fingers in a patient with finger agnosia.

    Science.gov (United States)

    Anema, Helen A; Overvliet, Krista E; Smeets, Jeroen B J; Brenner, Eli; Dijkerman, H Chris

    2011-01-01

    Finger agnosia has been described as an inability to explicitly individuate between the fingers, which is possibly due to fused neural representations of these fingers. Hence, are patients with finger agnosia unable to keep tactile information perceived over several fingers separate? Here, we tested a finger agnosic patient (GO) on two tasks that measured the ability to keep tactile information simultaneously perceived by individual fingers separate. In experiment 1 GO performed a haptic search task, in which a target (the absence of a protruded line) needed to be identified among distracters (protruded lines). The lines were presented simultaneously to the fingertips of both hands. Similarly to the controls, her reaction time decreased when her fingers were aligned as compared to when her fingers were stretched and in an unaligned position. This suggests that she can keep tactile input from different fingers separate. In experiment two, GO was required to judge the position of a target tactile stimulus to the index finger, relatively to a reference tactile stimulus to the middle finger, both in fingers uncrossed and crossed position. GO was able to indicate the relative position of the target stimulus as well as healthy controls, which indicates that she was able to keep tactile information perceived by two neighbouring fingers separate. Interestingly, GO performed better as compared to the healthy controls in the finger crossed condition. Together, these results suggest the GO is able to implicitly distinguish between tactile information perceived by multiple fingers. We therefore conclude that finger agnosia is not caused by minor disruptions of low-level somatosensory processing. These findings further underpin the idea of a selective impaired higher order body representation restricted to the fingers as underlying cause of finger agnosia. Copyright © 2010 Elsevier Ltd. All rights reserved.

  15. KLF2 in Regulation of NF-κB-Mediated Immune Cell Function and Inflammation

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    Prerana Jha

    2017-11-01

    Full Text Available KLF2 (Kruppel-like factor 2 is a member of the zinc finger transcription factor family, which critically regulates embryonic lung development, function of endothelial cells and maintenance of quiescence in T-cells and monocytes. It is expressed in naïve T-cells and monocytes, however its level of expression decreases during activation and differentiation. KLF2 also plays critical regulatory role in various inflammatory diseases and their pathogenesis. Nuclear factor-kappaB (NF-κB is an important inducer of inflammation and the inflammation is mediated through the transcription of several proinflammatory cytokines, chemokines and adhesion molecules. So, both transcriptional factors KLF2 and NF-κB are being associated with the similar cellular functions and their maintenance. It was shown that KLF2 regulates most of the NF-κB-mediated activities. In this review, we focused on emphasizing the involvement of KLF2 in health and disease states and how they interact with transcriptional master regulator NF-κB.

  16. Factor requirements for transcription in the Archaeon Sulfolobus shibatae.

    Science.gov (United States)

    Qureshi, S A; Bell, S D; Jackson, S P

    1997-05-15

    Archaea (archaebacteria) constitute a domain of life that is distinct from Bacteria (eubacteria) and Eucarya (eukaryotes). Although archaeal cells share many morphological features with eubacteria, their transcriptional apparatus is more akin to eukaryotic RNA polymerases I, II and III than it is to eubacterial transcription systems. Thus, in addition to possessing a 10 subunit RNA polymerase and a homologue of the TATA-binding protein (TBP), Archaea possess a polypeptide termed TFB that is homologous to eukaryotic TFIIB. Here, we investigate the factor requirements for transcription of several promoters of the archaeon Sulfolobus shibatae and its associated virus SSV. Through in vitro transcription and immunodepletion, we demonstrate that S. shibatae TBP, TFB and RNA polymerase are not complexed tightly with one another and that each is required for efficient transcription of all promoters tested. Furthermore, full transcription is restored by supplementing respective depleted extracts with recombinant TBP or TFB, indicating that TBP-associated factors or TFB-associated factors are not required. Indeed, gel-filtration suggests that Sulfolobus TBP and TFB are not associated stably with other proteins. Finally, all promoters analysed are transcribed accurately and efficiently in an in vitro system comprising recombinant TBP and TFB, together with essentially homogeneous preparation of RNA polymerase. Transcription in Archaea is therefore fundamentally homologous to that in eukaryotes, although factor requirements appear to be much less complex.

  17. Transcription factor-based biosensor

    Science.gov (United States)

    Dietrich, Jeffrey A; Keasling, Jay D

    2013-10-08

    The present invention provides for a system comprising a BmoR transcription factor, a .sigma..sup.54-RNA polymerase, and a pBMO promoter operatively linked to a reporter gene, wherein the pBMO promoter is capable of expression of the reporter gene with an activated form of the BmoR and the .sigma..sup.54-RNA polymerase.

  18. Detecting Differential Transcription Factor Activity from ATAC-Seq Data

    Directory of Open Access Journals (Sweden)

    Ignacio J. Tripodi

    2018-05-01

    Full Text Available Transcription factors are managers of the cellular factory, and key components to many diseases. Many non-coding single nucleotide polymorphisms affect transcription factors, either by directly altering the protein or its functional activity at individual binding sites. Here we first briefly summarize high-throughput approaches to studying transcription factor activity. We then demonstrate, using published chromatin accessibility data (specifically ATAC-seq, that the genome-wide profile of TF recognition motifs relative to regions of open chromatin can determine the key transcription factor altered by a perturbation. Our method of determining which TFs are altered by a perturbation is simple, is quick to implement, and can be used when biological samples are limited. In the future, we envision that this method could be applied to determine which TFs show altered activity in response to a wide variety of drugs and diseases.

  19. Expression of zinc transporter genes in rice as influenced by zinc-solubilizing Enterobacter cloacae strain ZSB14

    Directory of Open Access Journals (Sweden)

    Selvaraj eKrithika

    2016-04-01

    Full Text Available Zinc (Zn deficiency in major food crops has been considered as an important factor affecting the crop production and subsequently the human health. Rice (Oryza sativa is sensitive to Zn deficiency and thereby causes malnutrition to most of the rice-eating Asian populations. Application of zinc solubilizing bacteria (ZSB could be a sustainable agronomic approach to increase the soil available Zn which can mitigate the yield loss and consequently the nutritional quality of rice. Understanding the molecular interactions between rice and unexplored ZSB is useful for overcoming Zn deficiency problems. In the present study, the role of zinc solubilizing bacterial strain Enterobacter cloacae strain ZSB14 on regulation of Zn-regulated transporters and iron (Fe-regulated transporter-like protein (ZIP genes in rice under iron sufficient and deficient conditions was assessed by quantitative real-time reverse transcription PCR. The expression patterns of OsZIP1, OsZIP4 and OsZIP5 in root and shoot of rice were altered due to the Zn availability as dictated by Zn sources and ZSB inoculation. Fe sufficiency significantly reduced the root and shoot OsZIP1 expression, but not the OsZIP4 and OsZIP5 levels. Zinc oxide in the growth medium up-regulated all the assessed ZIP genes in root and shoot of rice seedlings. When ZSB was inoculated to rice seedlings grown with insoluble zinc oxide in the growth medium, the expression of root and shoot OsZIP1, OsZIP4 and OsZIP5 was reduced. In the absence of zinc oxide, ZSB inoculation up-regulated OsZIP1 and OsZIP5 expressions. Zinc nutrition provided to the rice seedling through ZSB-bound zinc oxide solubilization was comparable to the soluble zinc sulphate application which was evident through the ZIP genes’ expression and the Zn accumulation in root and shoot of rice seedlings. These results demonstrate that zinc solubilizing bacteria could play a crucial role in zinc fertilization and fortification of rice.

  20. Cyclin D3 interacts with human activating transcription factor 5 and potentiates its transcription activity

    International Nuclear Information System (INIS)

    Liu Wenjin; Sun Maoyun; Jiang Jianhai; Shen Xiaoyun; Sun Qing; Liu Weicheng; Shen Hailian; Gu Jianxin

    2004-01-01

    The Cyclin D3 protein is a member of the D-type cyclins. Besides serving as cell cycle regulators, D-type cyclins have been reported to be able to interact with several transcription factors and modulate their transcriptional activations. Here we report that human activating transcription factor 5 (hATF5) is a new interacting partner of Cyclin D3. The interaction was confirmed by in vivo coimmunoprecipitation and in vitro binding analysis. Neither interaction between Cyclin D1 and hATF5 nor interaction between Cyclin D2 and hATF5 was observed. Confocal microscopy analysis showed that Cyclin D3 could colocalize with hATF5 in the nuclear region. Cyclin D3 could potentiate hATF5 transcriptional activity independently of its Cdk4 partner. But Cyclin D1 and Cyclin D2 had no effect on hATF5 transcriptional activity. These data provide a new clue to understand the new role of Cyclin D3 as a transcriptional regulator

  1. Modulation of DNA binding by gene-specific transcription factors.

    Science.gov (United States)

    Schleif, Robert F

    2013-10-01

    The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

  2. Transcriptional repression of BODENLOS by HD-ZIP transcription factor HB5 in Arabidopsis thaliana.

    NARCIS (Netherlands)

    Smet, De I.; Lau, S.; Ehrismann, J.S.; Axiotis, I.; Kolb, M.; Kientz, M.; Weijers, D.; Jürgens, G.

    2013-01-01

    In Arabidopsis thaliana, the phytohormone auxin is an important patterning agent during embryogenesis and post-embryonic development, exerting effects through transcriptional regulation. The main determinants of the transcriptional auxin response machinery are AUXIN RESPONSE FACTOR (ARF)

  3. Zinc finger AN1-type containing 4 is a novel marker for predicting metastasis and poor prognosis in oral squamous cell carcinoma.

    Science.gov (United States)

    Kurihara-Shimomura, Miyako; Sasahira, Tomonori; Nakamura, Hiroshi; Nakashima, Chie; Kuniyasu, Hiroki; Kirita, Tadaaki

    2018-05-01

    Head and neck cancer, including oral squamous cell carcinoma (OSCC), is the sixth most common cancer worldwide and has a high potential for locoregional invasion and nodal metastasis. Therefore, discovery of a useful molecular biomarker capable of predicting tumour progression and metastasis of OSCC is crucial. We have previously reported zinc finger AN1-type containing 4 (ZFAND4) as one of the most upregulated genes in recurrent OSCC using a cDNA microarray analysis. Although ZFAND4 has been shown to promote cell proliferation of gastric cancer, its expression and clinicopathological roles in OSCC remain unclear. In this study, we examined ZFAND4 expression by immunohistochemistry in 214 cases of OSCC. High cytoplasmic expression of ZFAND4 was observed in 45 out of 214 (21%) patients with OSCC. Expression levels of ZFAND4 were strongly associated with metastasis to the lymph nodes (p=0.0429) and distant organs (p=0.0068). Cases with high expression of ZFAND4 had a significantly unfavourable prognosis compared with patients with low expression of ZFAND4 (p<0.0001). Furthermore, ZFAND4 overexpression was an independent poor prognostic factor for OSCC as determined by multivariate analysis using the Cox proportional hazards model (p<0.0001). These results suggest that ZFAND4 is a useful marker for predicting metastasis and poor prognosis in patients with OSCC. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  4. A critique on nuclear factor-kappa B and signal transducer and activator of transcription 3: The key transcription factors in periodontal pathogenesis

    Directory of Open Access Journals (Sweden)

    Ranjith Ambili

    2017-01-01

    Full Text Available Periodontal disease is initiated by microorganisms in dental plaque, and host immunoinflammatory response to the microbial challenge helps in disease progression. Conventional periodontal therapy was mainly targeted on the elimination of microbial component. However, a better understanding of molecular aspects in host response will enable the clinicians to formulate effective host modulation therapy (HMT for the periodontal management. Inflammatory mediators were the main targets for HMT in the past. Transcription factors can regulate the production of multiple mediators simultaneously, and inhibition of these factors will be more beneficial than blocking individual molecule. Two important transcription factors implicated in chronic inflammatory diseases are nuclear factor kappa B (NF-κB and signal transducers and activators of transcription 3. The role of these factors in periodontal disease is a less explored area. This comprehensive review is aimed at unveiling the critical role of NF-κB and signal transducers and activators of transcription 3 in periodontal pathogenesis. An online search was performed using MEDLINE/PubMed database. All publications till 2016 related to NF-κB, signal transducer and activator of transcription 3 (STAT3, and inflammation were included in writing this review. A total of 27,390 references were published based on the search terms used. Out of these, 507 were related to the periodontal research published in English till 2016. Relevant papers were chosen after carefully reading the abstract. This review has attempted to comprehend the existing knowledge regarding the role of transcription factors NF-κB and STAT3 in periodontal disease. Moreover, it also provides a connecting molecular link for the periodontal medicine concept.

  5. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes.

    Science.gov (United States)

    Riechmann, J L; Heard, J; Martin, G; Reuber, L; Jiang, C; Keddie, J; Adam, L; Pineda, O; Ratcliffe, O J; Samaha, R R; Creelman, R; Pilgrim, M; Broun, P; Zhang, J Z; Ghandehari, D; Sherman, B K; Yu, G

    2000-12-15

    The completion of the Arabidopsis thaliana genome sequence allows a comparative analysis of transcriptional regulators across the three eukaryotic kingdoms. Arabidopsis dedicates over 5% of its genome to code for more than 1500 transcription factors, about 45% of which are from families specific to plants. Arabidopsis transcription factors that belong to families common to all eukaryotes do not share significant similarity with those of the other kingdoms beyond the conserved DNA binding domains, many of which have been arranged in combinations specific to each lineage. The genome-wide comparison reveals the evolutionary generation of diversity in the regulation of transcription.

  6. Genome Wide Identification of Orthologous ZIP Genes Associated with Zinc and Iron Translocation in Setaria italica.

    Science.gov (United States)

    Alagarasan, Ganesh; Dubey, Mahima; Aswathy, Kumar S; Chandel, Girish

    2017-01-01

    Genes in the ZIP family encode transcripts to store and transport bivalent metal micronutrient, particularly iron (Fe) and or zinc (Zn). These transcripts are important for a variety of functions involved in the developmental and physiological processes in many plant species, including most, if not all, Poaceae plant species and the model species Arabidopsis. Here, we present the report of a genome wide investigation of orthologous ZIP genes in Setaria italica and the identification of 7 single copy genes. RT-PCR shows 4 of them could be used to increase the bio-availability of zinc and iron content in grains. Of 36 ZIP members, 25 genes have traces of signal peptide based sub-cellular localization, as compared to those of plant species studied previously, yet translocation of ions remains unclear. In silico analysis of gene structure and protein nature suggests that these two were preeminent in shaping the functional diversity of the ZIP gene family in S. italica . NAC, bZIP and bHLH are the predominant Fe and Zn responsive transcription factors present in SiZIP genes. Together, our results provide new insights into the signal peptide based/independent iron and zinc translocation in the plant system and allowed identification of ZIP genes that may be involved in the zinc and iron absorption from the soil, and thus transporting it to the cereal grain underlying high micronutrient accumulation.

  7. BACH transcription factors in innate and adaptive immunity.

    Science.gov (United States)

    Igarashi, Kazuhiko; Kurosaki, Tomohiro; Roychoudhuri, Rahul

    2017-07-01

    BTB and CNC homology (BACH) proteins are transcriptional repressors of the basic region leucine zipper (bZIP) transcription factor family. Recent studies indicate widespread roles of BACH proteins in controlling the development and function of the innate and adaptive immune systems, including the differentiation of effector and memory cells of the B and T cell lineages, CD4 + regulatory T cells and macrophages. Here, we emphasize similarities at a molecular level in the cell-type-specific activities of BACH factors, proposing that competitive interactions of BACH proteins with transcriptional activators of the bZIP family form a common mechanistic theme underlying their diverse actions. The findings contribute to a general understanding of how transcriptional repressors shape lineage commitment and cell-type-specific functions through repression of alternative lineage programmes.

  8. Thirty-seven transcription factor genes differentially respond to a ...

    Indian Academy of Sciences (India)

    Plant transcription factors and insect defence si. Thirty-seven transcription factor genes differentially respond to a harpin protein and affect resistance to the green peach aphid in Arabidopsis. HUNLIN. PIN. RUOXUE LIŲ, BEIBEI LÜ, XIAOMENG WANG, CHUNLING ZHANG, SHUPING ZHANG, JUN QIAN, LEI CHEN,.

  9. Integration of tactile input across fingers in a patient with finger agnosia.

    NARCIS (Netherlands)

    Anema, H.A.; Overvliet, K.E.; Smeets, J.B.J.; Brenner, E.; Dijkerman, H.C.

    2011-01-01

    Finger agnosia has been described as an inability to explicitly individuate between the fingers, which is possibly due to fused neural representations of these fingers. Hence, are patients with finger agnosia unable to keep tactile information perceived over several fingers separate? Here, we tested

  10. Structural Fingerprints of Transcription Factor Binding Site Regions

    Directory of Open Access Journals (Sweden)

    Peter Willett

    2009-03-01

    Full Text Available Fourier transforms are a powerful tool in the prediction of DNA sequence properties, such as the presence/absence of codons. We have previously compiled a database of the structural properties of all 32,896 unique DNA octamers. In this work we apply Fourier techniques to the analysis of the structural properties of human chromosomes 21 and 22 and also to three sets of transcription factor binding sites within these chromosomes. We find that, for a given structural property, the structural property power spectra of chromosomes 21 and 22 are strikingly similar. We find common peaks in their power spectra for both Sp1 and p53 transcription factor binding sites. We use the power spectra as a structural fingerprint and perform similarity searching in order to find transcription factor binding site regions. This approach provides a new strategy for searching the genome data for information. Although it is difficult to understand the relationship between specific functional properties and the set of structural parameters in our database, our structural fingerprints nevertheless provide a useful tool for searching for function information in sequence data. The power spectrum fingerprints provide a simple, fast method for comparing a set of functional sequences, in this case transcription factor binding site regions, with the sequences of whole chromosomes. On its own, the power spectrum fingerprint does not find all transcription factor binding sites in a chromosome, but the results presented here show that in combination with other approaches, this technique will improve the chances of identifying functional sequences hidden in genomic data.

  11. Zinc Deficiency in Humans and its Amelioration

    Directory of Open Access Journals (Sweden)

    Yashbir Singh Shivay

    2015-01-01

    Full Text Available Zinc (Zn deficiency in humans has recently received considerable attention. Global mortality in children under 5 years of age in 2004 due to Zn deficiency was estimated at 4,53,207 as against 6,66,771 for vitamin A deficiency; 20,854 for iron deficiency and 3,619 for iodine deficiency. In humans 2800-3000 proteins contain Zn prosthetic group and Zn is an integral component of zinc finger prints that regulate DNA transcription. Zinc is a Type-2 nutrient, which means that its concentration in blood does not decrease in proportion of the Zn deficiency. Adverse effects of Zn deficiency vary with age: low weight gain, diarrhoea, aneroxia and neurobehavioral disturbances are observed in infants, while skin changes and dwarfism are frequent in toddlers and adolescents. Common manifestations of Zn deficiency among elderly include hypogeusia, chronic non-healing ulcers and recurrent infections.Ameliorative measures of Zn deficiency in humans can be classified in two groups, namely, nutraceutical and biofortification of food grains. Nutraceutical interventions include pharmaceutical supplements, dietary supplements and dietary diversification, while biofortification of food grains can be achieved by genetic modification (GM of crops or by agronomic techniques that include soil or/and foliar fertilization of crops.The major disadvantage of nutraceutical approaches is that the major beneficiaries are urban people and the poor rural masses that need adequate Zn nutrition most are left out. Genetic biofortification of food grains requires large amounts of funds and a fairly long-period of time. Further, a large number of countries have not yet accepted genetically modified (GM foods. On the other hand agronomic biofortification of food grains yields immediate effects and rural and urban people are equally benefitted. Our studies have shown that Zn concentration in cereals (rice, wheat etc and pulses can be considerably increased by soil or/and foliar

  12. Zinc Deficiency in Humans and its Amelioration

    Directory of Open Access Journals (Sweden)

    Yashbir Singh Shivay

    2015-12-01

    Full Text Available Zinc (Zn deficiency in humans has recently received considerable attention. Global mortality in children under 5 years of age in 2004 due to Zn deficiency was estimated at 4,53,207 as against 6,66,771 for vitamin A deficiency; 20,854 for iron deficiency and 3,619 for iodine deficiency. In humans 2800-3000 proteins contain Zn prosthetic group and Zn is an integral component of zinc finger prints that regulate DNA transcription. Zinc is a Type-2 nutrient, which means that its concentration in blood does not decrease in proportion of the Zn deficiency. Adverse effects of Zn deficiency vary with age: low weight gain, diarrhoea, aneroxia and neurobehavioral disturbances are observed in infants, while skin changes and dwarfism are frequent in toddlers and adolescents. Common manifestations of Zn deficiency among elderly include hypogeusia, chronic non-healing ulcers and recurrent infections. Ameliorative measures of Zn deficiency in humans can be classified in two groups, namely, nutraceutical and biofortification of food grains. Nutraceutical interventions include pharmaceutical supplements, dietary supplements and dietary diversification, while biofortification of food grains can be achieved by genetic modification (GM of crops or by agronomic techniques that include soil or/and foliar fertilization of crops. The major disadvantage of nutraceutical approaches is that the major beneficiaries are urban people and the poor rural masses that need adequate Zn nutrition most are left out. Genetic biofortification of food grains requires large amounts of funds and a fairly long-period of time. Further, a large number of countries have not yet accepted genetically modified (GM foods. On the other hand agronomic biofortification of food grains yields immediate effects and rural and urban people are equally benefitted. Our studies have shown that Zn concentration in cereals (rice, wheat etc and pulses can be considerably increased by soil or/and foliar

  13. NAC Transcription Factors in Stress Responses and Senescence

    DEFF Research Database (Denmark)

    O'Shea, Charlotte

    Plant-specific NAM/ATAF/CUC (NAC) transcription factors have recently received considerable attention due to their significant roles in plant development and stress signalling. This interest has resulted in a number of physiological, genetic and cell biological studies of their functions. Some...... of these studies have also revealed emerging gene regulatory networks and protein-protein interaction networks. However, structural studies relating structure to function are lagging behind. Structure-function analysis of the NAC transcription factors has therefore been the main focus of this PhD thesis...... not involve significant folding-upon-binding but fuzziness or an extended ANAC046 region. The ANAC046 regulatory domain functions as an entropic chain with a bait for interactions with for example RCD1. RCD1 interacts with transcription factors from several different families, and the large stress...

  14. Cross-Family Transcription Factor Interactions

    NARCIS (Netherlands)

    Bemer, Marian; Dijk, van Aalt-Jan; Immink, Richard G.H.; Angenent, Gerco C.

    2017-01-01

    Specific and dynamic gene expression strongly depends on transcription factor (TF) activity and most plant TFs function in a combinatorial fashion. They can bind to DNA and control the expression of the corresponding gene in an additive fashion or cooperate by physical interactions, forming larger

  15. A moonlighting function of Mycobacterium smegmatis Ku in zinc homeostasis?

    Science.gov (United States)

    Kushwaha, Ambuj K; Deochand, Dinesh K; Grove, Anne

    2015-02-01

    Ku protein participates in DNA double-strand break repair via the nonhomologous end-joining pathway. The three-dimensional structure of eukaryotic Ku reveals a central core consisting of a β-barrel domain and pillar and bridge regions that combine to form a ring-like structure that encircles DNA. Homologs of Ku are encoded by a subset of bacterial species, and they are predicted to conserve this core domain. In addition, the bridge region of Ku from some bacteria is predicted from homology modeling and sequence analyses to contain a conventional HxxC and CxxC (where x is any residue) zinc-binding motif. These potential zinc-binding sites have either deteriorated or been entirely lost in Ku from other organisms. Using an in vitro metal binding assay, we show that Mycobacterium smegmatis Ku binds two zinc ions. Zinc binding modestly stabilizes the Ku protein (by ∼3°C) and prevents cysteine oxidation, but it has little effect on DNA binding. In vivo, zinc induces significant upregulation of the gene encoding Ku (∼sixfold) as well as a divergently oriented gene encoding a predicted zinc-dependent MarR family transcription factor. Notably, overexpression of Ku confers zinc tolerance on Escherichia coli. We speculate that zinc-binding sites in Ku proteins from M. smegmatis and other mycobacterial species have been evolutionarily retained to provide protection against zinc toxicity without compromising the function of Ku in DNA double-strand break repair. © 2014 The Protein Society.

  16. Transcription factor interplay in T helper cell differentiation

    Science.gov (United States)

    Evans, Catherine M.

    2013-01-01

    The differentiation of CD4 helper T cells into specialized effector lineages has provided a powerful model for understanding immune cell differentiation. Distinct lineages have been defined by differential expression of signature cytokines and the lineage-specifying transcription factors necessary and sufficient for their production. The traditional paradigm of differentiation towards Th1 and Th2 subtypes driven by T-bet and GATA3, respectively, has been extended to incorporate additional T cell lineages and transcriptional regulators. Technological advances have expanded our view of these lineage-specifying transcription factors to the whole genome and revealed unexpected interplay between them. From these data, it is becoming clear that lineage specification is more complex and plastic than previous models might have suggested. Here, we present an overview of the different forms of transcription factor interplay that have been identified and how T cell phenotypes arise as a product of this interplay within complex regulatory networks. We also suggest experimental strategies that will provide further insight into the mechanisms that underlie T cell lineage specification and plasticity. PMID:23878131

  17. Transcription factor interplay in T helper cell differentiation.

    Science.gov (United States)

    Evans, Catherine M; Jenner, Richard G

    2013-11-01

    The differentiation of CD4 helper T cells into specialized effector lineages has provided a powerful model for understanding immune cell differentiation. Distinct lineages have been defined by differential expression of signature cytokines and the lineage-specifying transcription factors necessary and sufficient for their production. The traditional paradigm of differentiation towards Th1 and Th2 subtypes driven by T-bet and GATA3, respectively, has been extended to incorporate additional T cell lineages and transcriptional regulators. Technological advances have expanded our view of these lineage-specifying transcription factors to the whole genome and revealed unexpected interplay between them. From these data, it is becoming clear that lineage specification is more complex and plastic than previous models might have suggested. Here, we present an overview of the different forms of transcription factor interplay that have been identified and how T cell phenotypes arise as a product of this interplay within complex regulatory networks. We also suggest experimental strategies that will provide further insight into the mechanisms that underlie T cell lineage specification and plasticity.

  18. Transcription factor 19 interacts with histone 3 lysine 4 trimethylation and controls gluconeogenesis via the nucleosome-remodeling-deacetylase complex.

    Science.gov (United States)

    Sen, Sabyasachi; Sanyal, Sulagna; Srivastava, Dushyant Kumar; Dasgupta, Dipak; Roy, Siddhartha; Das, Chandrima

    2017-12-15

    Transcription factor 19 (TCF19) has been reported as a type 1 diabetes-associated locus involved in maintenance of pancreatic β cells through a fine-tuned regulation of cell proliferation and apoptosis. TCF19 also exhibits genomic association with type 2 diabetes, although the precise molecular mechanism remains unknown. It harbors both a plant homeodomain and a forkhead-associated domain implicated in epigenetic recognition and gene regulation, a phenomenon that has remained unexplored. Here, we show that TCF19 selectively interacts with histone 3 lysine 4 trimethylation through its plant homeodomain finger. Knocking down TCF19 under high-glucose conditions affected many metabolic processes, including gluconeogenesis. We found that TCF19 overexpression represses de novo glucose production in HepG2 cells. The transcriptional repression of key genes, induced by TCF19, coincided with NuRD (nucleosome-remodeling-deacetylase) complex recruitment to the promoters of these genes. TCF19 interacted with CHD4 (chromodomain helicase DNA-binding protein 4), which is a part of the NuRD complex, in a glucose concentration-independent manner. In summary, our results show that TCF19 interacts with an active transcription mark and recruits a co-repressor complex to regulate gluconeogenic gene expression in HepG2 cells. Our study offers critical insights into the molecular mechanisms of transcriptional regulation of gluconeogenesis and into the roles of chromatin readers in metabolic homeostasis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Enhanceosomes as integrators of hypoxia inducible factor (HIF) and other transcription factors in the hypoxic transcriptional response.

    Science.gov (United States)

    Pawlus, Matthew R; Hu, Cheng-Jun

    2013-09-01

    Hypoxia is a prevalent attribute of the solid tumor microenvironment that promotes the expression of genes through posttranslational modifications and stabilization of alpha subunits (HIF1α and HIF2α) of hypoxia-inducible factors (HIFs). Despite significant similarities, HIF1 (HIF1α/ARNT) and HIF2 (HIF2α/ARNT) activate common as well as unique target genes and exhibit different functions in cancer biology. More surprisingly, accumulating data indicates that the HIF1- and/or HIF2-mediated hypoxia responses can be oncogenic as well as tumor suppressive. While the role of HIF in the hypoxia response is well established, recent data support the concept that HIF is necessary, but not sufficient for the hypoxic response. Other transcription factors that are activated by hypoxia are also required for the HIF-mediated hypoxia response. HIFs, other transcription factors, co-factors and RNA poll II recruited by HIF and other transcription factors form multifactorial enhanceosome complexes on the promoters of HIF target genes to activate hypoxia inducible genes. Importantly, HIF1 or HIF2 requires distinct partners in activating HIF1 or HIF2 target genes. Because HIF enhanceosome formation is required for the gene activation and distinct functions of HIF1 and HIF2 in tumor biology, disruption of the HIF1 or HIF2 specific enhanceosome complex may prove to be a beneficial strategy in tumor treatment in which tumor growth is specifically dependent upon HIF1 or HIF2 activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. DNA Binding by the Ribosomal DNA Transcription Factor Rrn3 Is Essential for Ribosomal DNA Transcription*

    Science.gov (United States)

    Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H.; Rothblum, Katrina; Schneider, David A.; Rothblum, Lawrence I.

    2013-01-01

    The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382–400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I. PMID:23393135

  1. DNA binding by the ribosomal DNA transcription factor rrn3 is essential for ribosomal DNA transcription.

    Science.gov (United States)

    Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H; Rothblum, Katrina; Schneider, David A; Rothblum, Lawrence I

    2013-03-29

    The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382-400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I.

  2. Expression of the potential therapeutic target CXXC5 in primary acute myeloid leukemia cells - high expression is associated with adverse prognosis as well as altered intracellular signaling and transcriptional regulation.

    Science.gov (United States)

    Bruserud, Øystein; Reikvam, Håkon; Fredly, Hanne; Skavland, Jørn; Hagen, Karen-Marie; van Hoang, Tuyen Thy; Brenner, Annette K; Kadi, Amir; Astori, Audrey; Gjertsen, Bjørn Tore; Pendino, Frederic

    2015-02-20

    The CXXC5 gene encodes a transcriptional activator with a zinc-finger domain, and high expression in human acute myeloid leukemia (AML) cells is associated with adverse prognosis. We now characterized the biological context of CXXC5 expression in primary human AML cells. The global gene expression profile of AML cells derived from 48 consecutive patients was analyzed; cells with high and low CXXC5 expression then showed major differences with regard to extracellular communication and intracellular signaling. We observed significant differences in the phosphorylation status of several intracellular signaling mediators (CREB, PDK1, SRC, STAT1, p38, STAT3, rpS6) that are important for PI3K-Akt-mTOR signaling and/or transcriptional regulation. High CXXC5 expression was also associated with high mRNA expression of several stem cell-associated transcriptional regulators, the strongest associations being with WT1, GATA2, RUNX1, LYL1, DNMT3, SPI1, and MYB. Finally, CXXC5 knockdown in human AML cell lines caused significantly increased expression of the potential tumor suppressor gene TSC22 and genes encoding the growth factor receptor KIT, the cytokine Angiopoietin 1 and the selenium-containing glycoprotein Selenoprotein P. Thus, high CXXC5 expression seems to affect several steps in human leukemogenesis, including intracellular events as well as extracellular communication.

  3. TrSDB: a proteome database of transcription factors

    Science.gov (United States)

    Hermoso, Antoni; Aguilar, Daniel; Aviles, Francesc X.; Querol, Enrique

    2004-01-01

    TrSDB—TranScout Database—(http://ibb.uab.es/trsdb) is a proteome database of eukaryotic transcription factors based upon predicted motifs by TranScout and data sources such as InterPro and Gene Ontology Annotation. Nine eukaryotic proteomes are included in the current version. Extensive and diverse information for each database entry, different analyses considering TranScout classification and similarity relationships are offered for research on transcription factors or gene expression. PMID:14681387

  4. Using TESS to predict transcription factor binding sites in DNA sequence.

    Science.gov (United States)

    Schug, Jonathan

    2008-03-01

    This unit describes how to use the Transcription Element Search System (TESS). This Web site predicts transcription factor binding sites (TFBS) in DNA sequence using two different kinds of models of sites, strings and positional weight matrices. The binding of transcription factors to DNA is a major part of the control of gene expression. Transcription factors exhibit sequence-specific binding; they form stronger bonds to some DNA sequences than to others. Identification of a good binding site in the promoter for a gene suggests the possibility that the corresponding factor may play a role in the regulation of that gene. However, the sequences transcription factors recognize are typically short and allow for some amount of mismatch. Because of this, binding sites for a factor can typically be found at random every few hundred to a thousand base pairs. TESS has features to help sort through and evaluate the significance of predicted sites.

  5. Microarray-Based Identification of Transcription Factor Target Genes

    NARCIS (Netherlands)

    Gorte, M.; Horstman, A.; Page, R.B.; Heidstra, R.; Stromberg, A.; Boutilier, K.A.

    2011-01-01

    Microarray analysis is widely used to identify transcriptional changes associated with genetic perturbation or signaling events. Here we describe its application in the identification of plant transcription factor target genes with emphasis on the design of suitable DNA constructs for controlling TF

  6. Repression of meiotic genes by antisense transcription and by Fkh2 transcription factor in Schizosaccharomyces pombe.

    Science.gov (United States)

    Chen, Huei-Mei; Rosebrock, Adam P; Khan, Sohail R; Futcher, Bruce; Leatherwood, Janet K

    2012-01-01

    In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the "unspliced" signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.

  7. Zinc Status Biomarkers and Cardiometabolic Risk Factors in Metabolic Syndrome: A Case Control Study

    Directory of Open Access Journals (Sweden)

    Erika P. S. Freitas

    2017-02-01

    Full Text Available Metabolic syndrome (MS involves pathophysiological alterations that might compromise zinc status. The aim of this study was to evaluate zinc status biomarkers and their associations with cardiometabolic factors in patients with MS. Our case control study included 88 patients with MS and 37 controls. We performed clinical and anthropometric assessments and obtained lipid, glycemic, and inflammatory profiles. We also evaluated zinc intake, plasma zinc, erythrocyte zinc, and 24-h urinary zinc excretion. The average zinc intake was significantly lower in the MS group (p < 0.001. Regression models indicated no significant differences in plasma zinc concentration (all p > 0.05 between the two groups. We found significantly higher erythrocyte zinc concentration in the MS group (p < 0.001 independent from co-variable adjustments. Twenty-four hour urinary zinc excretion was significantly higher in the MS group (p = 0.008, and adjustments for age and sex explained 21% of the difference (R2 = 0.21, p < 0.001. There were significant associations between zincuria and fasting blood glucose concentration (r = 0.479, waist circumference (r = 0.253, triglyceride concentration (r = 0.360, glycated hemoglobin concentration (r = 0.250, homeostatic model assessment—insulin resistance (r = 0.223, and high-sensitivity C-reactive protein concentration (r = 0.427 (all p < 0.05 in the MS group. Patients with MS had alterations in zinc metabolism mainly characterized by an increase in erythrocyte zinc and higher zincuria.

  8. Evaluation of the nutritional characteristics of a finger millet based complementary food.

    Science.gov (United States)

    Mbithi-Mwikya, Stephen; Van Camp, John; Mamiro, Peter R S; Ooghe, Wilfried; Kolsteren, Patrick; Huyghebaert, Andre

    2002-05-08

    Finger millet (Eleusine coracana), kidney beans (Phaseolus vulgaris), peanuts (Arachis hypogoea), and mango (Mangifera indica) were processed separately and then combined, on the basis of their amino acid scores and energy content, into a complementary food for children of weaning age. The finger millet and kidney beans were processed by germination, autoclaving, and lactic acid fermentation. A mixture containing, on a dry matter basis, 65.2, 19.1, 8.0, and 7.7% of the processed finger millet, kidney beans, peanuts, and mango, respectively, gave a composite protein with an in vitro protein digestibility of 90.2% and an amino acid chemical score of 0.84. This mixture had an energy density of 16.3 kJ.g(-1) of dry matter and a decreased antinutrient content and showed a measurable improvement in the in vitro extractability for calcium, iron, and zinc. A 33% (w/v) pap made from a mix of the processed ingredients had an energy density of 5.4 kJ.g(-1) of pap, which is sufficient to meet the energy requirements of well-nourished children of 6-24 months of age at three servings a day and at the FAO average breast-feeding frequency.

  9. Exploring the utility of organo-polyoxometalate hybrids to inhibit SOX transcription factors.

    Science.gov (United States)

    Narasimhan, Kamesh; Micoine, Kevin; Lacôte, Emmanuel; Thorimbert, Serge; Cheung, Edwin; Hasenknopf, Bernold; Jauch, Ralf

    2014-01-01

    SOX transcription factors constitute an attractive target class for intervention with small molecules as they play a prominent role in the field of regenerative biomedicine and cancer biology. However, rationally engineering specific inhibitors that interfere with transcription factor DNA interfaces continues to be a monumental challenge in the field of transcription factor chemical biology. Polyoxometalates (POMs) are inorganic compounds that were previously shown to target the high-mobility group (HMG) of SOX proteins at nanomolar concentrations. In continuation of this work, we carried out an assessment of the selectivity of a panel of newly synthesized organo-polyoxometalate hybrids in targeting different transcription factor families to enable the usage of polyoxometalates as specific SOX transcription factor drugs. The residual DNA-binding activities of 15 different transcription factors were measured after treatment with a panel of diverse polyoxometalates. Polyoxometalates belonging to the Dawson structural class were found to be more potent inhibitors than the Keggin class. Further, organically modified Dawson polyoxometalates were found to be the most potent in inhibiting transcription factor DNA binding activity. The size of the polyoxometalates and its derivitization were found to be the key determinants of their potency. Polyoxometalates are highly potent, nanomolar range inhibitors of the DNA binding activity of the Sox-HMG family. However, binding assays involving a limited subset of structurally diverse polyoxometalates revealed a low selectivity profile against different transcription factor families. Further progress in achieving selectivity and deciphering structure-activity relationship of POMs require the identification of POM binding sites on transcription factors using elaborate approaches like X-ray crystallography and multidimensional NMR. In summary, our report reaffirms that transcription factors are challenging molecular architectures

  10. TcoF-DB v2: update of the database of human and mouse transcription co-factors and transcription