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  1. The GATA transcription factor gene gtaG is required for terminal differentiation in Dictyostelium.

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    Katoh-Kurasawa, Mariko; Santhanam, Balaji; Shaulsky, Gad

    2016-03-09

    The GATA transcription factor GtaG is conserved in Dictyostelids and essential for terminal differentiation in Dictyostelium discoideum, but its function is not well understood. Here we show that gtaG is expressed in prestalk cells at the anterior region of fingers and in the extending stalk during culmination. The gtaG(-) phenotype is cell-autonomous in prestalk cells and non-cell-autonomous in prespore cells. Transcriptome analyses reveal that GtaG regulates prestalk gene expression during cell differentiation before culmination and is required for progression into culmination. GtaG-dependent genes include genetic suppressors of the Dd-STATa-defective phenotype as well as Dd-STATa target-genes, including extra cellular matrix genes. We show that GtaG may be involved in the production of two culmination-signaling molecules, cyclic di-GMP and the spore differentiation factor SDF-1 and that addition of c-di-GMP rescues the gtaG(-) culmination and spore formation deficiencies. We propose that GtaG is a regulator of terminal differentiation that functions in concert with Dd-STATa and controls culmination through regulating c-di-GMP and SDF-1 production in prestalk cells.

  2. Nuclear localization domains of GATA activator Gln3 are required for transcription of target genes through dephosphorylation in Saccharomyces cerevisiae.

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    Numamoto, Minori; Tagami, Shota; Ueda, Yusuke; Imabeppu, Yusuke; Sasano, Yu; Sugiyama, Minetaka; Maekawa, Hiromi; Harashima, Satoshi

    2015-08-01

    The GATA transcription activator Gln3 in the budding yeast (Saccharomyces cerevisiae) activates transcription of nitrogen catabolite repression (NCR)-sensitive genes. In cells grown in the presence of preferred nitrogen sources, Gln3 is phosphorylated in a TOR-dependent manner and localizes in the cytoplasm. In cells grown in non-preferred nitrogen medium or treated with rapamycin, Gln3 is dephosphorylated and is transported from the cytoplasm to the nucleus, thereby activating the transcription of NCR-sensitive genes. Caffeine treatment also induces dephosphorylation of Gln3 and its translocation to the nucleus and transcription of NCR-sensitive genes. However, the details of the mechanism by which phosphorylation controls Gln3 localization and transcriptional activity are unknown. Here, we focused on two regions of Gln3 with nuclear localization signal properties (NLS-K, and NLS-C) and one with nuclear export signal (NES). We constructed various mutants for our analyses: gln3 containing point mutations in all potential phosphoacceptor sites (Thr-339, Ser-344, Ser-347, Ser-355, Ser-391) in the NLS and NES regions to produce non-phosphorylatable (alanine) or mimic-phosphorylatable (aspartic acid) residues; and deletion mutants. We found that phosphorylation of Gln3 was impaired in all of these mutations and that the aspartic acid substitution mutants showed drastic reduction of Gln3-mediated transcriptional activity despite the fact that the mutations had no effect on nuclear localization of Gln3. Our observations suggest that these regions are required for transcription of target genes presumably through dephosphorylation.

  3. An Ebox element in the proximal Gata4 promoter is required for Gata4 expression in vivo.

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    Alain Boulende Sab

    Full Text Available GATA4 is an essential transcription factor required for the development and function of multiple tissues, including a major role in gonadogenesis. Despite its crucial role, the molecular mechanisms that regulate Gata4 expression in vivo remain poorly understood. We recently found that the Gata4 gene is expressed as multiple transcripts with distinct 5' origins. These co-expressed alternative transcripts are generated by different non-coding first exons with transcripts E1a and E1b being the most prominent. Moreover, we previously showed that an Ebox element, located in Gata4 5' flanking sequences upstream of exon 1a, is important for the promoter activity of these sequences in cell lines. To confirm the importance of this element in vivo, we generated and characterized Gata4 Ebox knockout mice. Quantitative PCR analyses realized on gonads, heart and liver at three developmental stages (embryonic, pre-pubertal and adult revealed that the Ebox mutation leads to a robust and specific decrease (up to 89% of Gata4 E1a transcript expression in all tissues and stages examined. However, a detailed characterization of the gonads revealed normal morphology and GATA4 protein levels in these mutants. Our qPCR data further indicate that this outcome is most likely due to the presence of Gata4 E1b mRNA, whose expression levels were not decreased by the Ebox mutation. In conclusion, our work clearly confirms the importance of the proximal Ebox element and suggests that adequate GATA4 protein expression is likely protected by a compensation mechanism between Gata4 E1a and E1b transcripts operating at the translational level.

  4. The chicken vitellogenin II gene is flanked by a GATA factor-dependent estrogen response unit.

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    Davis, D L; Burch, J B

    1996-08-01

    The chicken vitellogenin II (VTGII) gene is flanked by an imperfect estrogen response element (ERE) at -350 and a perfect ERE at -620. In the present study we show that this imperfect ERE lies within an estrogen response unit (ERU) that requires a GATA factor and the estrogen receptor to function as an estrogen-dependent enhancer. We infer that GATA-6 contributes to the estrogen-dependent and liver-specific regulation of the endogenous VTGII gene since this is the predominant GATA factor expressed in adult liver. Our analysis of the VTGII ERU revealed four salient points. First, this ERU is comprised of an ERE and a bank of functionally redundant GATA-binding sites. Second, the GATA-6 transactivation domain is necessary (and sufficient, when tethered near the ERE) to render this ERU functional. Third, ERU enhancer activity is dependent on GATA 6, regardless of whether the resident ERE is imperfect or perfect. Fourth, in contrast to a report that the estrogen receptor antagonizes the activity of another GATA factor (GATA-1), we show that these two factors can function in a synergistic manner within the context of the VTGII ERU.

  5. GATA4 mediates gene repression in the mature mouse small intestine through interactions with friend of GATA (FOG) cofactors

    NARCIS (Netherlands)

    E. Beuling (Eva); T. Bosse (Tjalling); D.J. Kerk (Daniel); C.M. Piaseckyj (Christina); Y. Fujiwara (Yuko); S.G. Katz (Samuel); S.H. Orkin (Stuart); R.J. Grand (Richard); S.D. Krasinski (Stephen)

    2008-01-01

    textabstractGATA4, a transcription factor expressed in the proximal small intestine but not in the distal ileum, maintains proximal-distal distinctions by multiple processes involving gene repression, gene activation, and cell fate determination. Friend of GATA (FOG) is an evolutionarily conserved f

  6. Gata4 is required for formation of the genital ridge in mice.

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    Yueh-Chiang Hu

    Full Text Available In mammals, both testis and ovary arise from a sexually undifferentiated precursor, the genital ridge, which first appears during mid-gestation as a thickening of the coelomic epithelium on the ventromedial surface of the mesonephros. At least four genes (Lhx9, Sf1, Wt1, and Emx2 have been demonstrated to be required for subsequent growth and maintenance of the genital ridge. However, no gene has been shown to be required for the initial thickening of the coelomic epithelium during genital ridge formation. We report that the transcription factor GATA4 is expressed in the coelomic epithelium of the genital ridge, progressing in an anterior-to-posterior (A-P direction, immediately preceding an A-P wave of epithelial thickening. Mouse embryos conditionally deficient in Gata4 show no signs of gonadal initiation, as their coelomic epithelium remains a morphologically undifferentiated monolayer. The failure of genital ridge formation in Gata4-deficient embryos is corroborated by the absence of the early gonadal markers LHX9 and SF1. Our data indicate that GATA4 is required to initiate formation of the genital ridge in both XX and XY fetuses, prior to its previously reported role in testicular differentiation of the XY gonad.

  7. Mi2β is required for γ-globin gene silencing: temporal assembly of a GATA-1-FOG-1-Mi2 repressor complex in β-YAC transgenic mice.

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    Flávia C Costa

    Full Text Available Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the -566 GATA motif of the (Aγ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of (Aγ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the -566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1-mediated repressor complex was disrupted by the -566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting -566 (Aγ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis.

  8. Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart.

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    Tarradas, Anna; Pinsach-Abuin, Mel Lina; Mackintosh, Carlos; Llorà-Batlle, Oriol; Pérez-Serra, Alexandra; Batlle, Montserrat; Pérez-Villa, Félix; Zimmer, Thomas; Garcia-Bassets, Ivan; Brugada, Ramon; Beltran-Alvarez, Pedro; Pagans, Sara

    2017-01-01

    Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. Lrrc10 is a novel cardiac-specific target gene of Nkx2-5 and GATA4

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    Brody, Matthew J.; Cho, Eunjin; Mysliwiec, Matthew R.; Kim, Tae-gyun; Carlson, Clayton D.; Lee, Kyu-Ho; Lee, Youngsook

    2013-01-01

    Cardiac gene expression is precisely regulated and its perturbation causes developmental defects and heart disease. Leucine-rich repeat containing 10 (Lrrc10) is a cardiac-specific factor that is crucial for proper cardiac development and deletion of Lrrc10 in mice results in dilated cardiomyopathy. However, the mechanisms regulating Lrrc10 expression in cardiomyocytes remain unknown. Therefore, we set out to determine trans-acting factors and cis-elements critical for mediating Lrrc10 expression. We identify Lrrc10 as a transcriptional target of Nkx2-5 and GATA4. The Lrrc10 promoter region contains two highly conserved cardiac regulatory elements, which are functional in cardiomyocytes but not in fibroblasts. In vivo, Nkx2-5 and GATA4 endogenously occupy the proximal and distal cardiac regulatory elements of Lrrc10 in the heart. Moreover, embryonic hearts of Nkx2-5 knockout mice have dramatically reduced expression of Lrrc10. These data demonstrate the importance of Nkx2-5 and GATA4 in regulation of Lrrc10 expression in vivo. The proximal cardiac regulatory element located at around −200 bp is synergistically activated by Nkx2-5 and GATA4 while the distal cardiac regulatory element present around −3 Kb requires SRF in addition to Nkx2-5 and GATA4 for synergistic activation. Mutational analyses identify a pair of adjacent Nkx2-5 and GATA binding sites within the proximal cardiac regulatory element that are necessary to induce expression of Lrrc10. In contrast, only the GATA site is functional in the distal regulatory element. Taken together, our data demonstrate that the transcription factors Nkx2-5 and GATA4 cooperatively regulate cardiac-specific expression of Lrrc10. PMID:23751912

  10. PIAS1 is a GATA4 SUMO ligase that regulates GATA4-dependent intestinal promoters independent of SUMO ligase activity and GATA4 sumoylation.

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    Narasimhaswamy S Belaguli

    Full Text Available GATA4 confers cell type-specific gene expression on genes expressed in cardiovascular, gastro-intestinal, endocrine and neuronal tissues by interacting with various ubiquitous and cell-type-restricted transcriptional regulators. By using yeast two-hybrid screening approach, we have identified PIAS1 as an intestine-expressed GATA4 interacting protein. The physical interaction between GATA4 and PIAS1 was confirmed in mammalian cells by coimmunoprecipitation and two-hybrid analysis. The interacting domains were mapped to the second zinc finger and the adjacent C-terminal basic region of GATA4 and the RING finger and the adjoining C-terminal 60 amino acids of PIAS1. PIAS1 and GATA4 synergistically activated IFABP and SI promoters but not LPH promoters suggesting that PIAS1 differentially activates GATA4 targeted promoters. In primary murine enterocytes PIAS1 was recruited to the GATA4-regulated IFABP promoter. PIAS1 promoted SUMO-1 modification of GATA4 on lysine 366. However, sumoylation was not required for the nuclear localization and stability of GATA4. Further, neither GATA4 sumoylation nor the SUMO ligase activity of PIAS1 was required for coactivation of IFABP promoter by GATA4 and PIAS1. Together, our results demonstrate that PIAS1 is a SUMO ligase for GATA4 that differentially regulates GATA4 transcriptional activity independent of SUMO ligase activity and GATA4 sumoylation.

  11. Decreased expression of the GATA3 gene is associated with poor prognosis in primary gastric adenocarcinoma.

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    Rajiv Prasad Keshari

    Full Text Available BACKGROUND: GATA binding protein 3 (GATA3 was recently proposed to function as a tumor suppressor gene in some types of human cancer. This study aims to investigate GATA3 expression and its prognostic significance in primary gastric adenocarcinoma. METHODOLOGY/PRINCIPAL FINDINGS: Using real-time quantitative PCR (RT-qPCR and immunohistochemical staining methods, GATA3 expression was analyzed in tissue samples from a consecutive series of 402 gastric adenocarcinoma patients who underwent resections between 2003 and 2006. The relationship between GATA3 expression, clinicopathological factors, and patient survival was investigated. The expression status of GATA3 was shown to be clearly reduced in the tumor tissue samples compared with that in the matched adjacent non-tumor tissue samples by RT-qPCR (P = 0.0014. Immunohistochemistry analysis indicated that GATA3 expression was significantly decreased in 225 of the 402 (56% gastric adenocarcinoma cases. Reduced GATA3 expression was also observed in patients with large tumors (P = 0.017, signet ring cell carcinoma or mucinous carcinoma (P = 0.005 and tumors with lymphatic or venous invasion (P = 0.040. Additionally, reduced expression of GATA3 was more commonly observed in tumors that were staged as T4a/b (P<0.001, N3 (P<0.001, or M1 (P<0.001. Kaplan-Meier survival curves revealed that reduced expression of GATA3 was associated with poor prognosis in gastric adenocarcinoma patients (P<0.001. Multivariate Cox analysis identified GATA3 expression as an independent prognostic factor for overall survival (HR = 5.375, 95% CI = 3.647-7.921, P<0.001. To investigate the predictive ability of the models with and without containing GATA3 gene expression, Harrell's c-index was calculated as a measure of predictive accuracy of survival outcome. The c-index values revealed that model containing GATA3 expression (c-index = 0.897 had superior discrimination ability to the model without containg it (c-index = 0

  12. Novel and functional DNA sequence variants within the GATA5 gene promoter in ventricular septal defects

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    Ji-Ping Shan; Xiao-Li Wang; Yuan-Gang Qiao; Hong-Xin Wan Yan; Wen-Hui Huang; Shu-Chao Pang; Bo Yan

    2014-01-01

    Background: Congenital heart disease (CHD) is the most common human birth defect. Genetic causes for CHD remain largely unknown. GATA transcription factor 5 (GATA 5) is an essential regulator for the heart development. Mutations in the GATA5 gene have been reported in patients with a variety of CHD. Since misregulation of gene expression have been associated with human diseases, we speculated that changed levels of cardiac transcription factors, GATA5, may mediate the development of CHD. Methods: In this study, GATA5 gene promoter was genetically and functionally analyzed in large cohorts of patients with ventricular septal defect (VSD) (n=343) and ethnic-matched healthy controls (n=348). Results: Two novel and heterozygous DNA sequence variants (DSVs), g.61051165A>G and g.61051463delC, were identified in three VSD patients, but not in the controls. In cultured cardiomyocytes, GATA5 gene promoter activities were significantly decreased by DSV g.61051165A>G and increased by DSV g.61051463delC. Moreover, fathers of the VSD patients carrying the same DSVs had reduced diastolic function of left ventricles. Three SNPs, g.61051279C>T (rs77067995), g.61051327A>C (rs145936691) and g.61051373G>A (rs80197101), and one novel heterozygous DSV, g.61051227C>T, were found in both VSD patients and controls with similar frequencies. Conclusion: Our data suggested that the DSVs in the GATA5 gene promoter may increase the susceptibility to the development of VSD as a risk factor.

  13. GATA factor switching from GATA2 to GATA1 contributes to erythroid differentiation.

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    Suzuki, Mikiko; Kobayashi-Osaki, Maki; Tsutsumi, Shuichi; Pan, Xiaoqing; Ohmori, Shin'ya; Takai, Jun; Moriguchi, Takashi; Ohneda, Osamu; Ohneda, Kinuko; Shimizu, Ritsuko; Kanki, Yasuharu; Kodama, Tatsuhiko; Aburatani, Hiroyuki; Yamamoto, Masayuki

    2013-11-01

    Transcription factor GATA2 is highly expressed in hematopoietic stem cells and progenitors, whereas its expression declines after erythroid commitment of progenitors. In contrast, the start of GATA1 expression coincides with the erythroid commitment and increases along with the erythroid differentiation. We refer this dynamic transition of GATA factor expression to as the 'GATA factor switching'. Here, we examined contribution of the GATA factor switching to the erythroid differentiation. In Gata1-knockdown embryos that concomitantly express Gata2-GFP reporter, high-level expression of GFP reporter was detected in accumulated immature hematopoietic cells with impaired differentiation, demonstrating that GATA1 represses Gata2 gene expression in hematopoietic progenitors in vivo. We have conducted chromatin immunoprecipitation (ChIP) on microarray analyses of GATA2 and GATA1, and results indicate that the GATA1-binding sites widely overlap with the sites pre-occupied by GATA2 before the GATA1 expression. Importantly, erythroid genes harboring GATA boxes bound by both GATA1 and GATA2 tend to be expressed in immature erythroid cells, whereas those harboring GATA boxes to which GATA1 binds highly but GATA2 binds only weakly are important for the mature erythroid cell function. Our results thus support the contention that preceding binding of GATA2 helps the following binding of GATA1 and thereby secures smooth expression of the transient-phase genes. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  14. Regulation of Cardiac Gene Expression by GATA-4/5/6.

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    Evans, T

    1997-04-01

    The identification of nuclear regulatory proteins provides great promise for advancing our understanding of the transcriptional control of cardiac gene expression. Three new members of the GATA family of DNA-binding transcription factors were recently discovered and designated GATA-4/5/6. On the basis of expression patterns, the identification of candidate cardiac target genes and the current understanding of how other GATA factors function in the hematopoietic system, it appears that these genes are important for regulating programs of cardiac development and terminal differentiation. Indeed, a functional role for GATA-4/5/6 in activating the cardiac differentiation program was demonstrated in cell culture and embryonic systems; however, recent results obtained in embryonic stem (ES) cells with a targeted mutation of GATA-4 raise new questions regarding specificity of action among the three genes. The future direction of research in the field is discussed; understanding GATA-4/5/6 function and regulation is likely to provide important insight into the specification and/or differentiation of cardiac progenitors, development and morphogenesis of the heart, and regulation of cardiac-specific gene expression. (Trends Cardiovasc Med 1997;7:75-83). © 1997, Elsevier Science Inc.

  15. GATA4 gene mutation in congenital heart disease%先天性心脏病GATA4基因突变研究进展

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    张艳丽

    2015-01-01

    GATA4 gene mutations are common in children with congenital heart disease.More than 120 mutations have been found in ventricular septal defect,atrial septal defects and the tetralogy of fallot.But the relationship between genotype and clinical phenotype has not yet been elucidated.This article summarizes the published germline and somatic mutations of GATA4 gene in human congenital heart disease,and provides insight into the phenotypic spectrum of GATA4 gene mutation.%GATA4基因突变在先天性心脏病患儿中较为常见.目前为止,已发现超过120个突变位点,其中在室间隔缺损、房室间隔缺损或法洛四联症患者中多见,但突变基因型与临床表型间的关系至今仍未阐明.该文总结了在人类先天性心脏病中已发表的GATA4基因突变位点,并绘制出功能性改变的基因突变图谱.

  16. MGMT, GATA6, CD81, DR4, and CASP8 gene promoter methylation in glioblastoma

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    Skiriute Daina

    2012-06-01

    Full Text Available Abstract Background Methylation of promoter region is the major mechanism affecting gene expression in tumors. Recent methylome studies of brain tumors revealed a list of new epigenetically modified genes. Our aim was to study promoter methylation of newly identified epigenetically silenced genes together with already known epigenetic markers and evaluate its separate and concomitant role in glioblastoma genesis and patient outcome. Methods The methylation status of MGMT, CD81, GATA6, DR4, and CASP8 in 76 patients with primary glioblastomas was investigated. Methylation-specific PCR reaction was performed using bisulfite treated DNA. Evaluating glioblastoma patient survival time after operation, patient data and gene methylation effect on survival was estimated using survival analysis. Results The overwhelming majority (97.3% of tumors were methylated in at least one of five genes tested. In glioblastoma specimens gene methylation was observed as follows: MGMT in 51.3%, GATA6 in 68.4%, CD81 in 46.1%, DR4 in 41.3% and CASP8 in 56.8% of tumors. Methylation of MGMT was associated with younger patient age (p CASP8 with older (p MGMT methylation was significantly more frequent event in patient group who survived longer than 36 months after operation (p CASP8 was more frequent in patients who survived shorter than 36 months (p MGMT, GATA6 and CASP8 as independent predictors for glioblastoma patient outcome (p MGMT and GATA6 were independent predictors for patient survival in younger patients’ group, while there were no significant associations observed in older patients’ group when adjusted for therapy. Conclusions High methylation frequency of tested genes shows heterogeneity of glioblastoma epigenome and the importance of MGMT, GATA6 and CASP8 genes methylation in glioblastoma patient outcome.

  17. GATA transcription factor required for immunity to bacterial and fungal pathogens.

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    Samantha Kerry

    Full Text Available In the past decade, Caenorhabditis elegans has been used to dissect several genetic pathways involved in immunity; however, little is known about transcription factors that regulate the expression of immune effectors. C. elegans does not appear to have a functional homolog of the key immune transcription factor NF-kappaB. Here we show that that the intestinal GATA transcription factor ELT-2 is required for both immunity to Salmonella enterica and expression of a C-type lectin gene, clec-67, which is expressed in the intestinal cells and is a good marker of S. enterica infection. We also found that ELT-2 is required for immunity to Pseudomonas aeruginosa, Enterococcus faecalis, and Cryptococcus neoformans. Lack of immune inhibition by DAF-2, which negatively regulates the FOXO transcription factor DAF-16, rescues the hypersusceptibility to pathogens phenotype of elt-2(RNAi animals. Our results indicate that ELT-2 is part of a multi-pathogen defense pathway that regulates innate immunity independently of the DAF-2/DAF-16 signaling pathway.

  18. The GATA3 gene is involved in leprosy susceptibility in Brazilian patients.

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    Medeiros, Priscila; da Silva, Weber Laurentino; de Oliveira Gimenez, Bruna Beatriz; Vallezi, Keren Bastos; Moraes, Milton Ozório; de Souza, Vânia Niéto Brito; Latini, Ana Carla Pereira

    2016-04-01

    Leprosy outcome is a complex trait and the host-pathogen-environment interaction defines the emergence of the disease. Host genetic risk factors have been successfully associated to leprosy. The 10p13 chromosomal region was linked to leprosy in familial studies and GATA3 gene is a strong candidate to be part of this association. Here, we tested tag single nucleotide polymorphisms at GATA3 in two case-control samples from Brazil comprising a total of 1633 individuals using stepwise strategy. The A allele of rs10905284 marker was associated with leprosy resistance. Then, a functional analysis was conducted and showed that individuals carrying AA genotype express higher levels of GATA-3 protein in lymphocytes. So, we confirmed that the rs10905284 is a locus associated to leprosy and influences the levels of this transcription factor in the Brazilian population.

  19. GATA3 Gene Polymorphisms Associated with Allergic Rhinitis in an Iranian Population

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    Shirkani, Afshin; Mansouri, Atena; Abbaszadegan, Mohammad Reza; Faridhosseini, Reza; Jabbari Azad, Farahzad; Gholamin, Mehran

    2017-01-01

    Background: The development of allergic rhinitis (AR) is caused by the interaction between genetic predisposition and environmental factors. In this study, the association between GATA3 single nucleotide polymorphisms and AR in an Iranian population was identified. Methods: This case-control study was performed on 86 patients with AR and 86 healthy subjects. This study aimed to evaluate a potential association between two GATA3 SNPs, rs1269486 and rs2229360, and AR. Blood samples were collected and DNA was extracted for the evaluation of these SNPs by RFLP-PCR. Results: A statistically-significant association was found between rs1269486 and AR (Ppopulation. Because of the significance of this gene in AR, studying the association between GATA3 polymorphisms and AR is recommended for other populations. PMID:28367470

  20. Transcription factor GATA1 is dispensable for mast cell differentiation in adult mice.

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    Ohneda, Kinuko; Moriguchi, Takashi; Ohmori, Shin'ya; Ishijima, Yasushi; Satoh, Hironori; Philipsen, Sjaak; Yamamoto, Masayuki

    2014-05-01

    Although previous studies have shown that GATA1 is required for mast cell differentiation, the effects of the complete ablation of GATA1 in mast cells have not been examined. Using conditional Gata1 knockout mice (Gata1(-/y)), we demonstrate here that the complete ablation of GATA1 has a minimal effect on the number and distribution of peripheral tissue mast cells in adult mice. The Gata1(-/y) bone marrow cells were capable of differentiating into mast cells ex vivo. Microarray analyses showed that the repression of GATA1 in bone marrow mast cells (BMMCs) has a small impact on the mast cell-specific gene expression in most cases. Interestingly, however, the expression levels of mast cell tryptases in the mouse chromosome 17A3.3 were uniformly reduced in the GATA1 knockdown cells, and GATA1 was found to bind to a 500-bp region at the 5' end of this locus. Revealing a sharp contrast to that observed in the Gata1-null BMMCs, GATA2 deficiency resulted in a significant loss of the c-Kit(+) FcεRIα(+) mast cell fraction and a reduced expression of several mast cell-specific genes. Collectively, GATA2 plays a more important role than GATA1 in the regulation of most mast cell-specific genes, while GATA1 might play specific roles in mast cell functions.

  1. The Oxidative Stress Response in Caenorhabditis elegans Requires the GATA Transcription Factor ELT-3 and SKN-1/Nrf2.

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    Hu, Queenie; D'Amora, Dayana R; MacNeil, Lesley T; Walhout, Albertha J M; Kubiseski, Terrance J

    2017-08-01

    Cellular damage caused by reactive oxygen species is believed to be a major contributor to age-associated diseases. Previously, we characterized the Caenorhabditis elegans Brap2 ortholog (BRAP-2) and found that it is required to prevent larval arrest in response to elevated levels of oxidative stress. Here, we report that C. elegans brap-2 mutants display increased expression of SKN-1-dependent, phase II detoxification enzymes that is dependent on PMK-1 (a p38 MAPK C. elegans ortholog). An RNA-interference screen was conducted using a transcription factor library to identify genes required for increased expression of the SKN-1 target gst-4 in brap-2 mutants. We identified ELT-3, a member of the GATA transcription factor family, as a positive regulator of gst-4p::gfp expression. We found that ELT-3 interacts with SKN-1 to activate gst-4 transcription in vitro and that elt-3 is required for enhanced gst-4 expression in the brap-2(ok1492) mutant in vivo Furthermore, nematodes overexpressing SKN-1 required ELT-3 for life-span extension. Taken together, these results suggest a model where BRAP-2 acts as negative regulator of SKN-1 through inhibition of p38 MAPK activity, and that the GATA transcription factor ELT-3 is required along with SKN-1 for the phase II detoxification response in C. elegans. Copyright © 2017 by the Genetics Society of America.

  2. The HMG-box transcription factor Sox4b is required for pituitary expression of gata2a and specification of thyrotrope and gonadotrope cells in zebrafish.

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    Quiroz, Yobhana; Lopez, Mauricio; Mavropoulos, Anastasia; Motte, Patrick; Martial, Joseph A; Hammerschmidt, Matthias; Muller, Marc

    2012-06-01

    The pituitary is a complex gland comprising different cell types each secreting specific hormones. The extensive network of signaling molecules and transcription factors required for determination and terminal differentiation of specific cell types is still not fully understood. The SRY-like HMG-box (SOX) transcription factor Sox4 plays important roles in many developmental processes and has two homologs in zebrafish, Sox4a and Sox4b. We show that the sox4b gene is expressed in the pituitary anlagen starting at 24 h after fertilization (hpf) and later in the entire head region including the pituitary. At 48 hpf, sox4b mRNA colocalizes with that for TSH (tshβ), glycoprotein subunit α (gsuα), and the Zn finger transcription factor Gata2a. Loss of Sox4b function, using morpholino knockdown or expression of a dominant-negative Sox4 mutant, leads to a drastic decrease in tshβ and gsuα expression and reduced levels of gh, whereas other anterior pituitary gland markers including prl, slβ, pomc, and lim3 are not affected. Sox4b is also required for expression of gata2a in the pituitary. Knockdown of gata2a leads to decreased tshβ and gsuα expression at 48 hpf, similar to sox4b morphants. Injection of gata2a mRNA into sox4b morphants rescued tshβ and gsuα expression in thyrotrope cells. Finally, sox4b or gata2a knockdown causes a significant decrease of gonadotropin expression (lhβ and fshβ) at 4 d after fertilization. In summary, our results indicate that Sox4b is expressed in zebrafish during pituitary development and plays a crucial role in the differentiation of thyrotrope and gonadotrope cells through induction of gata2a expression in the developing pituitary.

  3. Activation of GATA4 gene expression at the early stage of cardiac specification

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    Ayse eYilbas

    2014-03-01

    Full Text Available Currently, there are no effective treatments to directly repair damaged heart tissue after cardiac injury since existing therapies focus on rescuing or preserving reversibly damaged tissue. Cell-based therapies using cardiomyocytes generated from stem cells present a promising therapeutic approach to directly replace damaged myocardium with new healthy tissue. However, the molecular mechanisms underlying the commitment of stem cells into cardiomyocytes are not fully understood and will be critical to guide this new technology into the clinic. Since GATA4 is a critical regulator of cardiac differentiation, we examined the molecular basis underlying the early activation of GATA4 gene expression during cardiac differentiation of pluripotent stem cells. Our studies demonstrate the direct involvement of histone acetylation and transcriptional coactivator p300 in the regulation of GATA4 gene expression. More importantly, we show that histone acetyltransferase (HAT activity is important for GATA4 gene expression with the use of curcumin, a HAT inhibitor. In addition, the widely used histone deacetylase inhibitor valproic acid enhances both histone acetylation and cardiac specification.

  4. Activation of GATA4 gene expression at the early stage of cardiac specification

    Science.gov (United States)

    Yilbas, Ayse; Hamilton, Alison; Wang, Yingjian; Mach, Hymn; Lacroix, Natascha; Davis, Darryl; Chen, Jihong; LI, Qiao

    2014-03-01

    Currently, there are no effective treatments to directly repair damaged heart tissue after cardiac injury since existing therapies focus on rescuing or preserving reversibly damaged tissue. Cell-based therapies using cardiomyocytes generated from stem cells present a promising therapeutic approach to directly replace damaged myocardium with new healthy tissue. However, the molecular mechanisms underlying the commitment of stem cells into cardiomyocytes are not fully understood and will be critical to guide this new technology into the clinic. Since GATA4 is a critical regulator of cardiac differentiation, we examined the molecular basis underlying the early activation of GATA4 gene expression during cardiac differentiation of pluripotent stem cells. Our studies demonstrate the direct involvement of histone acetylation and transcriptional coactivator p300 in the regulation of GATA4 gene expression. More importantly, we show that histone acetyltransferase (HAT) activity is important for GATA4 gene expression with the use of curcumin, a HAT inhibitor. In addition, the widely used histone deacetylase inhibitor valproic acid enhances both histone acetylation and cardiac specification.

  5. Amino-terminal extension of 146 residues of L-type GATA-6 is required for transcriptional activation but not for self-association.

    Science.gov (United States)

    Takada, Kayoko; Obayashi, Kanako; Ohashi, Kazuaki; Ohashi-Kobayashi, Ayako; Nakanishi-Matsui, Mayumi; Maeda, Masatomo

    2014-10-01

    Transcription factor GATA-6 plays essential roles in developmental processes and tissue specific functions through regulation of gene expression. GATA-6 mRNA utilizes two Met-codons in frame as translational initiation codons. Deletion of the nucleotide sequence encoding the PEST sequence (Glu(31)-Cys(46)) between the two initiation codons unusually reduced the protein molecular size on SDS-polyacrylamide gel-electrophoresis, and re-introduction of this sequence reversed this change. The long-type (L-type) GATA-6 containing this PEST sequence self-associated similarly to the short-type (S-type) GATA-6, as determined on co-immunoprecipitation of Myc-tagged GATA-6 with HA-tagged GATA-6. The L-type and S-type GATA-6 also interacted mutually. The L-type GATA-6 without the PEST sequence also self-associated and interacted with the S-type GATA-6. The transcriptional activation potential of L-type GATA-6 is higher than that of S-type GATA-6. When the PEST sequence (Glu(31)-Cys(46)) was inserted into the L-type GATA-6 without Arg(13)-Gly(101), the resultant recombinant protein showed significantly higher transcriptional activity, while the construct with an unrelated sequence exhibited lower activity. These results suggest that the Glu(31)-Cys(46) segment plays an important role in the transcriptional activation, although it does not participate in the self-association.

  6. Global transcriptome and chromatin occupancy analysis reveal the short isoform of GATA1 is deficient for erythroid specification and gene expression.

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    Chlon, Timothy M; McNulty, Maureen; Goldenson, Benjamin; Rosinski, Alexander; Crispino, John D

    2015-05-01

    GATA1 is a master transcriptional regulator of the differentiation of several related myeloid blood cell types, including erythrocytes and megakaryocytes. Germ-line mutations that cause loss of full length GATA1, but allow for expression of the short isoform (GATA1s), are associated with defective erythropoiesis in a subset of patients with Diamond Blackfan Anemia. Despite extensive studies of GATA1s in megakaryopoiesis, the mechanism by which GATA1s fails to support normal erythropoiesis is not understood. In this study, we used global gene expression and chromatin occupancy analysis to compare the transcriptional activity of GATA1s to GATA1. We discovered that compared to GATA1, GATA1s is less able to activate the erythroid gene expression program and terminal differentiation in cells with dual erythroid-megakaryocytic differentiation potential. Moreover, we found that GATA1s bound to many of its erythroid-specific target genes less efficiently than full length GATA1. These results suggest that the impaired ability of GATA1s to promote erythropoiesis in DBA may be caused by failure to occupy erythroid-specific gene regulatory elements. Copyright© Ferrata Storti Foundation.

  7. GATA4 and GATA6 control mouse pancreas organogenesis

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    Carrasco, Manuel; Delgado, Irene; Soria, Bernat; Martín, Francisco; Rojas, Anabel

    2012-01-01

    Recently, heterozygous mutations in GATA6 have been found in neonatal diabetic patients with failed pancreatic organogenesis. To investigate the roles of GATA4 and GATA6 in mouse pancreas organogenesis, we conditionally inactivated these genes within the pancreas. Single inactivation of either gene did not have a major impact on pancreas formation, indicating functional redundancy. However, double Gata4/Gata6 mutant mice failed to develop pancreata, died shortly after birth, and displayed hyperglycemia. Morphological defects in Gata4/Gata6 mutant pancreata were apparent during embryonic development, and the epithelium failed to expand as a result of defects in cell proliferation and differentiation. The number of multipotent pancreatic progenitors, including PDX1+ cells, was reduced in the Gata4/Gata6 mutant pancreatic epithelium. Remarkably, deletion of only 1 Gata6 allele on a Gata4 conditional knockout background severely reduced pancreatic mass. In contrast, a single WT allele of Gata4 in Gata6 conditional knockout mice was sufficient for normal pancreatic development, indicating differential contributions of GATA factors to pancreas formation. Our results place GATA factors at the top of the transcriptional network hierarchy controlling pancreas organogenesis. PMID:23006330

  8. Methylation of Gata3 protein at Arg-261 regulates transactivation of the Il5 gene in T helper 2 cells.

    Science.gov (United States)

    Hosokawa, Hiroyuki; Kato, Miki; Tohyama, Hiroyuki; Tamaki, Yuuki; Endo, Yusuke; Kimura, Motoko Y; Tumes, Damon John; Motohashi, Shinichiro; Matsumoto, Masaki; Nakayama, Keiichi I; Tanaka, Tomoaki; Nakayama, Toshinori

    2015-05-22

    Gata3 acts as a master regulator for T helper 2 (Th2) cell differentiation by inducing chromatin remodeling of the Th2 cytokine loci, accelerating Th2 cell proliferation, and repressing Th1 cell differentiation. Gata3 also directly transactivates the interleukin-5 (Il5) gene via additional mechanisms that have not been fully elucidated. We herein identified a mechanism whereby the methylation of Gata3 at Arg-261 regulates the transcriptional activation of the Il5 gene in Th2 cells. Although the methylation-mimicking Gata3 mutant retained the ability to induce IL-4 and repress IFNγ production, the IL-5 production was selectively impaired. We also demonstrated that heat shock protein (Hsp) 60 strongly associates with the methylation-mimicking Gata3 mutant and negatively regulates elongation of the Il5 transcript by RNA polymerase II. Thus, arginine methylation appears to play a pivotal role in the organization of Gata3 complexes and the target gene specificity of Gata3.

  9. Furin gene (fur) regulation in differentiating human megakaryoblastic Dami cells: involvement of the proximal GATA recognition motif in the P1 promoter and impact on the maturation of furin substrates.

    Science.gov (United States)

    Laprise, Marie-Hélène; Grondin, Francine; Cayer, Pauline; McDonald, Patrick P; Dubois, Claire M

    2002-11-15

    The convertase furin is involved in the maturation of key growth/aggregation mediators synthesized by the platelet producers, megakaryocytes, but the regulation of furin in these cells remains unknown. Computer-assisted search of the furin promoter sequence revealed multiple potential binding motifs for GATA-1, suggesting that furin is expressed and regulated in these cells. Using megakaryoblastic Dami cells, we observed that fur mRNA expression increased gradually on phorbol 12-myristate 13-acetate-induced differentiation, reaching maximum levels (8.3-fold increase) at 10 days. Transient transfections with P1, P1A, or P1B fur-LUC-promoter constructs revealed that in Dami cells, the P1 promoter is the strongest and the most sensitive to forced expression of GATA-1. Coexpression of GATA-1 and its comodulator, Friend of GATA-1 (FOG-1), resulted in a cooperative increase in P1 activity. Deletion analysis indicated that important GATA-1-regulated sequences are located in the most proximal region of the P1 promoter. Further analysis revealed 2 potential GATA-binding motifs at positions -66 and +62. Point mutation of each of the 2 motifs indicated that the intactness of the first GATA site is required for full basal and GATA-1-stimulated promoter activity. Finally, the inhibition of furin activity through gene transfer of the inhibitor alpha1-AT-PDX led to a block in maturation of the furin substrates transforming growth factor-beta1 and platelet-derived growth factor. Taken together, these results indicate that the most proximal GATA element in the P1 promoter is needed for fur gene expression in megakaryoblastic cells. They also suggest that proper regulation of the fur gene in megakaryocytes has an impact on the activation of furin substrates involved in megakaryocyte maturation and platelet functions.

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

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    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.

  11. Transcription factor gene GATA2: Association of leukemia and nonsynonymous to the synonymous substitution rate across five mammals.

    Science.gov (United States)

    Mazumder, Tarikul Huda; Uddin, Arif; Chakraborty, Supriyo

    2016-04-01

    GATA2 gene encodes a member of the GATA family of zinc-finger transcription factors that play a pivotal role during the transition of primitive blood forming cells into white blood cells. Mutation in GATA2 results in the loss of function or even gain of function, including abnormal proliferation of white blood cells that may predispose to acute myeloid leukemia. Our results showed that the codon usage in GATA2 has been influenced by GC mutation bias where nature has highly favored fourteen most over represented codons but disfavored the ATA codon across five mammals. Purifying natural selection has affected GATA2 gene in human and other mammals to maintain its protein function during the period of evolution. Our findings report an insight into the codon usage patterns in gaining the clues for codon optimization to alter the translational efficiency as well as for the functional conservation of gene expression and the significance of nucleotide composition in GATA2 gene within mammals.

  12. Recovery from an acute infection in C. elegans requires the GATA transcription factor ELT-2.

    Science.gov (United States)

    Head, Brian; Aballay, Alejandro

    2014-10-01

    The mechanisms involved in the recognition of microbial pathogens and activation of the immune system have been extensively studied. However, the mechanisms involved in the recovery phase of an infection are incompletely characterized at both the cellular and physiological levels. Here, we establish a Caenorhabditis elegans-Salmonella enterica model of acute infection and antibiotic treatment for studying biological changes during the resolution phase of an infection. Using whole genome expression profiles of acutely infected animals, we found that genes that are markers of innate immunity are down-regulated upon recovery, while genes involved in xenobiotic detoxification, redox regulation, and cellular homeostasis are up-regulated. In silico analyses demonstrated that genes altered during recovery from infection were transcriptionally regulated by conserved transcription factors, including GATA/ELT-2, FOXO/DAF-16, and Nrf/SKN-1. Finally, we found that recovery from an acute bacterial infection is dependent on ELT-2 activity.

  13. Recovery from an acute infection in C. elegans requires the GATA transcription factor ELT-2.

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    Brian Head

    2014-10-01

    Full Text Available The mechanisms involved in the recognition of microbial pathogens and activation of the immune system have been extensively studied. However, the mechanisms involved in the recovery phase of an infection are incompletely characterized at both the cellular and physiological levels. Here, we establish a Caenorhabditis elegans-Salmonella enterica model of acute infection and antibiotic treatment for studying biological changes during the resolution phase of an infection. Using whole genome expression profiles of acutely infected animals, we found that genes that are markers of innate immunity are down-regulated upon recovery, while genes involved in xenobiotic detoxification, redox regulation, and cellular homeostasis are up-regulated. In silico analyses demonstrated that genes altered during recovery from infection were transcriptionally regulated by conserved transcription factors, including GATA/ELT-2, FOXO/DAF-16, and Nrf/SKN-1. Finally, we found that recovery from an acute bacterial infection is dependent on ELT-2 activity.

  14. GATA4 and NKX2.5 gene analysis in Chinese Uygur patients with congenital heart disease

    Institute of Scientific and Technical Information of China (English)

    ZHANG Wei-min; LI Xiao-feng; MA Zhong-yuan; ZHANG Jing; ZHOU Si-hai; LI Tao; SHI Lin; LI Zhong-zhi

    2009-01-01

    Background Congenital heart disease (CHD) is the most common developmental anomaly in newborns. The germline mutations in GATA4 and NKX2.5 genes have been identified as responsible for CHD. The frequency of GATA4 and NKX2.5 mutations in Chinese Uygur patients with CHD and the correlation between their genotype and CHD phenotype are unknown.Methods We examined the coding region of GATA4 and NKX2.5 genes in 62 Chinese Uygur patients with CHD and 117 Chinese Uygur individuals as the controls by denaturing high performance liquid chromatography (DHPLC) and sequencing.Results Two heterozygous missense mutations of c.1220C>A and c.1273G>A in GATA4 gene, which cause the amino acid residue changes of P407Q and D425N in GATA4, were found in a patient with tetralogy of Fallot and a patient with ventricular septal defect, respectively. The two patients did not have atrioventricular conduct defects or non-cardiac abnormalities. The two mutations are expected to affect the protein function. There were no reported NKX2.5 mutations in the patients.Conclusion Our results provided the primary data on CHD phenotype associated with GATA4 mutation in the Chinese Uygur population.

  15. The leukemia associated ETO nuclear repressor gene is regulated by the GATA-1 transcription factor in erythroid/megakaryocytic cells

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    Gullberg Urban

    2010-05-01

    Full Text Available Abstract Background The Eight-Twenty-One (ETO nuclear co-repressor gene belongs to the ETO homologue family also containing Myeloid Translocation Gene on chromosome 16 (MTG16 and myeloid translocation Gene-Related protein 1 (MTGR1. By chromosomal translocations ETO and MTG16 become parts of fusion proteins characteristic of morphological variants of acute myeloid leukemia. Normal functions of ETO homologues have as yet not been examined. The goal of this work was to identify structural and functional promoter elements upstream of the coding sequence of the ETO gene in order to explore lineage-specific hematopoietic expression and get hints to function. Results A putative proximal ETO promoter was identified within 411 bp upstream of the transcription start site. Strong ETO promoter activity was specifically observed upon transfection of a promoter reporter construct into erythroid/megakaryocytic cells, which have endogeneous ETO gene activity. An evolutionary conserved region of 228 bp revealed potential cis-elements involved in transcription of ETO. Disruption of the evolutionary conserved GATA -636 consensus binding site repressed transactivation and disruption of the ETS1 -705 consensus binding site enhanced activity of the ETO promoter. The promoter was stimulated by overexpression of GATA-1 into erythroid/megakaryocytic cells. Electrophoretic mobility shift assay with erythroid/megakaryocytic cells showed specific binding of GATA-1 to the GATA -636 site. Furthermore, results from chromatin immunoprecipitation showed GATA-1 binding in vivo to the conserved region of the ETO promoter containing the -636 site. The results suggest that the GATA -636 site may have a role in activation of the ETO gene activity in cells with erythroid/megakaryocytic potential. Leukemia associated AML1-ETO strongly suppressed an ETO promoter reporter in erythroid/megakaryocytic cells. Conclusions We demonstrate that the GATA-1 transcription factor binds and

  16. Suppression of erythropoietin gene expression by cadmium depends on inhibition of HIF-1, not stimulation of GATA-2

    Energy Technology Data Exchange (ETDEWEB)

    Obara, Naoshi; Imagawa, Shigehiko; Nakano, Yoko; Nagasawa, Toshiro [Division of Hematology, Institute of Clinical Medicine, University of Tsukuba, 305-8575, Tsukuba, Ibaraki (Japan); Suzuki, Norio; Yamamoto, Masayuki [Center for Tsukuba Advanced Research Alliance and Institute of Basic Medical Sciences, University of Tsukuba, 305-8577, Tsukuba, Ibaraki (Japan)

    2003-05-01

    Long-term exposure of rats to cadmium (Cd) resulted in a marked suppression of erythropoietin (Epo) mRNA expression in the kidneys and the development of severe anemia. A recent report revealed that Cd inhibited hypoxia-inducible factor 1 (HIF-1) binding activity and Epo mRNA expression and protein production. However, Epo gene expression is also regulated by transcription factor GATA-2, which binds to the GATA binding site of the Epo promoter. To elucidate the mechanism of suppression of Epo by Cd, the effect of Cd on GATA-2 function was studied. Epo promoter/enhancer luciferase constructs, one with the wild-type promoter and another with a promoter with a mutant GATA site, were transfected into Hep3B cells. No significant difference in Epo promoter activity in these two types of cells was observed in the presence of Cd. The binding activity of GATA-2 was not affected by Cd. This study showed that Cd inhibited HIF-1 binding activity and Epo promoter activity, and then suppressed Epo protein production. Inhibition of Epo gene expression by Cd depends on suppression of HIF-1 binding activity, not on alteration of GATA function. (orig.)

  17. The STAT5-GATA2 pathway is critical in basophil and mast cell differentiation and maintenance.

    Science.gov (United States)

    Li, Yapeng; Qi, Xiaopeng; Liu, Bing; Huang, Hua

    2015-05-01

    Transcription factor GATA binding protein 2 (GATA2) plays critical roles in hematopoietic stem cell survival and proliferation, granulocyte-monocyte progenitor differentiation, and basophil and mast cell differentiation. However, precise roles of GATA2 in basophil and mast cell differentiation and maintenance have not been delineated. We have identified GATA2 as an essential transcription factor in differentiation of newly identified common basophil and mast cell progenitors into basophils and mast cells. We observed Gata2 haploinsufficiency for mast cell differentiation, but not for basophil differentiation. We examined the precise role of GATA2 in maintaining the expression of a wide range of genes that are important for performing basophil or mast cell functions. The effects of GATA2 on gene expression were broadly based. We demonstrated that GATA2 was required for maintaining Fcer1a mRNA and FcεRIα protein expression on both basophils and mast cells, as well as for maintaining Kit mRNA and c-Kit protein expression on mast cells. GATA2 was required for histamine synthesis and was also critical for Il4 mRNA expression in basophils and Il13 mRNA expression in mast cells. We demonstrate a STAT5-GATA2 connection, showing that the STAT5 transcription factor directly bound to the promoter and an intronic region of the Gata2 gene. Overexpression of the Gata2 gene was sufficient to direct basophil and mast cell differentiation in the absence of the Stat5 gene. Our study reveals that the STAT5-GATA2 pathway is critical for basophil and mast cell differentiation and maintenance.

  18. Single-gene association between GATA-2 and autoimmune hepatitis: A novel genetic insight highlighting immunologic pathways to disease.

    Science.gov (United States)

    Webb, Gwilym; Chen, Yung-Yi; Li, Ka-Kit; Neil, Desley; Oo, Ye Htun; Richter, Alex; Bigley, Venetia; Collin, Matthew; Adams, David H; Hirschfield, Gideon M

    2016-05-01

    Background & Aims Autoimmune hepatitis (AIH), an immune-mediated liver disease, originates as a consequence of interacting genetic and environmental risk factors. Treatment remains non-specific and prone to side effects. Deficiencies in regulatory T cell (Treg) function are hypothesized to contribute to the pathogenesis of AIH. Methods We describe an adult patient who presented with AIH in the context of monocytopenia. The patient was characterized by GATA2 gene sequencing, flow cytometry of peripheral blood for leucocyte subsets, ELISA for serum Flt-3 ligand, and immunohistochemistry of liver biopsy tissue. Results Sequencing confirmed a GATA2 mutation. Peripheral Treg were absent in the context of a preserved total T cell count. Immunostaining for the Treg transcription factor FOXP3 was reduced in liver tissue as compared to a control AIH specimen. There were marked deficiencies in multiple antigen-presenting cell subsets and Flt-3 ligand was elevated. These findings are consistent with previous reports of GATA2 dysfunction. Conclusions The association of a GATA2 mutation with AIH is previously unrecognized. GATA2 encodes a hematopoietic cell transcription factor, and mutations may manifest as monocytopenia, dendritic and B cell deficiencies, myelodysplasia, and immunodeficiency. Tregs may be depleted as in this case. Our findings provide support for the role of Tregs in AIH, complement reports of other deficiencies in T cell regulation causing AIH-like syndromes, and support the rationale of attempting to modulate the Treg axis for the therapeutic benefit of AIH patients.

  19. A study of the role of GATA2 gene polymorphism in coronary artery disease risk traits.

    Science.gov (United States)

    Muiya, Nzioka P; Wakil, Salma; Al-Najai, Mohammed; Tahir, Asma I; Baz, Batoul; Andres, Editha; Al-Boudari, Olyan; Al-Tassan, Nada; Al-Shahid, Maie; Meyer, Brian F; Dzimiri, Nduna

    2014-07-10

    The GATA2 is a multi-catalytic transcription factor believed to play an important role in regulating inflammatory processes, largely contributory to cardiovascular-related events. However, its role in coronary artery disease (CAD) risk traits remains poorly understood. In a preliminary study using Affymetrix 250K, we established a link on chromosome (chr) 3, which harbors the GATA2 gene, to early onset of CAD in two families with heterozygous familial hyperlipidemia (HFH), suggesting a role for the gene in metabolic-related CAD in the general population. We then sequenced the gene in the families and an additional 200 individuals in the general population, followed by an association study for 8 SNPs on CAD metabolic risk traits in a total of 4557 individuals (2386 CAD cases versus 2171 angiographed controls) by the Applied Biosystems real-time PCR system. The rs1573949_C [1.15(1.00-1.32); p=0.049] was associated with MI, rs7431368_AA [5.2(1.05-26.60); p=0.43] conferred risk for harboring low high density lipoprotein, and obesity was linked to rs10934857_AA [5.69(1.04-30.98); p=0.045] following Bonferroni corrections and multivariate adjustments for confounders. Furthermore, a haplotype CCCGGGTC (χ(2)=4.23; p=0.04) constructed from the eight studied SNPs and its 6-mer derivative CGGGTC (χ(2)=5.05; p=0.025) were associated with CAD. Obesity was associated with the 6-mer CATAAA (χ(2)=3.66; p=0.049), and hypercholesterolemia was linked to the 8-mer CCTGGACC (χ(2)=6.02; p=0.014), but most significantly so with its 5-mer derivative, CTGGA (χ(2)=6.75; p=0.009). On the other hand, high low density lipoprotein was linked to TGG (χ(2)=4.48; p=0.034). Our study points to an association of GATA2 at both SNP and haplotype levels with important metabolic risk traits for atherosclerosis.

  20. Multiplex Ligation-Dependent Probe Amplification Analysis of GATA4 Gene Copy Number Variations in Patients with Isolated Congenital Heart Disease

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    Valentina Guida

    2010-01-01

    Full Text Available GATA4 mutations are found in patients with different isolated congenital heart defects (CHDs, mostly cardiac septal defects and tetralogy of Fallot. In addition, GATA4 is supposed to be the responsible gene for the CHDs in the chromosomal 8p23 deletion syndrome, which is recognized as a malformation syndrome with clinical symptoms of facial anomalies, microcephaly, mental retardation, and congenital heart defects. Thus far, no study has been carried out to investigate the role of GATA4 copy number variations (CNVs in non-syndromic CHDs. To explore the possible occurrence of GATA4 gene CNVs in isolated CHDs, we analyzed by multiplex ligation-dependent probe amplification (MLPA a cohort of 161 non-syndromic patients with cardiac anomalies previously associated with GATA4 gene mutations. The patients were mutation-negative for GATA4, NKX2.5, and FOG2 genes after screening with denaturing high performance liquid chromatography. MLPA analysis revealed that normalized MLPA signals were all found within the normal range values for all exons in all patients, excluding a major contribution of GATA4 gene CNVs in CHD pathogenesis.

  1. Cardiac expression of ms1/STARS, a novel gene involved in cardiac development and disease, is regulated by GATA4.

    Science.gov (United States)

    Ounzain, Samir; Kobayashi, Satoru; Peterson, Richard E; He, Aibin; Motterle, Anna; Samani, Nilesh J; Menick, Donald R; Pu, William T; Liang, Qiangrong; Chong, Nelson W

    2012-05-01

    Ms1/STARS is a novel muscle-specific actin-binding protein that specifically modulates the myocardin-related transcription factor (MRTF)-serum response factor (SRF) regulatory axis within striated muscle. This ms1/STARS-dependent regulatory axis is of central importance within the cardiac gene regulatory network and has been implicated in cardiac development and postnatal cardiac function/homeostasis. The dysregulation of ms1/STARS is associated with and causative of pathological cardiac phenotypes, including cardiac hypertrophy and cardiomyopathy. In order to gain an understanding of the mechanisms governing ms1/STARS expression in the heart, we have coupled a comparative genomic in silico analysis with reporter, gain-of-function, and loss-of-function approaches. Through this integrated analysis, we have identified three evolutionarily conserved regions (ECRs), α, SINA, and DINA, that act as cis-regulatory modules and confer differential cardiac cell-specific activity. Two of these ECRs, α and DINA, displayed distinct regulatory sensitivity to the core cardiac transcription factor GATA4. Overall, our results demonstrate that within embryonic, neonatal, and adult hearts, GATA4 represses ms1/STARS expression with the pathologically associated depletion of GATA4 (type 1/type 2 diabetic models), resulting in ms1/STARS upregulation. This GATA4-dependent repression of ms1/STARS expression has major implications for MRTF-SRF signaling in the context of cardiac development and disease.

  2. Lineage-affiliated transcription factors bind the Gata3 Tce1 enhancer to mediate lineage-specific programs.

    Science.gov (United States)

    Ohmura, Sakie; Mizuno, Seiya; Oishi, Hisashi; Ku, Chia-Jui; Hermann, Mary; Hosoya, Tomonori; Takahashi, Satoru; Engel, James Douglas

    2016-03-01

    The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell-specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte-specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte-specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell-regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell-specific transcriptional activity.

  3. Transcriptional repression of Caveolin-1 (CAV1) gene expression by GATA-6 in bladder smooth muscle hypertrophy in mice and human beings.

    Science.gov (United States)

    Boopathi, Ettickan; Gomes, Cristiano Mendes; Goldfarb, Robert; John, Mary; Srinivasan, Vittala Gopal; Alanzi, Jaber; Malkowicz, S Bruce; Kathuria, Hasmeena; Zderic, Stephen A; Wein, Alan J; Chacko, Samuel

    2011-05-01

    Hypertrophy occurs in urinary bladder wall smooth muscle (BSM) in men with partial bladder outlet obstruction (PBOO) caused by benign prostatic hyperplasia (BPH) and in animal models of PBOO. Hypertrophied BSM from the rabbit model exhibits down-regulation of caveolin-1, a structural and functional protein of caveolae that function as signaling platforms to mediate interaction between receptor proteins and adaptor and effector molecules to regulate signal generation, amplification, and diversification. Caveolin-1 expression is diminished in PBOO-induced BSM hypertrophy in mice and in men with BPH. The proximal promoter of the human and mouse caveolin-1 (CAV1) gene was characterized, and it was observed that the transcription factor GATA-6 binds this promoter, causing reduced expression of caveolin-1. Furthermore, caveolin-1 expression levels inversely correlate with the abundance of GATA-6 in BSM hypertrophy in mice and human beings. Silencing of GATA6 gene expression up-regulates caveolin-1 expression, whereas overexpression of GATA-6 protein sustains the transcriptional repression of caveolin-1 in bladder smooth muscle cells. Together, these data suggest that GATA-6 acts as a transcriptional repressor of CAV1 gene expression in PBOO-induced BSM hypertrophy in men and mice. GATA-6-induced transcriptional repression represents a new regulatory mechanism of CAV1 gene expression in pathologic BSM, and may serve as a target for new therapy for BPH-induced bladder dysfunction in aging men.

  4. Isolation, characterization and genetic analysis of canine GATA4 gene in a family of Doberman Pinschers with an atrial septal defect

    Indian Academy of Sciences (India)

    Shin-Aeh Lee; Seung-Gon Lee; Hyeong-Sun Moon; Lopeti Lavulo; Kyoung-Oh Cho; Changbaig Hyun

    2007-12-01

    GATA4 is expressed early in the developing heart where it plays a key role in regulating the expression of genes encoding myocardial contractile proteins. Gene mutations in the human GATA4 have been implicated in various congenital heart defects (CHD), including atrial septal defect (ASD). Although ASD is the third most common CHD in humans, it is generally rare in dogs and cats. There is also no obvious predilection for ASD in dogs and cats, based on sex or breed. However, among dogs, the incidence rate of ASD is relatively high in Samoyeds and Doberman Pinschers, where its inheritance and genetic aetiology are not well understood. In this study, we identified and investigated the genetic aetiology of an ASD affected family in a pure breed dog population. Although the GATA4 gene was screened, we did not find any mutations that would result in the alteration of the coding sequence and hence, the predicted GATA4 structure and function. Although the aetiology of ASD is multifactorial, our findings indicate that GATA4 may not be responsible for the ASD in the dogs used in this study. However, this does not eliminate GATA4 as a candidate for ASD in other dog breeds.

  5. GATA4 and GATA6 regulate intestinal epithelial cytodifferentiation during development.

    Science.gov (United States)

    Walker, Emily M; Thompson, Cayla A; Battle, Michele A

    2014-08-15

    The intestinal epithelium performs vital roles in organ function by absorbing nutrients and providing a protective barrier. The zinc-finger containing transcription factors GATA4 and GATA6 regulate enterocyte gene expression and control regional epithelial cell identity in the adult intestinal epithelium. Although GATA4 and GATA6 are expressed in the developing intestine, loss of either factor alone during the period of epithelial morphogenesis and cytodifferentiation fails to disrupt these processes. Therefore, we tested the hypothesis that GATA4 and GATA6 function redundantly to control these aspects of intestinal development. We used Villin-Cre, which deletes specifically in the intestinal epithelium during the period of villus development and epithelial cytodifferentiation, to generate Gata4Gata6 double conditional knockout embryos. Mice lacking GATA4 and GATA6 in the intestinal epithelium died within 24h of birth. At E18.5, intestinal villus architecture and epithelial cell populations were altered. Enterocytes were lost, and goblet cells were increased. Proliferation was also increased in GATA4-GATA6 deficient intestinal epithelium. Although villus morphology appeared normal at E16.5, the first time at which both Gata4 and Gata6 were efficiently reduced, changes in expression of markers of enterocytes, goblet cells, and proliferative cells were detected. Moreover, goblet cell number was increased at E16.5. Expression of the Notch ligand Dll1 and the Notch target Olfm4 were reduced in mutant tissue indicating decreased Notch signaling. Finally, we found that GATA4 occupies chromatin near the Dll1 transcription start site suggesting direct regulation of Dll1 by GATA4. We demonstrate that GATA4 and GATA6 play an essential role in maintaining proper intestinal epithelial structure and in regulating intestinal epithelial cytodifferentiation. Our data highlight a novel role for GATA factors in fine tuning Notch signaling during intestinal epithelial development to

  6. Elevated expression of steroidogenesis pathway genes; CYP17, GATA6 and StAR in prenatally androgenized rats.

    Science.gov (United States)

    Jahromi, Marziyeh Salehi; Tehrani, Fahimeh Ramezani; Noroozzadeh, Mahsa; Zarkesh, Maryam; Ghasemi, Asghar; Zadeh-Vakili, Azita

    2016-11-15

    It is believed that excess androgen exposure of the fetus, via altered gene expression, causes hyperandrogenism a key feature of polycystic ovary syndrome (PCOS). The aim of this study was to evaluate expression of Cytochrome P450-17 (CYP17), GATA-binding protein (GAGT6) and Steroidogenic acute regulatory protein (StAR), genes of adult female rats prenatally exposed to androgen excess, closely reflect endocrine and ovarian disturbances of PCOS in women, by comparing them during different phases of estrus cycle with those of non-treated rats. Both the adult prenatally testosterone exposed and control rats (n=23, each) were divided into four groups based on their observed vaginal smear (proestrus, estrus, metestrus and diestrus) and the relative expression of CYP17, GATA6 and StAR genes was measured in ovarian theca cells using Cyber-green Real-Time PCR. Serum sex steroid hormones and gonadotropins levels were measured using the ELISA method; a comparison of these two groups showed that there was an overall increase in the studied genes (CYP17; 2.39 fold change, 95% CI: 1.23-3.55; P<0.05, GATA6; 2.08 fold change, 95% CI: 1.62-2.55; P<0.0001, and StAR; 1.4 fold change, 95% CI: 1.02-1.78; P<0.05), despite variations in different phases with maximum elevation for all genes in diestrus. The changes observed may impair the normal development of ovaries that mediate the programming of adult PCOS.

  7. Pancreas-specific deletion of mouse Gata4 and Gata6 causes pancreatic agenesis

    Science.gov (United States)

    Xuan, Shouhong; Borok, Matthew J.; Decker, Kimberly J.; Battle, Michele A.; Duncan, Stephen A.; Hale, Michael A.; Macdonald, Raymond J.; Sussel, Lori

    2012-01-01

    Pancreatic agenesis is a human disorder caused by defects in pancreas development. To date, only a few genes have been linked to pancreatic agenesis in humans, with mutations in pancreatic and duodenal homeobox 1 (PDX1) and pancreas-specific transcription factor 1a (PTF1A) reported in only 5 families with described cases. Recently, mutations in GATA6 have been identified in a large percentage of human cases, and a GATA4 mutant allele has been implicated in a single case. In the mouse, Gata4 and Gata6 are expressed in several endoderm-derived tissues, including the pancreas. To analyze the functions of GATA4 and/or GATA6 during mouse pancreatic development, we generated pancreas-specific deletions of Gata4 and Gata6. Surprisingly, loss of either Gata4 or Gata6 in the pancreas resulted in only mild pancreatic defects, which resolved postnatally. However, simultaneous deletion of both Gata4 and Gata6 in the pancreas caused severe pancreatic agenesis due to disruption of pancreatic progenitor cell proliferation, defects in branching morphogenesis, and a subsequent failure to induce the differentiation of progenitor cells expressing carboxypeptidase A1 (CPA1) and neurogenin 3 (NEUROG3). These studies address the conserved and nonconserved mechanisms underlying GATA4 and GATA6 function during pancreas development and provide a new mouse model to characterize the underlying developmental defects associated with pancreatic agenesis. PMID:23006325

  8. Gata4 potentiates second heart field proliferation and Hedgehog signaling for cardiac septation.

    Science.gov (United States)

    Zhou, Lun; Liu, Jielin; Xiang, Menglan; Olson, Patrick; Guzzetta, Alexander; Zhang, Ke; Moskowitz, Ivan P; Xie, Linglin

    2017-02-21

    GATA4, an essential cardiogenic transcription factor, provides a model for dominant transcription factor mutations in human disease. Dominant GATA4 mutations cause congenital heart disease (CHD), specifically atrial and atrioventricular septal defects (ASDs and AVSDs). We found that second heart field (SHF)-specific Gata4 heterozygote embryos recapitulated the AVSDs observed in germline Gata4 heterozygote embryos. A proliferation defect of SHF atrial septum progenitors and hypoplasia of the dorsal mesenchymal protrusion, rather than anlage of the atrioventricular septum, were observed in this model. Knockdown of the cell-cycle repressor phosphatase and tensin homolog (Pten) restored cell-cycle progression and rescued the AVSDs. Gata4 mutants also demonstrated Hedgehog (Hh) signaling defects. Gata4 acts directly upstream of Hh components: Gata4 activated a cis-regulatory element at Gli1 in vitro and occupied the element in vivo. Remarkably, SHF-specific constitutive Hh signaling activation rescued AVSDs in Gata4 SHF-specific heterozygous knockout embryos. Pten expression was unchanged in Smoothened mutants, and Hh pathway genes were unchanged in Pten mutants, suggesting pathway independence. Thus, both the cell-cycle and Hh-signaling defects caused by dominant Gata4 mutations were required for CHD pathogenesis, suggesting a combinatorial model of disease causation by transcription factor haploinsufficiency.

  9. Next-Generation Sequencing Approach in Methylation Analysis of HNF1B and GATA4 Genes: Searching for Biomarkers in Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Ivana Bubancova

    2017-02-01

    Full Text Available DNA methylation is well-known to be associated with ovarian cancer (OC and has great potential to serve as a biomarker in monitoring response to therapy and for disease screening. The purpose of this study was to investigate methylation of HNF1B and GATA4 and correlate detected methylation with clinicopathological characteristic of OC patients. The study group consisted of 64 patients with OC and 35 control patients. To determine the most important sites of HNF1B and GATA4, we used next-generation sequencing. For further confirmation of detected methylation of selected regions, we used high-resolution melting analysis and methylation-specific real-time polymerase chain reaction (PCR. Selected regions of HNF1B and GATA4 were completely methylation free in all control samples, whereas methylation-positive pattern was observed in 32.8% (HNF1B and 45.3% (GATA4 of OC samples. Evaluating both genes together, we were able to detect methylation in 65.6% of OC patients. We observed a statistically significant difference in HNF1B methylation between samples with different stages of OC. We also detected subtype specific methylation in GATA4 and a decrease of methylation in late stages of OC. The combination of unmethylated HNF1B and methylated GATA4 was associated with longer overall survival. In our study, we employed innovative approach of methylation analysis of HNF1B and GATA4 to search for possible epigenetic biomarkers. We confirmed the significance of the HNF1B and GATA4 hypermethylation with emphasis on the need of selecting the most relevant sites for analysis. We suggest selected CpGs to be further examined as a potential positive prognostic factor.

  10. Next-Generation Sequencing Approach in Methylation Analysis of HNF1B and GATA4 Genes: Searching for Biomarkers in Ovarian Cancer

    Science.gov (United States)

    Bubancova, Ivana; Kovarikova, Helena; Laco, Jan; Ruszova, Ema; Dvorak, Ondrej; Palicka, Vladimir; Chmelarova, Marcela

    2017-01-01

    DNA methylation is well-known to be associated with ovarian cancer (OC) and has great potential to serve as a biomarker in monitoring response to therapy and for disease screening. The purpose of this study was to investigate methylation of HNF1B and GATA4 and correlate detected methylation with clinicopathological characteristic of OC patients. The study group consisted of 64 patients with OC and 35 control patients. To determine the most important sites of HNF1B and GATA4, we used next-generation sequencing. For further confirmation of detected methylation of selected regions, we used high-resolution melting analysis and methylation-specific real-time polymerase chain reaction (PCR). Selected regions of HNF1B and GATA4 were completely methylation free in all control samples, whereas methylation-positive pattern was observed in 32.8% (HNF1B) and 45.3% (GATA4) of OC samples. Evaluating both genes together, we were able to detect methylation in 65.6% of OC patients. We observed a statistically significant difference in HNF1B methylation between samples with different stages of OC. We also detected subtype specific methylation in GATA4 and a decrease of methylation in late stages of OC. The combination of unmethylated HNF1B and methylated GATA4 was associated with longer overall survival. In our study, we employed innovative approach of methylation analysis of HNF1B and GATA4 to search for possible epigenetic biomarkers. We confirmed the significance of the HNF1B and GATA4 hypermethylation with emphasis on the need of selecting the most relevant sites for analysis. We suggest selected CpGs to be further examined as a potential positive prognostic factor. PMID:28241454

  11. Cardiac gene activation analysis in mammalian non-myoblasic cells by Nkx2-5, Tbx5, Gata4 and Myocd.

    Directory of Open Access Journals (Sweden)

    Lei Zhou

    Full Text Available Cardiac transcription factors are master regulators during heart development. Some were shown to transdifferentiate tail tip and cardiac fibroblasts into cardiomyocytes. However, recent studies have showed that controversies exist. Potential difference in tail tip and cardiac fibroblast isolation may possibly confound the observations. Moreover, due to the use of a cardiac reporter (Myh6 selection strategy for induced cardiomyocyte enrichment, and the lack of tracking signals for each transcription factors, individual roles of each transcription factors in activating cardiac gene expression in mammalian non-myoblastic cells have never been elucidated. Answers to these questions are an important step toward cardiomyocyte regeneration. Because mouse 10T1/2 fibroblasts are non-myoblastic in nature and can be induced to express genes of all three types of muscle cells, they are an ideal model for the analysis of cardiac and non-cardiac gene activation after induction. We constructed bi-cistronic lentiviral vectors, capable of expressing cardiac transcription factors along with different fluorescent tracking signals. By infecting 10T1/2 fibroblasts with Nkx2-5, Tbx5, Gata4 or Myocd cardiac transcription factor lentivirus alone or different combinations, we found that only Tbx5+Myocd and Tbx5+Gata4+Myocd combinations induced Myh6 and Tnnt2 cardiac marker protein expression. Microarray-based gene ontology analysis revealed that Tbx5 alone activated genes involved in the Wnt receptor signaling pathway and inhibited genes involved in a number of cardiac-related processes. Myocd alone activated genes involved in a number of cardiac-related processes and inhibited genes involved in the Wnt receptor signaling pathway and non-cardiac processes. Gata4 alone inhibited genes involved in non-cardiac processes. Tbx5+Gata4+Myocd was the most effective activator of genes associated with cardiac-related processes. Unlike Tbx5, Gata4, Myocd alone or Tbx5+Myocd, Tbx5

  12. Zinc fingers as protein recognition motifs: Structural basis for the GATA-1/Friend of GATA interaction

    OpenAIRE

    Liew, Chu Kong; Simpson, Raina J. Y.; Kwan, Ann H.Y.; Crofts, Linda A.; Loughlin, Fionna E.; Matthews, Jacqueline M; Crossley, Merlin; Mackay, Joel P.

    2005-01-01

    GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1/FOG interaction, we have determined the three-dimensional structure of a compl...

  13. Tissue-specific expression of GFP reporter gene in germline driven by GATA-2 promoter and enhancers in zebrafish

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    GATA-2,a transcription factor,is expressed in several types of blood cells and in the central nervous system (CNS),and regulates the differentiation of these cells.We have obtained five zebrafish transgenic germlines that carry and express the green fluorescent protein (GFP) gene ligated to various 5′flanking sequences of zebrafish GATA-2 gene.The spatial pattern of GFP expression varies,mainly depending on which regulatory sequence is used,among the germlines.In some of the germlines,the expression of GFP is restricted to the CNS and the enveloping layer (EVL) cells,while in some other lines GFP is observed only in the CNS.It is noted that the intensity of GFP in the transgenic fish remain unchanged after a six-generation passage of the transgenes.The transgenic fish could find its uses in the future in generating tissue-specific,even cellspecific mutant fish and in functional study of related genes through transgenesis.

  14. [Effects of acupoint injection of autoblood on expression of pulmonary transcription factor GATA 3 and T-bet proteins and genes in asthma rats].

    Science.gov (United States)

    Mo, Lü; Li, Jun-Xiong; Kravitz, Julia; Tang, Chun-Zhi; Guo, Ze-Kai; Li, Pei-Yong; Guo, Feng-Mei

    2012-10-01

    To observe the effect of autoblood acupoint-injection (ABAI) on expression levels of pulmonary transacting T-cell-specific transcription factor GATA 3 (involving Th 2 cytokine expression), Th 1-specific T-box transcription factor T-box expressed in T-cells (T-bet) proteins and genes in asthmatic rats so as to explore its mechanisms underlying asthma relief. Forty-eight male SD rats were randomized into normal control (n = 8), model (n = 10), saline acupoint-injection (SAI, n = 10), ABAI (n = 10), and Dexamethasone (DXM, n = 10) groups. Asthma model was established during 28 days by 10% Ovalbumin + 10% aluminium hydroxide solution injection (i. p.) and vapourized 2% Ovalbumin inhaling for 14 days. For rats of the ABAI group, 0.4 mL autoblood was injected into the bilateral "Feishu" (BL 13) or "Shenshu" (BL 23) alternately, once every other day for six times. For rats of the DXM group, 50% DXM solution (0.5 mg/kg, i. p.) was given from the 17th day on after starting the modeling, once every other day for 11 days. Pulmonary GATA 3 and T-bet protein expression was detected by immunohistochemistry, and GATA 3 mRNA and T-bet mRNA expression detected by real time-PCR. In comparison with the normal group, pulmonary GATA 3 protein and mRNA expression levels in the model group were up-regulated significantly (P model group was down-regulated obviously (P model group, GATA 3 protein and mRNA expression levels were down-regulated significantly in both ABAI and DXM groups (P model and SAI groups, and between ABAI and DXM groups in GATA 3 protein expression levels; and between ABAI and DXM groups in GATA 3 mRNA expression levels; between normal and model groups, and between SAI and ABAI groups in T-bet protein expression levels; between model and SAI groups and between ABAI and DXM groups in T-bet mRNA expression levels (P > 0.05). The ratio of GATA 3 mRNA/T-bet mRNA expression was significantly higher in the model group than in the normal group (P model group (P 0

  15. GATA factors in endocrine neoplasia.

    Science.gov (United States)

    Pihlajoki, Marjut; Färkkilä, Anniina; Soini, Tea; Heikinheimo, Markku; Wilson, David B

    2016-02-01

    GATA transcription factors are structurally-related zinc finger proteins that recognize the consensus DNA sequence WGATAA (the GATA motif), an essential cis-acting element in the promoters and enhancers of many genes. These transcription factors regulate cell fate specification and differentiation in a wide array of tissues. As demonstrated by genetic analyses of mice and humans, GATA factors play pivotal roles in the development, homeostasis, and function of several endocrine organs including the adrenal cortex, ovary, pancreas, parathyroid, pituitary, and testis. Additionally, GATA factors have been shown to be mutated, overexpressed, or underexpressed in a variety of endocrine tumors (e.g., adrenocortical neoplasms, parathyroid tumors, pituitary adenomas, and sex cord stromal tumors). Emerging evidence suggests that GATA factors play a direct role in the initiation, proliferation, or propagation of certain endocrine tumors via modulation of key developmental signaling pathways implicated in oncogenesis, such as the WNT/β-catenin and TGFβ pathways. Altered expression or function of GATA factors can also affect the metabolism, ploidy, and invasiveness of tumor cells. This article provides an overview of the role of GATA factors in endocrine neoplasms. Relevant animal models are highlighted.

  16. Function of GATA Factors in the Adult Mouse Liver

    Science.gov (United States)

    Zheng, Rena; Rebolledo-Jaramillo, Boris; Zong, Yiwei; Wang, Liqing; Russo, Pierre; Hancock, Wayne; Stanger, Ben Z.; Hardison, Ross C.; Blobel, Gerd A.

    2013-01-01

    GATA transcription factors and their Friend of Gata (FOG) cofactors control the development of diverse tissues. GATA4 and GATA6 are essential for the expansion of the embryonic liver bud, but their expression patterns and functions in the adult liver are unclear. We characterized the expression of GATA and FOG factors in whole mouse liver and purified hepatocytes. GATA4, GATA6, and FOG1 are the most prominently expressed family members in whole liver and hepatocytes. GATA4 chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) identified 4409 occupied sites, associated with genes enriched in ontologies related to liver function, including lipid and glucose metabolism. However, hepatocyte-specific excision of Gata4 had little impact on gross liver architecture and function, even under conditions of regenerative stress, and, despite the large number of GATA4 occupied genes, resulted in relatively few changes in gene expression. To address possible redundancy between GATA4 and GATA6, both factors were conditionally excised. Surprisingly, combined Gata4,6 loss did not exacerbate the phenotype resulting from Gata4 loss alone. This points to the presence of an unusually robust transcriptional network in adult hepatocytes that ensures the maintenance of liver function. PMID:24367609

  17. Identification of GATA2 and AP-1 activator elements within the enhancer VNTR occurring in intron 5 of the human SIRT3 gene

    Science.gov (United States)

    Human SIRT3 gene contains an intronic VNTR enhancer. A T > C transition occurring in the second repeat of each VNTR allele implies the presence/absence of a putative GATA binding motif. A partially overlapping AP-1 site, not affected by the transition, was also identified. Aims of the present study ...

  18. GATA-1 Inhibits PU.1 Gene via DNA and Histone H3K9 Methylation of Its Distal Enhancer in Erythroleukemia.

    Directory of Open Access Journals (Sweden)

    Pavel Burda

    Full Text Available GATA-1 and PU.1 are two important hematopoietic transcription factors that mutually inhibit each other in progenitor cells to guide entrance into the erythroid or myeloid lineage, respectively. PU.1 controls its own expression during myelopoiesis by binding to the distal URE enhancer, whose deletion leads to acute myeloid leukemia (AML. We herein present evidence that GATA-1 binds to the PU.1 gene and inhibits its expression in human AML-erythroleukemias (EL. Furthermore, GATA-1 together with DNA methyl Transferase I (DNMT1 mediate repression of the PU.1 gene through the URE. Repression of the PU.1 gene involves both DNA methylation at the URE and its histone H3 lysine-K9 methylation and deacetylation as well as the H3K27 methylation at additional DNA elements and the promoter. The GATA-1-mediated inhibition of PU.1 gene transcription in human AML-EL mediated through the URE represents important mechanism that contributes to PU.1 downregulation and leukemogenesis that is sensitive to DNA demethylation therapy.

  19. GATA-1 Inhibits PU.1 Gene via DNA and Histone H3K9 Methylation of Its Distal Enhancer in Erythroleukemia.

    Science.gov (United States)

    Burda, Pavel; Vargova, Jarmila; Curik, Nikola; Salek, Cyril; Papadopoulos, Giorgio Lucio; Strouboulis, John; Stopka, Tomas

    2016-01-01

    GATA-1 and PU.1 are two important hematopoietic transcription factors that mutually inhibit each other in progenitor cells to guide entrance into the erythroid or myeloid lineage, respectively. PU.1 controls its own expression during myelopoiesis by binding to the distal URE enhancer, whose deletion leads to acute myeloid leukemia (AML). We herein present evidence that GATA-1 binds to the PU.1 gene and inhibits its expression in human AML-erythroleukemias (EL). Furthermore, GATA-1 together with DNA methyl Transferase I (DNMT1) mediate repression of the PU.1 gene through the URE. Repression of the PU.1 gene involves both DNA methylation at the URE and its histone H3 lysine-K9 methylation and deacetylation as well as the H3K27 methylation at additional DNA elements and the promoter. The GATA-1-mediated inhibition of PU.1 gene transcription in human AML-EL mediated through the URE represents important mechanism that contributes to PU.1 downregulation and leukemogenesis that is sensitive to DNA demethylation therapy.

  20. Regulation of a vascular plexus by gata4 is mediated in zebrafish through the chemokine sdf1a.

    Directory of Open Access Journals (Sweden)

    Ingrid Torregroza

    Full Text Available Using the zebrafish model we describe a previously unrecognized requirement for the transcription factor gata4 controlling embryonic angiogenesis. The development of a vascular plexus in the embryonic tail, the caudal hematopoietic tissue (CHT, fails in embryos depleted of gata4. Rather than forming a normal vascular plexus, the CHT of gata4 morphants remains fused, and cells in the CHT express high levels of osteogenic markers ssp1 and runx1. Definitive progenitors emerge from the hemogenic aortic endothelium, but fail to colonize the poorly vascularized CHT. We also found abnormal patterns and levels for the chemokine sdf1a in gata4 morphants, which was found to be functionally relevant, since the embryos also show defects in development of the lateral line, a mechano-sensory organ system highly dependent on a gradient of sdf1a levels. Reduction of sdf1a levels was sufficient to rescue lateral line development, circulation, and CHT morphology. The result was surprising since neither gata4 nor sdf1a is obviously expressed in the CHT. Therefore, we generated transgenic fish that conditionally express a dominant-negative gata4 isoform, and determined that gata4 function is required during gastrulation, when it is co-expressed with sdf1a in lateral mesoderm. Our study shows that the gata4 gene regulates sdf1a levels during early embryogenesis, which impacts embryonic patterning and subsequently the development of the caudal vascular plexus.

  1. The +37 kb Cebpa Enhancer Is Critical for Cebpa Myeloid Gene Expression and Contains Functional Sites that Bind SCL, GATA2, C/EBPα, PU.1, and Additional Ets Factors.

    Science.gov (United States)

    Cooper, Stacy; Guo, Hong; Friedman, Alan D

    2015-01-01

    The murine Cebpa gene contains an evolutionarily conserved 453 bp enhancer located at +37 kb that, together with its promoter, directs expression to myeloid progenitors and to long-term hematopoietic stem cells in transgenic mice. In human acute myeloid leukemia cases, the enhancer lacks point mutations but binds the RUNX1-ETO oncoprotein. The enhancer contains the H3K4me1 and H3K27Ac histone modifications, denoting an active enhancer, at progressively increasing levels as long-term hematopoietic stem cells transition to granulocyte-monocyte progenitors. We previously identified four enhancer sites that bind RUNX1 and demonstrated that their integrity is required for maximal enhancer activity in 32Dcl3 myeloid cells. The +37 kb Cebpa enhancer also contains C/EBP, Ets factor, Myb, GATA, and E-box consensus sites conserved in the human +42 kb CEBPA enhancer. Mutation of the two C/EBP, seven Ets, one Myb, two GATA, or two E-box sites reduces activity of an enhancer-promoter reporter in 32Dcl3 cells. In 293T gel shift assays, exogenous C/EBPα binds both C/EBP sites, c-Myb binds the Myb site, PU.1 binds the second Ets site, PU.1, Fli-1, ERG, and Ets1 bind the sixth Ets site, GATA2 binds both GATA sites, and SCL binds the second E-box. Endogenous hematopoietic RUNX1, PU.1, Fli-1, ERG, C/EBPα, GATA2, and SCL were previously shown to bind the enhancer, and we find that endogenous PU.1 binds the second Ets site in 32Dcl3 cells. Using CRISPR/Cas9, we developed 32Dcl3 lines in which the wild-type enhancer alleles are replaced with a variant mutant in the seven Ets sites. These lines have 20-fold reduced Cebpa mRNA when cultured in IL-3 or G-CSF, demonstrating a critical requirement for enhancer integrity for optimal Cebpa expression. In addition, these results indicate that the +37 kb Cebpa enhancer is the focus of multiple regulatory transcriptional pathways that impact its expression during normal hematopoiesis and potentially during myeloid transformation.

  2. The +37 kb Cebpa Enhancer Is Critical for Cebpa Myeloid Gene Expression and Contains Functional Sites that Bind SCL, GATA2, C/EBPα, PU.1, and Additional Ets Factors.

    Directory of Open Access Journals (Sweden)

    Stacy Cooper

    Full Text Available The murine Cebpa gene contains an evolutionarily conserved 453 bp enhancer located at +37 kb that, together with its promoter, directs expression to myeloid progenitors and to long-term hematopoietic stem cells in transgenic mice. In human acute myeloid leukemia cases, the enhancer lacks point mutations but binds the RUNX1-ETO oncoprotein. The enhancer contains the H3K4me1 and H3K27Ac histone modifications, denoting an active enhancer, at progressively increasing levels as long-term hematopoietic stem cells transition to granulocyte-monocyte progenitors. We previously identified four enhancer sites that bind RUNX1 and demonstrated that their integrity is required for maximal enhancer activity in 32Dcl3 myeloid cells. The +37 kb Cebpa enhancer also contains C/EBP, Ets factor, Myb, GATA, and E-box consensus sites conserved in the human +42 kb CEBPA enhancer. Mutation of the two C/EBP, seven Ets, one Myb, two GATA, or two E-box sites reduces activity of an enhancer-promoter reporter in 32Dcl3 cells. In 293T gel shift assays, exogenous C/EBPα binds both C/EBP sites, c-Myb binds the Myb site, PU.1 binds the second Ets site, PU.1, Fli-1, ERG, and Ets1 bind the sixth Ets site, GATA2 binds both GATA sites, and SCL binds the second E-box. Endogenous hematopoietic RUNX1, PU.1, Fli-1, ERG, C/EBPα, GATA2, and SCL were previously shown to bind the enhancer, and we find that endogenous PU.1 binds the second Ets site in 32Dcl3 cells. Using CRISPR/Cas9, we developed 32Dcl3 lines in which the wild-type enhancer alleles are replaced with a variant mutant in the seven Ets sites. These lines have 20-fold reduced Cebpa mRNA when cultured in IL-3 or G-CSF, demonstrating a critical requirement for enhancer integrity for optimal Cebpa expression. In addition, these results indicate that the +37 kb Cebpa enhancer is the focus of multiple regulatory transcriptional pathways that impact its expression during normal hematopoiesis and potentially during myeloid

  3. 应用 TALEN 技术敲除 gata4基因建立斑马鱼先天性心脏病模型%Knockout gata4 gene and establish ment of a zebrafish model of congenital heart disease by TALEN

    Institute of Scientific and Technical Information of China (English)

    刘静; 何嘉玲; 暴国; 李楠; 王天奇; 张长勇; 孙德明

    2015-01-01

    Objective To establish a gata4 gene knockout zebrafish model of congenital heart disease, and construct transcription activator-like effector nuclease ( TALEN) vectors targeting gata4 gene.Method We construct TALEN vectors targeting zabrafish gata4 gene using unit assembly method and the in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage zebrafish embryos.The efficiency of TALEN was identified by injected embryos, and mutations of zebrafish were screened and confirmed the different types through PCR and enzyme digestion.Results We successfully constructed correct targeting vectors by enzyme digestion and sequencing, and the gene knockout efficiency was 35.18%.We screened the mutant zebrafish and confirmed different types of gata4 gene mutations.Conclusions A gata4 knockout zebrafish model is successfully established, it can provide a good animal model for further research of congenital heart diseases.%目的:人工构建TALEN靶向敲除载体敲除gata4基因,建立斑马鱼先天性心脏病动物模型。方法采用单元组装法构建靶向敲除gata4基因的载体,将其体外转录成TALEN mRNAs,通过显微注射的方式注入单细胞期受精卵中,胚胎检测TALEN的敲除效率,再通过剪尾、酶切、测序筛选发生基因突变的斑马鱼及突变的类型。结果酶切、测序结果证实成功构建出正确的 gata4靶向敲除载体,注入斑马鱼受精卵中,发生基因敲除的效率为35.18%,通过剪尾、PCR、酶切检测成功地筛选出了发生基因突变的斑马鱼,测序结果显示出不同种类的gata4基因突变。结论成功构建gata4基因敲除的斑马鱼动物模型,为进一步研究人类gata4基因突变引起先天性心脏病的发病机制提供了良好的动物模型。

  4. Solanum paniculatum L. decreases levels of inflammatory cytokines by reducing NFKB, TBET and GATA3 gene expression in vitro.

    Science.gov (United States)

    Rios, Raimon; Silva, Hugo Bernardino Ferreira da; Carneiro, Norma Vilany Queiroz; Pires, Anaque de Oliveira; Carneiro, Tamires Cana Brasil; Costa, Ryan Dos Santos; Marques, Cintia Rodrigues; Machado, Marta Santos Serafim; Velozo, Eudes da Silva; Silva, Telma M G da; Silva, Tania M S da; Conceição, Adilva de Souza; Alcântara-Neves, Neuza Maria; Figueiredo, Camila Alexandrina

    2017-09-14

    Solanum paniculatum L., popularly known as jurubeba, is a common subtropical plant from Brazil, Paraguay, Bolivia and Argentina, that is used in folk medicine for the treatment of anemia, gastrointestinal disorders and inflammatory conditions in general. In addition to that, an ethnobotanical survey in "Todos os Santos" Bay have pointed out S. paniculatum as an herb to treat asthma. Previous publications have shown that S. paniculatum possesses antibiotic, antioxidant and modulatory effects on gastric acid secretion; however, its anti-inflammatory potential remains unexplored. Herein, we analyzed the S. paniculatum fruits hexane extract (SpE) for the presence of stigmasterol and β-sitosterol and investigated the anti-inflammatory effect of SpE in vitro. SpE was subjected to high-performance liquid chromatography (HPLC) for standardization and quantification of stigmasterol and β-sitosterol. Spleen cells from BALB/c mice were cultivated and stimulated with pokeweed mitogen and also exposed to 15, 30 and 60µg/mL of SpE. Following treatment, levels of IFN-γ, IL-4 and IL-10 in the culture supernatants were assessed by ELISA. We also evaluated nitric oxide (NO) production by murine LPS-stimulated peritoneal macrophages using the Griess technique. In addition, the ability of SpE to stabilize membranes was assessed using a model of hemolysis induced by heat on murine erythrocytes. Gene expression of Th1-cell-specific Tbx21 transcription factor (TBET), zinc-finger transcription factor-3 (GATA3), and nuclear factor-κB (NFKB) in murine spleen cells were assessed by quantitative Polymerase Chain Reaction (qRT-PCR). SpE at 15, 30 and 60µg/mL significantly attenuated cell proliferation, decreased IL-4 release, reduced NO production and improved erythrocyte membrane stabilization in a concentration-dependent manner. SpE was also able to decrease the release of IFN-γ without altering IL-10 levels. The mechanism whereby SpE decreased inflammatory markers may be related to

  5. Structural basis of simultaneous recruitment of the transcriptional regulators LMO2 and FOG1/ZFPM1 by the transcription factor GATA1.

    Science.gov (United States)

    Wilkinson-White, Lorna; Gamsjaeger, Roland; Dastmalchi, Siavoush; Wienert, Beeke; Stokes, Philippa H; Crossley, Merlin; Mackay, Joel P; Matthews, Jacqueline M

    2011-08-30

    The control of red blood cell and megakaryocyte development by the regulatory protein GATA1 is a paradigm for transcriptional regulation of gene expression in cell lineage differentiation and maturation. Most GATA1-regulated events require GATA1 to bind FOG1, and essentially all GATA1-activated genes are cooccupied by a TAL1/E2A/LMO2/LDB1 complex; however, it is not known whether FOG1 and TAL1/E2A/LMO2/LDB1 are simultaneously recruited by GATA1. Our structural data reveal that the FOG1-binding domain of GATA1, the N finger, can also directly contact LMO2 and show that, despite the small size (structural snapshot of multiprotein complex formation at GATA1-dependent genes and support a model in which FOG1 and TAL1/E2A/LMO2/LDB1 can cooccupy E-box/WGATAR sites to facilitate GATA1-mediated activation of gene activation.

  6. GATA Factor-G-Protein-Coupled Receptor Circuit Suppresses Hematopoiesis

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    Xin Gao

    2016-03-01

    Full Text Available Hematopoietic stem cells (HSCs originate from hemogenic endothelium within the aorta-gonad-mesonephros (AGM region of the mammalian embryo. The relationship between genetic circuits controlling stem cell genesis and multi-potency is not understood. A Gata2 cis element (+9.5 enhances Gata2 expression in the AGM and induces the endothelial to HSC transition. We demonstrated that GATA-2 rescued hematopoiesis in +9.5−/− AGMs. As G-protein-coupled receptors (GPCRs are the most common targets for FDA-approved drugs, we analyzed the GPCR gene ensemble to identify GATA-2-regulated GPCRs. Of the 20 GATA-2-activated GPCR genes, four were GATA-1-activated, and only Gpr65 expression resembled Gata2. Contrasting with the paradigm in which GATA-2-activated genes promote hematopoietic stem and progenitor cell genesis/function, our mouse and zebrafish studies indicated that GPR65 suppressed hematopoiesis. GPR65 established repressive chromatin at the +9.5 site, restricted occupancy by the activator Scl/TAL1, and repressed Gata2 transcription. Thus, a Gata2 cis element creates a GATA-2-GPCR circuit that limits positive regulators that promote hematopoiesis.

  7. Cx30.2 enhancer analysis identifies Gata4 as a novel regulator of atrioventricular delay

    Science.gov (United States)

    Munshi, Nikhil V.; McAnally, John; Bezprozvannaya, Svetlana; Berry, Jeff M.; Richardson, James A.; Hill, Joseph A.; Olson, Eric N.

    2009-01-01

    Summary The cardiac conduction system comprises a specialized tract of electrically coupled cardiomyocytes responsible for impulse propagation through the heart. Abnormalities in cardiac conduction are responsible for numerous forms of cardiac arrhythmias, but relatively little is known about the gene regulatory mechanisms that control the formation of the conduction system. We demonstrate that a distal enhancer for the connexin 30.2 (Cx30.2, also known as Gjd3) gene, which encodes a gap junction protein required for normal atrioventricular (AV) delay in mice, is necessary and sufficient to direct expression to the developing AV conduction system (AVCS). Moreover, we show that this enhancer requires Tbx5 and Gata4 for proper expression in the conduction system, and Gata4+/- mice have short PR intervals indicative of accelerated AV conduction. Thus, our results implicate Gata4 in conduction system function and provide a clearer understanding of the transcriptional pathways that impact normal AV delay. PMID:19592579

  8. Zinc fingers as protein recognition motifs: structural basis for the GATA-1/friend of GATA interaction.

    Science.gov (United States)

    Liew, Chu Kong; Simpson, Raina J Y; Kwan, Ann H Y; Crofts, Linda A; Loughlin, Fionna E; Matthews, Jacqueline M; Crossley, Merlin; Mackay, Joel P

    2005-01-18

    GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1/FOG interaction, we have determined the three-dimensional structure of a complex comprising the interaction domains of these proteins. The structure reveals how zinc fingers can act as protein recognition motifs. Details of the architecture of the contact domains and their physical properties provide a molecular explanation for how the GATA-1 mutations contribute to distinct but related genetic diseases.

  9. A high-resolution map of genes, microsatellite markers, and new dinucleotide repeats from UBE1 to the GATA locus in the region Xp11.23

    Energy Technology Data Exchange (ETDEWEB)

    Kwan, Sau-Ping; Hagemann, T.L. [Rush Medical School, Chicago, IL (United States); Rosen, F.S. [Harvard Medical School, Boston, MA (United States)] [and others

    1995-09-01

    Several new genes and markers have recently been identified on the proximal short arm of the human X chromosome in the area of Xp11.23. We had previously generated at YAC contig in this region extending from UBE1 to the OATL1 locus. In this report two polymorphic dinucleotide repeats, DXS6949 and DXS6950, were isolated and characterized from the OATL1 locus. A panel of YAC deletion derivatives from the distal portion of the contig was used in conjunction with the rest of the YAC map to position the new microsatellites and order other markers localizing to this interval. The marker order was determined to be DXS1367-ZNF81-DXS6849-ZNF21-DXS6616-DXS6950-DXS6949. In the proximal region below OATL1, we have isolated a pair of YACs from the GATA locus, B1026 and C01160. Mapping within these YACs indicates the orientation of DXS1126 and DXS1240, while a cosmid near the OATL1 region reveals the overlap between the YAC contigs from the two loci. This cosmid contains the gene responsible for Wiskott-Aldrich syndrome (WAS) and localizes the disease gene between OATL1 and GATA. These data enable the expansion of the present physical map of the X chromosome from UBE1 to the GATA locus, covering a large portion of the Xp11.23 region. Genetic crossovers in Xp11.23 support the marker orientation and the position of WAS, contrary to previous reports. With the integration of both physical and genetic maps we have predicted the following marker order: Xpter-UBE1-SYN1/ARAF1/TIMP1/DXS1367-ZNF81-DXS-6849-ZNF21-DXSy6616-(OATL1, DXS6950-DXS6949)-WAS-(GATA,DXS1126)-DXS12410-Xcen. This orientation identifies DXS6949 and DXS1126 as the nearest flanking polymorphic markers for WAS and provides useful anchor positions for the analysis of other disease genes that have been localized to this area including three different retinal defects and X-linked nephrolithiasis. 39 refs., 3 figs., 1 tab.

  10. Transcription factors GATA/ELT-2 and forkhead/HNF-3/PHA-4 regulate the tropomyosin gene expression in the pharynx and intestine of Caenorhabditis elegans.

    Science.gov (United States)

    Anokye-Danso, Frederick; Anyanful, Akwasi; Sakube, Yasuji; Kagawa, Hiroaki

    2008-05-30

    Gene regulation during development is an important biological activity that leads to synthesis of biomolecules at specific locations and specific times. The single tropomyosin gene of Caenorhabditis elegans, tmy-1/lev-11, produces four isoforms of protein: two from the external promoter and two from the internal promoter. We investigated the internal promoter of tropomyosin to identify sequences that regulate expression of tmy-1 in the pharynx and intestine. By promoter deletion of tmy-1 reporters as well as by database analyses, a 100-bp fragment that contained binding sequences for a GATA factor, for a chicken CdxA homolog, and for a forkhead factor was identified. Both the forkhead and CdxA binding sequences contributed to pharyngeal and intestinal expression. In addition, the GATA site also influenced intestinal expression of tmy-1 reporter. We showed that ELT-2 and PHA-4 proteins interact directly with the GATA and forkhead binding sequences, respectively, in gel mobility shift assays. RNA interference knockdown of elt-2 diminished tmy-1::gfp expression in the intestine. In contrast to RNA interference knockdown of pha-4, expression of tmy-1::gfp in pha-4;smg-1 mutants was slightly weaker than that of the wild type. Ectopic expression of PHA-4 and ELT-2 by heat shock was sufficient to elicit widespread expression of tmy-1::lacZ reporter in embryos. We found no indication of a synergistic relation between ELT-2 and PHA-4. Based on our data, PHA-4 and CdxA function as general transcription factors for pharyngeal and intestinal regulation of tmy-1. We present models by which ELT-2, PHA-4, and CdxA orchestrate expression from the internal promoter of tmy-1.

  11. A tissue-specific knockout reveals that Gata1 is not essential for Sertoli cell function in the mouse

    Science.gov (United States)

    Lindeboom, Fokke; Gillemans, Nynke; Karis, Alar; Jaegle, Martine; Meijer, Dies; Grosveld, Frank; Philipsen, Sjaak

    2003-01-01

    The transcription factor Gata1 is essential for the development of erythroid cells. Consequently, Gata1 null mutants die in utero due to severe anaemia. Outside the haematopoietic system, Gata1 is only expressed in the Sertoli cells of the testis. To elucidate the function of Gata1 in the testis, we made a Sertoli cell-specific knockout of the Gata1 gene in the mouse. We deleted a normally functioning ‘floxed’ Gata1 gene in pre-Sertoli cells in vivo through the expression of Cre from a transgene driven by the Desert Hedgehog promoter. Sur prisingly, Gata1 null testes developed to be morphologically normal, spermatogenesis was not obviously affected and expression levels of putative Gata1 target genes, and other Gata factors, were not altered. We conclude that expression of Gata1 in Sertoli cells is not essential for testis development or spermatogenesis in the mouse. PMID:12954777

  12. Berberine increases expression of GATA-2 and GATA-3 during inhibition of adipocyte differentiation.

    Science.gov (United States)

    Hu, Y; Davies, G E

    2009-09-01

    It is known that a number of transcription factors are key regulators in the complex process of adipocyte differentiation including peroxisome proliferator activated receptor gamma (PPARgamma) and the CCAAT enhancer binding protein alpha (C/EBPalpha). Studies have demonstrated that in pre-adipocyte 3T3-L1 cells constitutive expression of the DNA binding proteins GATA-2 and GATA-3 results in protein/protein interactions with C/EBPalpha resulting in down regulation of PPARgamma and subsequent suppressed adipocyte differentiation with cells trapped at the pre-adipocyte stage. Thus it appears that GATA-2 and GATA-3 are of critical importance in regulating adipocyte differentiation through molecular interactions with PPARgamma and C/EBPalpha. Recent reports suggest that berberine, an isoquinoline derivative alkaloid isolated from many medicinal herbs prevents differentiation of 3T3-L1 cells via a down regulation of PPARgamma and C/EBPalpha expression. The aim of this study was to determine the effect of berberine on GATA-2 and 3 gene and protein expression levels during differentiation of 3T3-L1 cells. MTT (Methylthiazolyldiphenyl-tetrazolium bromide) was used to detect the cytotoxic effects of berberine on the viability of 3T3-L1 cells during proliferation and differentiation. Differentiation of 3T3-L1 cells was monitored by Oil Red O staining and RT-PCR of PPARgamma and C/EBPalpha and the expression of GATA-2 and 3 was determined by RT-PCR and Western Blot. Results show that following treatment with 8microM berberine the mRNA and protein expression levels of GATA-2 and 3 were elevated and accompanied by inhibited adipocyte differentiation. These results may lead to the use of berberine to target the induction of specific genes such as GATA-2 and GATA-3 which affect adipocyte differentiation.

  13. GATA-3-Not Just for Th2 Cells Anymore

    Institute of Scientific and Technical Information of China (English)

    I-Cheng Ho; Sung-Yun Pai

    2007-01-01

    GATA-3 was first cloned as a T cell specific transcription factor in 1991 and its importance in the transcriptional control of T helper type 2 cell (Th2) differentiation was established in the mid to late 90's. A role for GATA-3during thymic development has long implied by its continuous and regulated expression through out T lineage development, but the absolute requirement for GATA-3 during early T lymphoid commitment/survival previously precluded definitive answers to this question. Several technical breakthroughs have fueled fruitful investigation in recent years and uncovered unexpected and critical roles for GATA-3 in CD4 thymocyte survival, invariant natural killer T cell generation and function, and also in beta selection. Not only does GATA-3 participate in nearly every stage of T cell development from common lymphoid progenitor to Th2, conditional knockout studies have indicated that the influence of GATA-3 also extends beyond the immune system.

  14. The GATA1-HS2 enhancer allows persistent and position-independent expression of a β-globin transgene.

    Directory of Open Access Journals (Sweden)

    Annarita Miccio

    Full Text Available Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene. Transcriptional enhancers binding chromatin-remodeling and modifying complexes may play a role in shielding transgenes from repressive chromatin effects. We tested the activity of the HS2 enhancer of the GATA1 gene in protecting the expression of a β-globin minigene delivered by a lentiviral vector in hematopoietic stem/progenitor cells. Gene expression from proviruses carrying GATA1-HS2 in both LTRs was persistent and resistant to silencing at most integration sites in the in vivo progeny of human hematopoietic progenitors and murine long-term repopulating stem cells. The GATA1-HS2-modified vector allowed correction of murine β-thalassemia at low copy number without inducing clonal selection of erythroblastic progenitors. Chromatin immunoprecipitation studies showed that GATA1 and the CBP acetyltransferase bind to GATA1-HS2, significantly increasing CBP-specific histone acetylations at the LTRs and β-globin promoter. Recruitment of CBP by the LTRs thus establishes an open chromatin domain encompassing the entire provirus, and increases the therapeutic efficacy of β-globin gene transfer by reducing expression variegation and epigenetic silencing.

  15. Characterization of hematopoietic GATA transcription factor expression in mouse and human dendritic cells.

    Science.gov (United States)

    Scheenstra, Maaike R; Salunkhe, Vishal; De Cuyper, Iris M; Hoogenboezem, Mark; Li, Eveline; Kuijpers, Taco W; van den Berg, Timo K; Gutiérrez, Laura

    2015-12-01

    Dendritic cells (DCs) are key initiators and regulators of the immune response. The development of the DC lineage and their subsets requires an orchestrated regulation of their transcriptional program. Gata1, a transcription factor expressed in several hematopoietic cell lineages, has been recently reported to be required for mouse DC development and function. In humans, GATA1 is involved in the lineage separation between monocyte-derived DCs and Langerhans cells (LC) and loss of GATA1 results in differentiation arrest at the monocyte stage. The hematopoietic GATA factors (i.e. Gata1, Gata2, Gata3) are known to regulate each other's expression and to function consecutively throughout lineage commitment (so-called GATA switch). In humans, mutations in GATA2 are causative of MonoMAC disease, a human immunodeficiency syndrome characterized by loss of DCs, monocytes, B and NK cells. However, additional data on the expression of hematopoietic GATA factors in the DC lineage is missing. In this study, we have characterized the expression of hematopoietic GATA factors in murine and human DCs and their expression dynamics upon TLR stimulation. We found that all hematopoietic GATA factors are expressed in DCs, but identified species-specific differences in the relative expression of each GATA factor, and how their expression fluctuates upon stimulation.

  16. GATA Factor-Dependent Positive-Feedback Circuit in Acute Myeloid Leukemia Cells

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    Koichi R. Katsumura

    2016-08-01

    Full Text Available The master regulatory transcription factor GATA-2 triggers hematopoietic stem and progenitor cell generation. GATA2 haploinsufficiency is implicated in myelodysplastic syndrome (MDS and acute myeloid leukemia (AML, and GATA2 overexpression portends a poor prognosis for AML. However, the constituents of the GATA-2-dependent genetic network mediating pathogenesis are unknown. We described a p38-dependent mechanism that phosphorylates GATA-2 and increases GATA-2 target gene activation. We demonstrate that this mechanism establishes a growth-promoting chemokine/cytokine circuit in AML cells. p38/ERK-dependent GATA-2 phosphorylation facilitated positive autoregulation of GATA2 transcription and expression of target genes, including IL1B and CXCL2. IL-1β and CXCL2 enhanced GATA-2 phosphorylation, which increased GATA-2-mediated transcriptional activation. p38/ERK-GATA-2 stimulated AML cell proliferation via CXCL2 induction. As GATA2 mRNA correlated with IL1B and CXCL2 mRNAs in AML-M5 and high expression of these genes predicted poor prognosis of cytogenetically normal AML, we propose that the circuit is functionally important in specific AML contexts.

  17. The effect of Gata3-Targeting gene silence on dendritic cells phenotype and the effector T cell imbalance expression in pregnancy asthma of mice%靶向沉默Gata3对妊娠期哮喘小鼠树突状细胞表型及效应T细胞失衡表达的影响

    Institute of Scientific and Technical Information of China (English)

    马佳佳; 陈必良; Nick Lu

    2014-01-01

    目的:研究慢病毒介导siRNA靶向沉默Gata3基因表达对妊娠哮喘模型小鼠骨髓来源树突状细胞( DC )抗原递呈及其免疫生物学特性的影响。方法:以脂质体DNA沉淀法将重组慢病毒干扰质粒与包装质粒共转染AD293细胞,包装生成慢病毒感染体外诱导DC,RT-PCR、Western blot分析其对siGata3基因的整合转录效果;流式细胞术观察细胞表型变化。通过尾静脉注射将pSH1/siGata3转移至小鼠体内,ELISA法测定其肺泡灌洗液( BALF)中的炎性因子含量;MTT法验证混合淋巴细胞反应( MLR)后T细胞增殖能力。结果:经重组、包装携有Gata3特异siRNA慢病毒感染DC成功,其siRNA靶点Gata3 mRNA及蛋白的转录表达分别下调87.30%和81.33%( P<0.05);同时降低DCs膜表面CD86荧光强度( P<0.05)。减少pSH1/siGata3组小鼠BALF中IL-5、IL-17分泌而提升IL-12水平( P<0.05);MLR 证实,共培养感染 DC 激活 T 细胞的能力明显不足( P<0.05)。结论:siRNA干扰可有效抑制模型小鼠免疫微环境特定Gata3通路,阻遏T细胞及其亚群分化由此来诱导DC免疫耐受,为妊娠期哮喘防治奠定基础并提供新的思路和手段。%Objective:To investigate the influence of lentivirus-mediated siRNA,targe-ting to Gata3 gene,on the antigen's presenting and its immune biological characteristics of bone marrow-derived dendritic cells ( DC) of mice pregnancy asthma model. Methods:The recombi-nant lentiviral vector and packaging plasmid were co-transfected to AD293 cells, by liposome DNA precipitation method,to induce lentivirus-infected DC in vitro. Its effects on the integration and transcription of siGata3 gene were detected by RT-PCR and Western blot assay. The pheno-typic changes of cells were observed by flow cytometry. Transferring pSH1/siGata3 to mice by tail intravenous injection,the inflammatory factor levels in alveolar lavage fluid ( BALF) were measured by ELISA method. After mixed lymphocyte reaction

  18. Role of the GATA family of transcription factors in endocrine development, function, and disease.

    Science.gov (United States)

    Viger, Robert S; Guittot, Séverine Mazaud; Anttonen, Mikko; Wilson, David B; Heikinheimo, Markku

    2008-04-01

    The WGATAR motif is a common nucleotide sequence found in the transcriptional regulatory regions of numerous genes. In vertebrates, these motifs are bound by one of six factors (GATA1 to GATA6) that constitute the GATA family of transcriptional regulatory proteins. Although originally considered for their roles in hematopoietic cells and the heart, GATA factors are now known to be expressed in a wide variety of tissues where they act as critical regulators of cell-specific gene expression. This includes multiple endocrine organs such as the pituitary, pancreas, adrenals, and especially the gonads. Insights into the functional roles played by GATA factors in adult organ systems have been hampered by the early embryonic lethality associated with the different Gata-null mice. This is now being overcome with the generation of tissue-specific knockout models and other knockdown strategies. These approaches, together with the increasing number of human GATA-related pathologies have greatly broadened the scope of GATA-dependent genes and, importantly, have shown that GATA action is not necessarily limited to early development. This has been particularly evident in endocrine organs where GATA factors appear to contribute to the transcription of multiple hormone-encoding genes. This review provides an overview of the GATA family of transcription factors as they relate to endocrine function and disease.

  19. NECK LEAF 1, a GATA type transcription factor, modulates organogenesis by regulating the expression of multiple regulatory genes during reproductive development in rice

    Institute of Scientific and Technical Information of China (English)

    Liping Wang; Hengfu Yin; Qian Qian; Jun Yang; Chaofeng Huang; Xiaohe Hu; Da Luo

    2009-01-01

    In the monocot rice species Oryza sativa L., one of the most striking morphological processes during reproductive development is the concurrence of panicle development with the sequential elongation of upper internodes (UPIs). To elucidate the underlying molecular mechanisms, we cloned the rice gene NECK LEAF 1 (NL1), which when mutated results in delays in flowering time, smaller panicles with overgrown bracts and abnormal UPI elongation patterns. The NL1 gene encodes a GATA-type transcription factor with a single zinc finger domain, and its transcripts are de-tected predominantly in the bract primordia, which normally degenerate in the wild-type plants. Overexpression of NL1 in transgenic plants often gives rise to severe growth retardation, less vegetative phytomers and smaller leaves, suggesting that NL1 plays an important role in organ differentiation. A novel mutant allele of PLASTOCHRON1 (PLA1), a gene known to play a key role in regulating leaf initiation, was identified in this study. Genetic analysis demonstrated an interaction between nll and plal, with NL1 acting upstream of PLA1. The expression level and spatial pattern of PLA1 were found to be altered in the nll mutant. Furthermore, the expression of two regulators of flowering, Hd3a and OsMADS1, was also affected in the nll mutant. On the basis of these findings, we propose that NL1 is an intrinsic factor that modulates and coordinates organogenesis through regulating the expression of PLA1 and other regulatory genes during reproductive development in rice.

  20. Endothelial GATA-6 Deficiency Promotes Pulmonary Arterial Hypertension

    Science.gov (United States)

    Ghatnekar, Angela; Chrobak, Izabela; Reese, Charlie; Stawski, Lukasz; Seta, Francesca; Wirrig, Elaine; Paez-Cortez, Jesus; Markiewicz, Margaret; Asano, Yoshihide; Harley, Russell; Silver, Richard; Feghali-Bostwick, Carol; Trojanowska, Maria

    2014-01-01

    Pulmonary arterial hypertension (PAH) is a chronic and progressive disease characterized by pulmonary vasculopathy with elevation of pulmonary artery pressure, often culminating in right ventricular failure. GATA-6, a member of the GATA family of zinc-finger transcription factors, is highly expressed in quiescent vasculature and is frequently lost during vascular injury. We hypothesized that endothelial GATA-6 may play a critical role in the molecular mechanisms underlying endothelial cell (EC) dysfunction in PAH. Here we report that GATA-6 is markedly reduced in pulmonary ECs lining both occluded and nonoccluded vessels in patients with idiopathic and systemic sclerosis-associated PAH. GATA-6 transcripts are also rapidly decreased in rodent PAH models. Endothelial GATA-6 is a direct transcriptional regulator of genes controlling vascular tone [endothelin-1, endothelin-1 receptor type A, and endothelial nitric oxide synthase (eNOS)], pro-inflammatory genes, CX3CL1 (fractalkine), 5-lipoxygenease-activating protein, and markers of vascular remodeling, including PAI-1 and RhoB. Mice with the genetic deletion of GATA-6 in ECs (Gata6-KO) spontaneously develop elevated pulmonary artery pressure and increased vessel muscularization, and these features are further exacerbated in response to hypoxia. Furthermore, innate immune cells including macrophages (CD11b+/F4/80+), granulocytes (Ly6G+/CD45+), and dendritic cells (CD11b+/CD11c+) are significantly increased in normoxic Gata6-KO mice. Together, our findings suggest a critical role of endothelial GATA-6 deficiency in development and disease progression in PAH. PMID:23583651

  1. Altered translation of GATA1 in Diamond-Blackfan anemia.

    Science.gov (United States)

    Ludwig, Leif S; Gazda, Hanna T; Eng, Jennifer C; Eichhorn, Stephen W; Thiru, Prathapan; Ghazvinian, Roxanne; George, Tracy I; Gotlib, Jason R; Beggs, Alan H; Sieff, Colin A; Lodish, Harvey F; Lander, Eric S; Sankaran, Vijay G

    2014-07-01

    Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA), congenital asplenia and T cell leukemia. Yet, how mutations in genes encoding ubiquitously expressed proteins such as these result in cell-type- and tissue-specific defects remains unknown. Here, we identify mutations in GATA1, encoding the critical hematopoietic transcription factor GATA-binding protein-1, that reduce levels of full-length GATA1 protein and cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can lead to decreased GATA1 mRNA translation, possibly resulting from a higher threshold for initiation of translation of this mRNA in comparison with other mRNAs. In primary hematopoietic cells from patients with mutations in RPS19, encoding ribosomal protein S19, the amplitude of a transcriptional signature of GATA1 target genes was globally and specifically reduced, indicating that the activity, but not the mRNA level, of GATA1 is decreased in patients with DBA associated with mutations affecting ribosomal proteins. Moreover, the defective hematopoiesis observed in patients with DBA associated with ribosomal protein haploinsufficiency could be partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations affecting ubiquitous ribosomal proteins can result in human disease.

  2. GATA6 is a crucial regulator of Shh in the limb bud.

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    Elena Kozhemyakina

    2014-01-01

    Full Text Available In the limb bud, patterning along the anterior-posterior (A-P axis is controlled by Sonic Hedgehog (Shh, a signaling molecule secreted by the "Zone of Polarizing Activity", an organizer tissue located in the posterior margin of the limb bud. We have found that the transcription factors GATA4 and GATA6, which are key regulators of cell identity, are expressed in an anterior to posterior gradient in the early limb bud, raising the possibility that GATA transcription factors may play an additional role in patterning this tissue. While both GATA4 and GATA6 are expressed in an A-P gradient in the forelimb buds, the hindlimb buds principally express GATA6 in an A-P gradient. Thus, to specifically examine the role of GATA6 in limb patterning we generated Prx1-Cre; GATA6(fl/fl mice, which conditionally delete GATA6 from their developing limb buds. We found that these animals display ectopic expression of both Shh and its transcriptional targets specifically in the anterior mesenchyme of the hindlimb buds. Loss of GATA6 in the developing limbs results in the formation of preaxial polydactyly in the hindlimbs. Conversely, forced expression of GATA6 throughout the limb bud represses expression of Shh and results in hypomorphic limbs. We have found that GATA6 can bind to chromatin (isolated from limb buds encoding either Shh or Gli1 regulatory elements that drive expression of these genes in this tissue, and demonstrated that GATA6 works synergistically with FOG co-factors to repress expression of luciferase reporters driven by these sequences. Most significantly, we have found that conditional loss of Shh in limb buds lacking GATA6 prevents development of hindlimb polydactyly in these compound mutant embryos, indicating that GATA6 expression in the anterior region of the limb bud blocks hindlimb polydactyly by repressing ectopic expression of Shh.

  3. GATA6 is a crucial regulator of Shh in the limb bud.

    Science.gov (United States)

    Kozhemyakina, Elena; Ionescu, Andreia; Lassar, Andrew B

    2014-01-01

    In the limb bud, patterning along the anterior-posterior (A-P) axis is controlled by Sonic Hedgehog (Shh), a signaling molecule secreted by the "Zone of Polarizing Activity", an organizer tissue located in the posterior margin of the limb bud. We have found that the transcription factors GATA4 and GATA6, which are key regulators of cell identity, are expressed in an anterior to posterior gradient in the early limb bud, raising the possibility that GATA transcription factors may play an additional role in patterning this tissue. While both GATA4 and GATA6 are expressed in an A-P gradient in the forelimb buds, the hindlimb buds principally express GATA6 in an A-P gradient. Thus, to specifically examine the role of GATA6 in limb patterning we generated Prx1-Cre; GATA6(fl/fl) mice, which conditionally delete GATA6 from their developing limb buds. We found that these animals display ectopic expression of both Shh and its transcriptional targets specifically in the anterior mesenchyme of the hindlimb buds. Loss of GATA6 in the developing limbs results in the formation of preaxial polydactyly in the hindlimbs. Conversely, forced expression of GATA6 throughout the limb bud represses expression of Shh and results in hypomorphic limbs. We have found that GATA6 can bind to chromatin (isolated from limb buds) encoding either Shh or Gli1 regulatory elements that drive expression of these genes in this tissue, and demonstrated that GATA6 works synergistically with FOG co-factors to repress expression of luciferase reporters driven by these sequences. Most significantly, we have found that conditional loss of Shh in limb buds lacking GATA6 prevents development of hindlimb polydactyly in these compound mutant embryos, indicating that GATA6 expression in the anterior region of the limb bud blocks hindlimb polydactyly by repressing ectopic expression of Shh.

  4. GATA family of transcription factors of vertebrates: phylogenetics and chromosomal synteny

    Indian Academy of Sciences (India)

    Chunjiang He; Hanhua Cheng; Rongjia Zhou

    2007-12-01

    GATA genes are an evolutionarily conserved family, which encode a group of important transcription factors involved in the regulation of diverse processes including the development of the heart, haematopoietic system and sex gonads. However, the evolutionary history of the GATA family has not been completely understood. We constructed a complete phylogenetic tree with functional domain information of the GATA genes of both vertebrates and several invertebrates, and mapped the GATA genes onto relevant chromosomes. Conserved synteny was observed around the GATA loci on the chromosomes. GATAs have a tendency to segregate onto different chromosomes during evolution. The phylogenetic tree is consistent with the relevant functions of GATA members. Analysis of the zinc finger domain showed that the domain tends to be duplicated during evolution from invertebrates to vertebrates. We propose that the balance between duplications of zinc finger domains and GATA members should be maintained to exert their physiological roles in each evolutionary stage. Therefore, evolutionary pressure on the GATAs must exist to maintain the balance during evolution from invertebrates to vertebrates. These results reveal the evolutionary characteristics of the GATA family and contribute to a better understanding of the relationship between evolution and biological functions of the gene family, which will help to uncover the GATAs’ biological roles, evolution and their relationship with associated diseases.

  5. Gata3 acts downstream of beta-catenin signaling to prevent ectopic metanephric kidney induction.

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    David Grote

    2008-12-01

    Full Text Available Metanephric kidney induction critically depends on mesenchymal-epithelial interactions in the caudal region of the nephric (or Wolffian duct. Central to this process, GDNF secreted from the metanephric mesenchyme induces ureter budding by activating the Ret receptor expressed in the nephric duct epithelium. A failure to regulate this pathway is believed to be responsible for a large proportion of the developmental anomalies affecting the urogenital system. Here, we show that the nephric duct-specific inactivation of the transcription factor gene Gata3 leads to massive ectopic ureter budding. This results in a spectrum of urogenital malformations including kidney adysplasia, duplex systems, and hydroureter, as well as vas deferens hyperplasia and uterine agenesis. The variability of developmental defects is reminiscent of the congenital anomalies of the kidney and urinary tract (CAKUT observed in human. We show that Gata3 inactivation causes premature nephric duct cell differentiation and loss of Ret receptor gene expression. These changes ultimately affect nephric duct epithelium homeostasis, leading to ectopic budding of interspersed cells still expressing the Ret receptor. Importantly, the formation of these ectopic buds requires both GDNF/Ret and Fgf signaling activities. We further identify Gata3 as a central mediator of beta-catenin function in the nephric duct and demonstrate that the beta-catenin/Gata3 pathway prevents premature cell differentiation independently of its role in regulating Ret expression. Together, these results establish a genetic cascade in which Gata3 acts downstream of beta-catenin, but upstream of Ret, to prevent ectopic ureter budding and premature cell differentiation in the nephric duct.

  6. 不同物种GATA-2基因编码区生物信息学分析%Bioinformatics Analysis of Coding Regions of GATA-2 Gene among Different Species

    Institute of Scientific and Technical Information of China (English)

    张天; 李祥龙; 周荣艳; 李兰会

    2013-01-01

    利用生物信息学方法分析了小家鼠(Mus musculus)、褐家鼠(Rattus norvegicus)、人(Homo sapiens)、黑猩猩(Pan troglodytes)、大猩猩(Gorilla)、倭黑猩猩(Pan paniscus)、猿(Nomascus leucogenys)、狨(Callithrix jacchus)、亚马逊松鼠猴(Saimiri boliviensis)、家马(Equus caballus)、小耳大婴猴(Otolemur garnettii)、家猫(Felis catus)、东非狒狒(Papio Anubis)、猕猴(Macaca mulatta)、犬(Cains lapus)、野猪(Sus scrofa)、大熊猫(Ailuropoda melanoleuca)等17个物种GA TA-2基因编码序列(Coding sequence,CDS),并对该基因的遗传多样性、信号肽、导肽、跨膜结构域、疏水性/亲水性、蛋白质二级结构、氨基酸序列进行了分析和预测.结果表明,在17个物种52条基因序列中共检测到344个多态位点,有25种单倍型,GA TA-2基因序列编码区的种内、种间存在丰富的遗传多样性.GATA-2蛋白N端无信号肽,不具有导肽,没有跨膜结构域,表现为亲水性,蛋白质二级结构主要结构元件是无规卷曲和d-螺旋,理论等电点为9.43,GATA-2蛋白呈碱性.

  7. GATA4 is a critical regulator of gonadectomy-induced adrenocortical tumorigenesis in mice.

    Science.gov (United States)

    Krachulec, Justyna; Vetter, Melanie; Schrade, Anja; Löbs, Ann-Kathrin; Bielinska, Malgorzata; Cochran, Rebecca; Kyrönlahti, Antti; Pihlajoki, Marjut; Parviainen, Helka; Jay, Patrick Y; Heikinheimo, Markku; Wilson, David B

    2012-06-01

    In response to gonadectomy certain inbred mouse strains develop sex steroidogenic adrenocortical neoplasms. One of the hallmarks of neoplastic transformation is expression of GATA4, a transcription factor normally present in gonadal but not adrenal steroidogenic cells of the adult mouse. To show that GATA4 directly modulates adrenocortical tumorigenesis and is not merely a marker of gonadal-like differentiation in the neoplasms, we studied mice with germline or conditional loss-of-function mutations in the Gata4 gene. Germline Gata4 haploinsufficiency was associated with attenuated tumor growth and reduced expression of sex steroidogenic genes in the adrenal glands of ovariectomized B6D2F1 and B6AF1 mice. At 12 months after ovariectomy, wild-type B6D2F1 mice had biochemical and histological evidence of adrenocortical estrogen production, whereas Gata4(+/-) B6D2F1 mice did not. Germline Gata4 haploinsufficiency exacerbated the secondary phenotype of postovariectomy obesity in B6D2F1 mice, presumably by limiting ectopic estrogen production in the adrenal glands. Amhr2-cre-mediated deletion of floxed Gata4 (Gata4(F)) in nascent adrenocortical neoplasms of ovariectomized B6.129 mice reduced tumor growth and the expression of gonadal-like markers in a Gata4(F) dose-dependent manner. We conclude that GATA4 is a key modifier of gonadectomy-induced adrenocortical neoplasia, postovariectomy obesity, and sex steroidogenic cell differentiation.

  8. GATA3 mutation in a family with hypoparathyroidism, deafness and renal dysplasia syndrome

    Institute of Scientific and Technical Information of China (English)

    Zi-Yang Zhu; Qiao-Li Zhou; Shi-Ning Ni; Wei Gu

    2014-01-01

    Background: The hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome is an autosomal dominant disorder primarily caused by GATA3 gene mutation. We report here a case that both of a Chinese boy and his father had HDR syndrome which caused by a novel mutation of GATA3. Methods: Polymerase chain reaction and DNA sequencing was performed to detect the exons of the GATA3 gene for mutation analysis. Results: Sequence analysis of GATA3 revealed a heterozygous nonsense mutation in this family: a mutation of GATA3 at exon 2 (c.515C >A) that resulted in a premature stop at codon 172 (p.S172X) with a loss of two zinc fi nger domains. Conclusion: We identifi ed a novel nonsense mutation which will expand the spectrum of HDR-associated GATA3 mutations.

  9. Functional interaction of CP2 with GATA-1 in the regulation of erythroid promoters.

    Science.gov (United States)

    Bosè, Francesca; Fugazza, Cristina; Casalgrandi, Maura; Capelli, Alessia; Cunningham, John M; Zhao, Quan; Jane, Stephen M; Ottolenghi, Sergio; Ronchi, Antonella

    2006-05-01

    We observed that binding sites for the ubiquitously expressed transcription factor CP2 were present in regulatory regions of multiple erythroid genes. In these regions, the CP2 binding site was adjacent to a site for the erythroid factor GATA-1. Using three such regulatory regions (from genes encoding the transcription factors GATA-1, EKLF, and p45 NF-E2), we demonstrated the functional importance of the adjacent CP2/GATA-1 sites. In particular, CP2 binds to the GATA-1 HS2 enhancer, generating a ternary complex with GATA-1 and DNA. Mutations in the CP2 consensus greatly impaired HS2 activity in transient transfection assays with K562 cells. Similar results were obtained by transfection of EKLF and p45 NF-E2 mutant constructs. Chromatin immunoprecipitation with K562 cells showed that CP2 binds in vivo to all three regulatory elements and that both GATA-1 and CP2 were present on the same GATA-1 and EKLF regulatory elements. Adjacent CP2/GATA-1 sites may represent a novel module for erythroid expression of a number of genes. Additionally, coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrated a physical interaction between GATA-1 and CP2. This may contribute to the functional cooperation between these factors and provide an explanation for the important role of ubiquitous CP2 in the regulation of erythroid genes.

  10. Genetic redundancy of GATA factors in the extraembryonic trophoblast lineage ensures the progression of preimplantation and postimplantation mammalian development

    Science.gov (United States)

    Home, Pratik; Kumar, Ram Parikshan; Ganguly, Avishek; Saha, Biswarup; Milano-Foster, Jessica; Bhattacharya, Bhaswati; Ray, Soma; Gunewardena, Sumedha; Paul, Arindam; Camper, Sally A.; Fields, Patrick E.

    2017-01-01

    GATA transcription factors are implicated in establishing cell fate during mammalian development. In early mammalian embryos, GATA3 is selectively expressed in the extraembryonic trophoblast lineage and regulates gene expression to promote trophoblast fate. However, trophoblast-specific GATA3 function is dispensable for early mammalian development. Here, using dual conditional knockout mice, we show that genetic redundancy of Gata3 with paralog Gata2 in trophoblast progenitors ensures the successful progression of both pre- and postimplantation mammalian development. Stage-specific gene deletion in trophoblasts reveals that loss of both GATA genes, but not either alone, leads to embryonic lethality prior to the onset of their expression within the embryo proper. Using ChIP-seq and RNA-seq analyses, we define the global targets of GATA2/GATA3 and show that they directly regulate a large number of common genes to orchestrate stem versus differentiated trophoblast fate. In trophoblast progenitors, GATA factors directly regulate BMP4, Nodal and Wnt signaling components that promote embryonic-extraembryonic signaling cross-talk, which is essential for the development of the embryo proper. Our study provides genetic evidence that impairment of trophoblast-specific GATA2/GATA3 function could lead to early pregnancy failure. PMID:28232602

  11. GATA3 inhibits breast cancer metastasis through the reversal of epithelial-mesenchymal transition.

    Science.gov (United States)

    Yan, Wei; Cao, Qing Jackie; Arenas, Richard B; Bentley, Brooke; Shao, Rong

    2010-04-30

    GATA3, a transcription factor that regulates T lymphocyte differentiation and maturation, is exclusively expressed in early stage well differentiated breast cancers but not in advanced invasive cancers. However, little is understood regarding its activity and the mechanisms underlying this differential expression in cancers. Here, we employed GATA3-positive, non-invasive (MCF-7) and GATA3-negative, invasive (MDA-MB-231) breast cancer cells to define its role in the transformation between these two distinct phenotypes. Ectopic expression of GATA3 in MDA-MB-231 cells led to a cuboidal-like epithelial phenotype and reduced cell invasive activity. These cells also increased E-cadherin expression but decreased levels of vimentin, N-cadherin, and MMP-9. Further, MDA-MB-231 cells expressing GATA3 grew smaller primary tumors without metastasis compared with larger metastatic tumors derived from control MDA-MB-231 cells in xenografted mice. GATA3 was found to induce E-cadherin expression through binding GATA-like motifs located in the E-cadherin promoter. Blockade of GATA3 using small interfering RNA gene knockdown in MCF-7 cells triggered fibroblastic transformation and cell invasion, resulting in distant metastasis. Studies of human breast cancer showed that GATA3 expression correlated with elevated E-cadherin levels, ER expression, and long disease-free survival. These data suggest that GATA3 drives invasive breast cancer cells to undergo the reversal of epithelial-mesenchymal transition, leading to the suppression of cancer metastasis.

  12. Chromosomal mapping of the major and minor ribosomal genes, (GATA)n and U2 snRNA gene by double-colour FISH in species of the Batrachoididae family.

    Science.gov (United States)

    Ubeda-Manzanaro, María; Merlo, Manuel A; Palazón, José L; Cross, Ismael; Sarasquete, Carmen; Rebordinos, Laureana

    2010-07-01

    In the present study dual-colour fluorescence in situ hybridization (FISH) was performed to study the chromosomal distribution of 18S and 5S rDNAs, (GATA)(n) and 5S rDNA, and U2 snRNA and 18S rDNA in four species of Batrachoididae family: Amphichthys cryptocentrus, Batrachoides manglae, Porichthys plectrodon and Thalassophryne maculosa. The 18S rDNA signals were present in only one pair of chromosomes in all the four Batrachoididae species. The 5S rDNA was mapped on one pair of chromosomes, except in B. manglae, which showed a hybridization signal in two pairs. The two ribosomal genes are located on different chromosome pairs, except in A. cryptocentrus, in which they appear co-located. In all the cases, the (GATA)(n) probe produced disperse hybridization signals in all four species. The U2 snRNA signals appear very widely scattered in A. cryptocentrus, P. plectrodon, but show a degree of clustering in a specific chromosome pair in B. manglae. In T. maculosa, they are thinly dispersed and strong hybridization signals are observed co-located to the 18S rDNA-bearing chromosomes. Finally, a double-colour FISH with U2 snRNA and 5S rDNA probes was performed in B. manglae, and this showed that these genes were not co-located. These results have been compared with those from another Batrachoididae species, and evolutive processes of these species are discussed.

  13. Overexpression of GATA1 confers resistance to chemotherapy in acute megakaryocytic Leukemia.

    Directory of Open Access Journals (Sweden)

    John Timothy Caldwell

    Full Text Available It has been previously shown that acute myeloid leukemia (AML patients with higher levels of GATA1 expression have poorer outcomes. Furthermore, pediatric Down syndrome (DS patients with acute megakaryocytic leukemia (AMKL, whose blast cells almost universally harbor somatic mutations in exon 2 of the transcription factor gene GATA1, demonstrate increased overall survival relative to non-DS pediatric patients, suggesting a potential role for GATA1 in chemotherapy response. In this study, we confirmed that amongst non-DS patients, GATA1 transcripts were significantly higher in AMKL blasts compared to blasts from other AML subgroups. Further, GATA1 transcript levels significantly correlated with transcript levels for the anti-apoptotic protein Bcl-xL in our patient cohort. ShRNA knockdown of GATA1 in the megakaryocytic cell line Meg-01 resulted in significantly increased cytarabine (ara-C and daunorubicin anti-proliferative sensitivities and decreased Bcl-xL transcript and protein levels. Chromatin immunoprecipitation (ChIP and reporter gene assays demonstrated that the Bcl-x gene (which transcribes the Bcl-xL transcripts is a bona fide GATA1 target gene in AMKL cells. Treatment of the Meg-01 cells with the histone deacetylase inhibitor valproic acid resulted in down-regulation of both GATA1 and Bcl-xL and significantly enhanced ara-C sensitivity. Furthermore, additional GATA1 target genes were identified by oligonucleotide microarray and ChIP-on-Chip analyses. Our findings demonstrate a role for GATA1 in chemotherapy resistance in non-DS AMKL cells, and identified additional GATA1 target genes for future studies.

  14. Effects of β-d-mannuronic acid, as a novel non-steroidal anti-inflammatory medication within immunosuppressive properties, on IL17, RORγt, IL4 and GATA3 gene expressions in rheumatoid arthritis patients

    Science.gov (United States)

    Barati, Anis; Jamshidi, Ahmad Reza; Ahmadi, Hossein; Aghazadeh, Zahra; Mirshafiey, Abbas

    2017-01-01

    Rheumatoid arthritis (RA) is the most common form of chronic inflammatory arthritis characterized by pain, swelling and destruction of joints, with a resultant disability. Disease-modifying anti-rheumatic drugs (DMARDs) and biological drugs can interfere with the disease process. In this study, the effect of β-d-mannuronic acid (M2000) as a novel non-steroidal anti-inflammatory drug (NSAID) with immunosuppressive and anti-inflammatory effects together with antioxidant effects was evaluated on IL17, RORγt, IL4 and GATA3 gene expression in 12 RA patients. Previously, M2000 driven from sodium alginate (natural product; patented, DEU: 102016113018.4) has shown a notable efficacy in experimental models of multiple sclerosis, RA and nephrotic syndrome. This study was performed on 12 patients with RA who had an inadequate response to conventional treatments. During this trial, patients were permitted to continue the conventional therapy excluding NSAIDs. M2000 was administered orally at a dose of 500 mg twice daily for 12 weeks. The peripheral blood mononuclear cells (PBMCs) were collected before and after treatment to evaluate the expression levels of IL4, GATA3, IL17 and RORγt. The gene expression results showed that M2000 has a potent efficacy, so that it could not only significantly decrease IL17 and RORγt levels but also increase IL4 and GATA3 levels after 12 weeks of treatment. Moreover, the gene expression results were in accordance with the clinical and preclinical assessments. In conclusion, M2000 as a natural novel agent has therapeutic and immunosuppressive properties on RA patients (identifier: IRCT2014011213739N2).

  15. Generation of embryos directly from embryonic stem cells by tetraploid embryo complementation reveals a role for GATA factors in organogenesis.

    Science.gov (United States)

    Duncan, S A

    2005-12-01

    Gene targeting in ES (embryonic stem) cells has been used extensively to study the role of proteins during embryonic development. In the traditional procedure, this requires the generation of chimaeric mice by introducing ES cells into blastocysts and allowing them to develop to term. Once chimaeric mice are produced, they are bred into a recipient mouse strain to establish germline transmission of the allele of interest. Although this approach has been used very successfully, the breeding cycles involved are time consuming. In addition, genes that are essential for organogenesis often have roles in the formation of extra-embryonic tissues that are essential for early stages of post-implantation development. For example, mice lacking the GATA transcription factors, GATA4 or GATA6, arrest during gastrulation due to an essential role for these factors in differentiation of extra-embryonic endoderm. This lethality has frustrated the study of these factors during the development of organs such as the liver and heart. Extraembryonic defects can, however, be circumvented by generating clonal mouse embryos directly from ES cells by tetraploid complementation. Here, we describe the usefulness and efficacy of this approach using GATA factors as an example.

  16. Cardiomyocyte protection by GATA-4 gene engineered mesenchymal stem cells is partially mediated by translocation of miR-221 in microvesicles.

    Directory of Open Access Journals (Sweden)

    Bin Yu

    Full Text Available INTRODUCTION: microRNAs (miRs, a novel class of small non-coding RNAs, are involved in cell proliferation, differentiation, development, and death. In this study, we found that miR-221 translocation by microvesicles (MVs plays an important role in cardioprotection mediated by GATA-4 overexpressed mesenchymal stem cells (MSC. METHODS AND RESULTS: Adult rat bone marrow MSC and neonatal rat ventricle cardiomyocytes (CM were harvested as primary cultures. MSC were transduced with GATA-4 (MSC(GATA-4 using the murine stem cell virus (pMSCV retroviral expression system. Empty vector transfection was used as a control (MSC(Null. The expression of miRs was assessed by real-time PCR and localized using in situ hybridization (ISH. MVs collected from MSC cultures were characterized by expression of CD9, CD63, and HSP70, and photographed with electron microscopy. Cardioprotection during hypoxia afforded by conditioned medium (CdM from MSC cultures was evaluated by lactate dehydrogenase (LDH release, MTS uptake by CM, and caspase 3/7 activity. Expression of miR-221/222 was significantly higher in MSC than in CM and miR-221 was upregulated in MSC(GATA-4. MSC overexpression of miR-221 significantly enhanced cardioprotection by reducing the expression of p53 upregulated modulator of apoptosis (PUMA. Moreover, expression of PUMA was significantly decreased in CM co-cultured with MSC. MVs derived from MSC expressed high levels of miR-221, and were internalized quickly by CM as documented in images obtained from a Time-Lapse Imaging System. CONCLUSIONS: Our results demonstrate that cardioprotection by MSC(GATA-4 may be regulated in part by a transfer of anti-apoptotic miRs contained within MVs.

  17. Downstream targets of GATA3 in the vestibular sensory organs of the inner ear.

    Science.gov (United States)

    Alvarado, David M; Veile, Rose; Speck, Judith; Warchol, Mark; Lovett, Michael

    2009-12-01

    Haploinsufficiency for the transcription factor GATA3 leads to hearing loss in humans. It is expressed throughout the auditory sensory epithelium (SE). In the vestibular organs, GATA3 is limited to the striola reversal zone of the utricle. Stereocilia orientation shifts 180 degrees at this region, which contains morphologically distinct type-I hair cells. The striola is conserved in all amniotes, its function is unknown, and GATA3 is the only known marker of the reversal zone. To identify downstream targets of GATA3 that might point to striolar function, we measured gene expression differences between striolar and extra-striolar SE. These were compared with profiles after GATA3 RNAi and GATA3 over-expression. We identified four genes (BMP2, FKHL18, LMO4, and MBNL2) that consistently varied with GATA3. Two of these (LMO4 and MBNL2) were shown to be direct targets of GATA3 by ChIP. Our results suggest that GATA3 impacts WNT signaling in this region of the sensory macula.

  18. A unique role of GATA1s in Down syndrome acute megakaryocytic leukemia biology and therapy.

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    Ana C Xavier

    Full Text Available BACKGROUND: Acute megakaryocytic leukemia (AMkL in Down syndrome (DS children is uniformly associated with somatic GATA1 mutations, which result in the synthesis of a shorter protein (GATA1s with altered transactivation activity compared to the wild-type GATA1. It is not fully established whether leukemogenesis and therapeutic responses in DS AMkL patients are due to loss of the wild-type GATA1 or due to a unique function of GATA1s. METHODOLOGY: Stable clones of CMK cells with decreased GATA1s or Bcl-2 levels were generated by using GATA1- or BCL-2-specific lentivirus shRNAs. In vitro ara-C, daunorubicin, and VP-16 cytotoxicities of the shRNA stable clones were determined by using the Cell Titer-blue reagent. Apoptosis and cell cycle distribution were determined by flow cytometry analysis. Changes in gene transcript levels were determined by gene expression microarray and/or real-time RT-PCR. Changes in protein levels were measured by Western blotting. In vivo binding of GATA1s to IL1A promoter was determined by chromatin immunoprecipitation assays. RESULTS: Lentivirus shRNA knockdown of the GATA1 gene in the DS AMkL cell line, CMK (harbors a mutated GATA1 gene and only expresses GATA1s, resulting in lower GATA1s protein levels, promoted cell differentiation towards the megakaryocytic lineage and repressed cell proliferation. Increased basal apoptosis and sensitivities to ara-C, daunorubicin, and VP-16 accompanied by down-regulated Bcl-2 were also detected in the CMK GATA1 shRNA knockdown clones. Essentially the same results were obtained when Bcl-2 was knocked down with lentivirus shRNA in CMK cells. Besides Bcl-2, down-regulation of GATA1s also resulted in altered expression of genes (e.g., IL1A, PF4, and TUBB1 related to cell death, proliferation, and differentiation. CONCLUSION: Our results suggest that GATA1s may facilitate leukemogenesis and potentially impact therapeutic responses in DS AMkL by promoting proliferation and survival, and by

  19. Novel GATA6 mutations in patients with pancreatic agenesis and congenital heart malformations.

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    Christina S Chao

    Full Text Available Patients with pancreatic agenesis are born without a pancreas, causing permanent neonatal diabetes and pancreatic enzyme insufficiency. These patients require insulin and enzyme replacement therapy to survive, grow, and maintain normal blood glucose levels. Pancreatic agenesis is an uncommon condition but high-throughput sequencing methods provide a rare opportunity to identify critical genes that are necessary for human pancreas development. Here we present the clinical history, evaluation, and the genetic and molecular analysis from two patients with pancreatic agenesis. Both patients were born with intrauterine growth restriction, minor heart defects and neonatal diabetes. In both cases, pancreatic agenesis was confirmed by imaging studies. The patients are clinically stable with pancreatic enzymes and insulin therapy. In order identify the etiology for their disease, we performed whole exome sequencing on both patients. For each proband we identified a de novo heterozygous mutation in the GATA6 gene. GATA6 is a homeobox containing transcription factor involved in both early development of the pancreas and heart. In vitro functional analysis of one of the variants revealed that the mutation creates a premature stop codon in the coding sequence resulting in the production of a truncated protein with loss of activity. These results show how genetic mutations in GATA6 may lead to functional inactivity and pancreatic agenesis in humans.

  20. Repercussion of Megakaryocyte-Specific Gata1 Loss on Megakaryopoiesis and the Hematopoietic Precursor Compartment.

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    Marjolein Meinders

    Full Text Available During hematopoiesis, transcriptional programs are essential for the commitment and differentiation of progenitors into the different blood lineages. GATA1 is a transcription factor expressed in several hematopoietic lineages and essential for proper erythropoiesis and megakaryopoiesis. Megakaryocyte-specific genes, such as GP1BA, are known to be directly regulated by GATA1. Mutations in GATA1 can lead to dyserythropoietic anemia and pseudo gray-platelet syndrome. Selective loss of Gata1 expression in adult mice results in macrothrombocytopenia with platelet dysfunction, characterized by an excess of immature megakaryocytes. To specifically analyze the impact of Gata1 loss in mature committed megakaryocytes, we generated Gata1-Lox|Pf4-Cre mice (Gata1cKOMK. Consistent with previous findings, Gata1cKOMK mice are macrothrombocytopenic with platelet dysfunction. Supporting this notion we demonstrate that Gata1 regulates directly the transcription of Syk, a tyrosine kinase that functions downstream of Clec2 and GPVI receptors in megakaryocytes and platelets. Furthermore, we show that Gata1cKOMK mice display an additional aberrant megakaryocyte differentiation stage. Interestingly, these mice present a misbalance of the multipotent progenitor compartment and the erythroid lineage, which translates into compensatory stress erythropoiesis and splenomegaly. Despite the severe thrombocytopenia, Gata1cKOMK mice display a mild reduction of TPO plasma levels, and Gata1cKOMK megakaryocytes show a mild increase in Pf4 mRNA levels; such a misbalance might be behind the general hematopoietic defects observed, affecting locally normal TPO and Pf4 levels at hematopoietic stem cell niches.

  1. Continued expression of GATA3 is necessary for cochlear neurosensory development.

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    Jeremy S Duncan

    Full Text Available Hair cells of the developing mammalian inner ear are progressively defined through cell fate restriction. This process culminates in the expression of the bHLH transcription factor Atoh1, which is necessary for differentiation of hair cells, but not for their specification. Loss of several genes will disrupt ear morphogenesis or arrest of neurosensory epithelia development. We previously showed in null mutants that the loss of the transcription factor, Gata3, results specifically in the loss of all cochlear neurosensory development. Temporal expression of Gata3 is broad from the otic placode stage through the postnatal ear. It therefore remains unclear at which stage in development Gata3 exerts its effect. To better understand the stage specific effects of Gata3, we investigated the role of Gata3 in cochlear neurosensory specification and differentiation utilizing a LoxP targeted Gata3 line and two Cre lines. Foxg1(Cre∶Gata3(f/f mice show recombination of Gata3 around E8.5 but continue to develop a cochlear duct without differentiated hair cells and spiral ganglion neurons. qRT-PCR data show that Atoh1 was down-regulated but not absent in the duct whereas other hair cell specific genes such as Pou4f3 were completely absent. In addition, while Sox2 levels were lower in the Foxg1(Cre:Gata3(f/f cochlea, Eya1 levels remained normal. We conclude that Eya1 is unable to fully upregulate Atoh1 or Pou4f3, and drive differentiation of hair cells without Gata3. Pax2-Cre∶Gata3(f/f mice show a delayed recombination of Gata3 in the ear relative to Foxg1(Cre:Gata3(f/f . These mice exhibited a cochlear duct containing patches of partially differentiated hair cells and developed only few and incorrectly projecting spiral ganglion neurons. Our conditional deletion studies reveal a major role of Gata3 in the signaling of prosensory genes and in the differentiation of cochlear neurosenory cells. We suggest that Gata3 may act in combination with Eya1, Six1, and

  2. Generation of mice harbouring a conditional loss-of-function allele of Gata6

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    Duncan Stephen A

    2006-04-01

    Full Text Available Abstract The zinc finger transcription factor GATA6 is believed to have important roles in the development of several organs including the liver, gastrointestinal tract and heart. However, analyses of the contribution of GATA6 toward organogenesis have been hampered because Gata6-/- mice fail to develop beyond gastrulation due to defects in extraembryonic endoderm function. We have therefore generated a mouse line harbouring a conditional loss-of-function allele of Gata6 using Cre/loxP technology. LoxP elements were introduced into introns flanking exon 2 of the Gata6 gene by homologous recombination in ES cells. Mice containing this altered allele were bred to homozygosity and were found to be viable and fertile. To assess the functional integrity of the loxP sites and to confirm that we had generated a Gata6 loss-of-function allele, we bred Gata6 'floxed' mice to EIIa-Cre mice in which Cre is ubiquitously expressed, and to Villin-Cre mice that express Cre in the epithelial cells of the intestine. We conclude that we have generated a line of mice in which GATA6 activity can be ablated in a cell type specific manner by expression of Cre recombinase. This line of mice can be used to establish the role of GATA6 in regulating embryonic development and various aspects of mammalian physiology.

  3. Cooperation of Gata3, c-Myc and Notch in malignant transformation of double positive thymocytes.

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    van Hamburg, Jan Piet; de Bruijn, Marjolein J W; Dingjan, Gemma M; Beverloo, H Berna; Diepstraten, Hans; Ling, Kam-Wing; Hendriks, Rudi W

    2008-06-01

    Gata transcription factors are critical regulators of proliferation and differentiation implicated in various human cancers, but specific genes activated by Gata proteins remain to be identified. We previously reported that enforced expression of Gata3 during T cell development in CD2-Gata3 transgenic mice induced CD4(+)CD8(+) double-positive (DP) T cell lymphoma. Here, we show that the presence of the DO11.10 T-cell receptor transgene, which directs DP cells towards the CD4 lineage, resulted in enhanced lymphoma development and a dramatic increase in thymocyte cell size in CD2-Gata3 transgenic mice. CD2-Gata3 DP cells expressed high levels of the proto-oncogene c-Myc but the Notch1 signaling pathway, which is known to induce c-Myc, was not activated. Gene expression profiling showed that in CD2-Gata3 lymphoma cells transcription of c-Myc and its target genes was further increased. A substantial fraction of CD2-Gata3 lymphomas had trisomy of chromosome 15, leading to an increased c-Myc gene dose. Interestingly, most lymphomas showed high expression of the Notch targets Deltex1 and Hes1, often due to activating Notch1 PEST domain mutations. Therefore, we conclude that enforced Gata3 expression converts DP thymocytes into a pre-malignant state, characterized by high c-Myc expression, whereby subsequent induction of Notch1 signaling cooperates to establish malignant transformation. The finding that Gata3 regulates c-Myc expression levels, in a direct or indirect fashion, may explain the parallel phenotypes of mice with overexpression or deficiency of either of the two transcription factors.

  4. The GATA transcription factor egl-27 delays aging by promoting stress resistance in Caenorhabditis elegans.

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    Xiao Xu

    Full Text Available Stress is a fundamental aspect of aging, as accumulated damage from a lifetime of stress can limit lifespan and protective responses to stress can extend lifespan. In this study, we identify a conserved Caenorhabditis elegans GATA transcription factor, egl-27, that is involved in several stress responses and aging. We found that overexpression of egl-27 extends the lifespan of wild-type animals. Furthermore, egl-27 is required for the pro-longevity effects from impaired insulin/IGF-1 like signaling (IIS, as reduced egl-27 activity fully suppresses the longevity of worms that are mutant for the IIS receptor, daf-2. egl-27 expression is inhibited by daf-2 and activated by pro-longevity factors daf-16/FOXO and elt-3/GATA, suggesting that egl-27 acts at the intersection of IIS and GATA pathways to extend lifespan. Consistent with its role in IIS signaling, we found that egl-27 is involved in stress response pathways. egl-27 expression is induced in the presence of multiple stresses, its targets are significantly enriched for many types of stress genes, and altering levels of egl-27 itself affects survival to heat and oxidative stress. Finally, we found that egl-27 expression increases between young and old animals, suggesting that increased levels of egl-27 in aged animals may act to promote stress resistance. These results identify egl-27 as a novel factor that links stress and aging pathways.

  5. Altered translation of GATA1 in Diamond-Blackfan anemia

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    Ludwig, Leif S.; Gazda, Hanna T.; Eng, Jennifer C.; Eichhorn, Stephen W.; Thiru, Prathapan; Ghazvinian, Roxanne; George, Tracy I.; Gotlib, Jason R.; Beggs, Alan H.; Sieff, Colin A.; Lodish, Harvey F.; Lander, Eric S.; Sankaran, Vijay G.

    2014-01-01

    Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA),1,2 congenital asplenia,3 and T-cell leukemia.4 Yet how mutations in such ubiquitously expressed proteins result in cell-type and tissue specific defects remains a mystery.5 Here, we show that GATA1 mutations that reduce full-length protein levels of this critical hematopoietic transcription factor can cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can similarly reduce translation of GATA1 mRNA - a phenomenon that appears to result from this mRNA having a higher threshold for initiation of translation. In primary hematopoietic cells from patients with RPS19 mutations, a transcriptional signature of GATA1 target genes is globally and specifically reduced, confirming that the activity, but not the mRNA level, of GATA1 is reduced in DBA patients with ribosomal protein mutations. The defective hematopoiesis observed in DBA patients with ribosomal protein haploinsufficiency can be at least partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations in ubiquitous ribosomal proteins can result in human disease. PMID:24952648

  6. PU.1 Suppresses Th2 Cytokine Expression via Silencing of GATA3 Transcription in Dendritic Cells.

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    Yashiro, Takuya; Kubo, Masato; Ogawa, Hideoki; Okumura, Ko; Nishiyama, Chiharu

    2015-01-01

    The transcription factor PU.1 is predominantly expressed in dendritic cells (DCs) and is essential for DC differentiation. Although there are several reports that PU.1 positively regulates the expression of DC-specific genes, whether PU.1 also has a suppressive effect on DCs is largely unknown. Here we demonstrate that PU.1 suppresses the expression of Th2 cytokines including IL-13 and IL-5 in bone marrow-derived DCs (BMDCs), through repression of the expression of GATA3, which is a master regulator of Th2 differentiations. When PU.1 siRNA was introduced into BMDCs, LPS-induced expression of IL-13 and IL-5 was increased along with upregulation of the constitutive expression of GATA2 and GATA3. The additional introduction of GATA3 siRNA but not of GATA2 siRNA abrogated PU.1 siRNA-mediated upregulation of IL-13 and IL-5. A chromatin immunoprecipitation assay showed that PU.1 bound to Gata3 proximal promoter region, which is more dominant than the distal promoter in driving GATA3 transcription in DCs. The degree of histone acetylation at the Gata3 promoter was decreased in PU.1 siRNA-introduced DCs, suggesting the involvement of PU.1 in chromatin modification of the Gata3 promoter. Treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A, increased the degree of histone H3 acetylation at the Gata3 promoter and induced the subsequent expression of GATA3. Experiments using HDAC inhibitors and siRNAs showed that HDAC3 suppressed GATA3 expression. The recruitment of HDAC3 to the Gata3 promoter was decreased by PU.1 knockdown. LPS-induced IL-13 expression was dramatically reduced in BMDCs generated from mice lacking the conserved GATA3 response element, termed CGRE, which is an essential site for the binding of GATA3 on the Il-13 promoter. The degree of H3K4me3 at CGRE was significantly increased in PU.1 siRNA-transfected stimulated DCs. Our results indicate that PU.1 plays pivotal roles in DC development and function, serving not only as a transcriptional

  7. Characterization of an Nmr homolog that modulates GATA factor-mediated nitrogen metabolite repression in Cryptococcus neoformans.

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    I Russel Lee

    Full Text Available Nitrogen source utilization plays a critical role in fungal development, secondary metabolite production and pathogenesis. In both the Ascomycota and Basidiomycota, GATA transcription factors globally activate the expression of catabolic enzyme-encoding genes required to degrade complex nitrogenous compounds. However, in the presence of preferred nitrogen sources such as ammonium, GATA factor activity is inhibited in some species through interaction with co-repressor Nmr proteins. This regulatory phenomenon, nitrogen metabolite repression, enables preferential utilization of readily assimilated nitrogen sources. In the basidiomycete pathogen Cryptococcus neoformans, the GATA factor Gat1/Are1 has been co-opted into regulating multiple key virulence traits in addition to nitrogen catabolism. Here, we further characterize Gat1/Are1 function and investigate the regulatory role of the predicted Nmr homolog Tar1. While GAT1/ARE1 expression is induced during nitrogen limitation, TAR1 transcription is unaffected by nitrogen availability. Deletion of TAR1 leads to inappropriate derepression of non-preferred nitrogen catabolic pathways in the simultaneous presence of favoured sources. In addition to exhibiting its evolutionary conserved role of inhibiting GATA factor activity under repressing conditions, Tar1 also positively regulates GAT1/ARE1 transcription under non-repressing conditions. The molecular mechanism by which Tar1 modulates nitrogen metabolite repression, however, remains open to speculation. Interaction between Tar1 and Gat1/Are1 was undetectable in a yeast two-hybrid assay, consistent with Tar1 and Gat1/Are1 each lacking the conserved C-terminus regions present in ascomycete Nmr proteins and GATA factors that are known to interact with each other. Importantly, both Tar1 and Gat1/Are1 are suppressors of C. neoformans virulence, reiterating and highlighting the paradigm of nitrogen regulation of pathogenesis.

  8. Effects of β-D-mannuronic acid, as a novel non-steroidal anti-inflammatory medication within immunosuppressive properties, on IL17, RORγt, IL4 and GATA3 gene expressions in rheumatoid arthritis patients

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    Barati A

    2017-03-01

    Full Text Available Anis Barati,1 Ahmad Reza Jamshidi,2,* Hossein Ahmadi,1 Zahra Aghazadeh,1 Abbas Mirshafiey1,* 1Department of Immunology, School of Public Health, 2Iranian Institute for Health Sciences Research, Rheumatology Research Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran *These authors contributed equally to this work Abstract: Rheumatoid arthritis (RA is the most common form of chronic inflammatory arthritis characterized by pain, swelling and destruction of joints, with a resultant disability. Disease-modifying anti-rheumatic drugs (DMARDs and biological drugs can interfere with the disease process. In this study, the effect of β-D-mannuronic acid (M2000 as a novel non-steroidal anti-inflammatory drug (NSAID with immunosuppressive and anti-inflammatory effects together with antioxidant effects was evaluated on IL17, RORγt, IL4 and GATA3 gene expression in 12 RA patients. Previously, M2000 driven from sodium alginate (natural product; patented, DEU: 102016113018.4 has shown a notable efficacy in experimental models of multiple sclerosis, RA and nephrotic syndrome. This study was performed on 12 patients with RA who had an inadequate response to conventional treatments. During this trial, patients were permitted to continue the conventional therapy excluding NSAIDs. M2000 was administered orally at a dose of 500 mg twice daily for 12 weeks. The peripheral blood mononuclear cells (PBMCs were collected before and after treatment to evaluate the expression levels of IL4, GATA3, IL17 and RORγt. The gene expression results showed that M2000 has a potent efficacy, so that it could not only significantly decrease IL17 and RORγt levels but also increase IL4 and GATA3 levels after 12 weeks of treatment. Moreover, the gene expression results were in accordance with the clinical and preclinical assessments. In conclusion, M2000 as a natural novel agent has therapeutic and immunosuppressive properties on RA patients (identifier

  9. BMP-mediated induction of GATA4/5/6 blocks somitic responsiveness to SHH.

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    Daoud, Georges; Kempf, Hervé; Kumar, Deepak; Kozhemyakina, Elena; Holowacz, Tamara; Kim, Dae-Won; Ionescu, Andreia; Lassar, Andrew B

    2014-10-01

    The relative timing of SHH and BMP signals controls whether presomitic mesoderm (PSM) cells will adopt either a chondrogenic or lateral plate mesoderm fate. Here we document that SHH-mediated induction of Nkx3.2 maintains the competence of somitic cells to initiate chondrogenesis in response to subsequent BMP signals by repressing BMP-dependent induction of GATA genes. Conversely, administration of BMP signals to PSM or forced expression of GATA family members in chick PSM explants blocks induction of hedgehog-dependent gene expression. We demonstrate that GATA factors can interact with Gli factors and can recruit the transcriptional co-factor FOG1 (ZFPM1) to the regulatory region of the mouse Gli1 gene, repressing the induction of Gli1 by SHH by binding to both GATA and Gli binding sites. Knockdown of FOG1 reverses the ability of GATA factors to repress Gli1 expression. Our findings uncover a novel role for GATA transcription factors as repressors of hedgehog signaling, and document that NKX3.2 maintains the ability of sclerotomal cells to express SHH transcriptional targets in the presence of BMP signals by repressing the induction of Gata4/5/6.

  10. Function of GATA transcription factors in hydroxyurea-induced HEL cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    HEL cells, a human erythroleukemia cell line, mainly express the fetal (γ)globin gene and trace amount of the embryonic (ε)globin gene, but not adult (β) globin gene. Here we show that hydroxyurea (HU) can induce HEL cells to express adult (β) globin gene and lead these cells to terminal differentiation. Results showed in Gel mobility shift assays that GATA factors could specifically bind to the regulatory elements of humanβ- globin gene, including the proximal regulatory element (theβ- promoter) and the distal regulatory elements (the DNase I hypersensitive sites in the LCR, HS2-HS4 core sequences). However, the DNA binding patterns of GATA factors were quite different between HU-induced and uninduced HEL cells. Western-blot analysis of nuclear extracts from both the uninduced and HU- induced HEL cells revealed that the level of GATA-2 transcription factor decreased, whereas the level of GATA-1 transcription factor increased following the time of hydroxyurea induction. Furthermore, using RT-PCR analysis the expression of human β-globin gene in HU-induced HEL cells could be blocked again when HEL cells were incubated in the presence of antisense oligonucleotides for hGATA-1, suggesting that the upregulation of hGATA-1 transcription factor might be critical for the expression of humanβ- globin gene in HU-induced HEL cells.

  11. Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 reduces cardiac fibroblast proliferation by suppressing GATA Binding Protein 4

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Bin; Liu, Ning-Ning; Liu, Wei-Hua; Zhang, Shuang-Wei; Zhang, Jing-Zhi; Li, Ai-Qun [Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Guangzhou Institute of Cardiovascular Disease, Guangzhou (China); Liu, Shi-Ming, E-mail: gzliushiming@126.com [Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Guangzhou Institute of Cardiovascular Disease, Guangzhou (China)

    2016-07-08

    Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and GATA Binding Protein 4 (GATA4) are important for the growth of cardiac fibroblasts (CFs). When deregulated, LOX-1 and GATA4 can cause cardiac remodeling. In the present study, we found novel evidence that GATA4 was required for the LOX-1 regulation of CF proliferation. The inhibition of LOX-1 by RNA interference LOX-1 lentivirus resulted in the loss of PI3K/Akt activation and GATA4 protein expression. The overexpression of LOX-1 by lentivirus rescued CF proliferation, PI3K/Akt activation, and GATA4 protein expression. Moreover, GATA4 overexpression enhanced CF proliferation with LOX-1 inhibition. We also found that the inhibition of PI3K/Akt activation by LY294002, a PI3K inhibitor, reduced cell proliferation and protein level of GATA4. In summary, GATA4 may play an important role in the LOX-1 and PI3K/Akt regulation of CF proliferation. -- Highlights: •GATA4 is regulated by LOX-1 signaling in CFs. •GATA4 is involved in LOX-1 regulating CF proliferation. •GATA4 is regulated by PI3K/Akt signaling in CFs.

  12. A GATA4-regulated tumor suppressor network represses formation of malignant human astrocytomas

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    Agnihotri, Sameer; Wolf, Amparo; Munoz, Diana M.; Smith, Christopher J.; Gajadhar, Aaron; Restrepo, Andres; Clarke, Ian D.; Fuller, Gregory N.; Kesari, Santosh; Dirks, Peter B.; McGlade, C. Jane; Stanford, William L.; Aldape, Kenneth; Mischel, Paul S.; Hawkins, Cynthia

    2011-01-01

    Glioblastoma Multiforme (GBM), the most common and lethal primary human brain tumor, exhibits multiple molecular aberrations. We report that loss of the transcription factor GATA4, a negative regulator of normal astrocyte proliferation, is a driver in glioma formation and fulfills the hallmarks of a tumor suppressor gene (TSG). Although GATA4 was expressed in normal brain, loss of GATA4 was observed in 94/163 GBM operative samples and was a negative survival prognostic marker. GATA4 loss occurred through promoter hypermethylation or novel somatic mutations. Loss of GATA4 in normal human astrocytes promoted high-grade astrocytoma formation, in cooperation with other relevant genetic alterations such as activated Ras or loss of TP53. Loss of GATA4 with activated Ras in normal astrocytes promoted a progenitor-like phenotype, formation of neurospheres, and the ability to differentiate into astrocytes, neurons, and oligodendrocytes. Re-expression of GATA4 in human GBM cell lines, primary cultures, and brain tumor–initiating cells suppressed tumor growth in vitro and in vivo through direct activation of the cell cycle inhibitor P21CIP1, independent of TP53. Re-expression of GATA4 also conferred sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in GBM therapy. This sensitivity was independent of MGMT (O-6-methylguanine-DNA-methyltransferase), the DNA repair enzyme which is often implicated in temozolomide resistance. Instead, GATA4 reduced expression of APNG (alkylpurine-DNA-N-glycosylase), a DNA repair enzyme which is poorly characterized in GBM-mediated temozolomide resistance. Identification and validation of GATA4 as a TSG and its downstream targets in GBM may yield promising novel therapeutic strategies. PMID:21464220

  13. Essential role for the planarian intestinal GATA transcription factor in stem cells and regeneration.

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    Flores, Natasha M; Oviedo, Néstor J; Sage, Julien

    2016-10-01

    The cellular turnover of adult tissues and injury-induced repair proceed through an exquisite integration of proliferation, differentiation, and survival signals that involve stem/progenitor cell populations, their progeny, and differentiated tissues. GATA factors are DNA binding proteins that control stem cells and the development of tissues by activating or repressing transcription. Here we examined the role of GATA transcription factors in Schmidtea mediterranea, a freshwater planarian that provides an excellent model to investigate gene function in adult stem cells, regeneration, and differentiation. Smed-gata4/5/6, the homolog of the three mammalian GATA-4,-5,-6 factors is expressed at high levels in differentiated gut cells but also at lower levels in neoblast populations, the planarian stem cells. Smed-gata4/5/6 knock-down results in broad differentiation defects, especially in response to injury. These defects are not restricted to the intestinal lineage. In particular, at late time points during the response to injury, loss of Smed-gata4/5/6 leads to decreased neoblast proliferation and to gene expression changes in several neoblast subpopulations. Thus, Smed-gata4/5/6 plays a key evolutionary conserved role in intestinal differentiation in planarians. These data further support a model in which defects in the intestinal lineage can indirectly affect other differentiation pathways in planarians.

  14. GATA transcription factors in adrenal development and tumors.

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    Parviainen, Helka; Kiiveri, Sanne; Bielinska, Malgorzata; Rahman, Nafis; Huhtaniemi, Ilpo T; Wilson, David B; Heikinheimo, Markku

    2007-02-01

    Of the six GATA transcription factors, GATA-4 and GATA-6 are expressed in the mouse and human adrenal with distinct developmental profiles. GATA-4 is confined to the fetal cortex, i.e. to the less differentiated proliferating cells, while GATA-6 is expressed both in the fetal and adult adrenal. In vitro, GATA-4 regulates inhibin-alpha and steroidogenic factor-1 implicated in normal adrenal function. GATA-6 probably has roles in the development and differentiation of adrenocortical cells, and in the regulation of steroidogenesis. GATA-4 expression is dramatically upregulated and GATA-6 downregulated in gonadotropin dependent mouse adrenocortical tumors. This is accompanied by the appearance of luteinizing hormone receptor (LHR). In vitro, GATA-4 transactivates LHR promoter, and gonadotropins upregulate GATA-4 levels. Human adrenal tumors occasionally express GATA-4, whereas GATA-6 levels are usually lower than normal.

  15. The N-terminal zinc finger of the erythroid transcription factor GATA-1 binds GATC motifs in DNA.

    Science.gov (United States)

    Newton, A; Mackay, J; Crossley, M

    2001-09-21

    The mammalian transcription factor GATA-1 is required for normal erythroid and megakaryocytic development. GATA-1 contains two zinc fingers, the C-terminal finger, which is known to bind (A/T)GATA(A/G) motifs in DNA and the N-finger, which is important for interacting with co-regulatory proteins such as Friend of GATA (FOG). We now show that, like the C-finger, the N-finger of GATA-1 is also capable of binding DNA but recognizes distinct sequences with the core GATC. We demonstrate that the GATA-1 N-finger can bind these sequences in vitro and that in cellular assays, GATA-1 can activate promoters containing GATC motifs. Experiments with mutant GATA-1 proteins confirm the importance of the N-finger, as the C-finger is not required for transactivation from GATC sites. Recently four naturally occurring mutations in GATA-1 have been shown to be associated with familial blood disorders. These mutations all map to the N-finger domain. We have investigated the effect of these mutations on the recognition of GATC sites by the N-finger and show that one mutation R216Q abolishes DNA binding, whereas the others have only minor effects.

  16. Genomic profiling identifies GATA6 as a candidate oncogene amplified in pancreatobiliary cancer.

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    Kevin A Kwei

    2008-05-01

    Full Text Available Pancreatobiliary cancers have among the highest mortality rates of any cancer type. Discovering the full spectrum of molecular genetic alterations may suggest new avenues for therapy. To catalogue genomic alterations, we carried out array-based genomic profiling of 31 exocrine pancreatic cancers and 6 distal bile duct cancers, expanded as xenografts to enrich the tumor cell fraction. We identified numerous focal DNA amplifications and deletions, including in 19% of pancreatobiliary cases gain at cytoband 18q11.2, a locus uncommonly amplified in other tumor types. The smallest shared amplification at 18q11.2 included GATA6, a transcriptional regulator previously linked to normal pancreas development. When amplified, GATA6 was overexpressed at both the mRNA and protein levels, and strong immunostaining was observed in 25 of 54 (46% primary pancreatic cancers compared to 0 of 33 normal pancreas specimens surveyed. GATA6 expression in xenografts was associated with specific microarray gene-expression patterns, enriched for GATA binding sites and mitochondrial oxidative phosphorylation activity. siRNA mediated knockdown of GATA6 in pancreatic cancer cell lines with amplification led to reduced cell proliferation, cell cycle progression, and colony formation. Our findings indicate that GATA6 amplification and overexpression contribute to the oncogenic phenotypes of pancreatic cancer cells, and identify GATA6 as a candidate lineage-specific oncogene in pancreatobiliary cancer, with implications for novel treatment strategies.

  17. Gata4 and Hnf1α are partially required for the expression of specific intestinal genes during development

    NARCIS (Netherlands)

    T. Bosse (Tjalling); J.J. Fialkovich (John); C.M. Piaseckyj (Christina); E. Beuling (Eva); H. Broekman (Henrike); R.J. Grand (Richard); R.K. Montgomery (Robert); S.D. Krasinski (Stephen)

    2007-01-01

    textabstractThe terminal differentiation phases of intestinal development in mice occur during cytodifferentiation and the weaning transition. Lactase-phlorizin hydrolase (LPH), liver fatty acid binding protein (Fabp1), and sucrase-isomaltase (SI) are well-characterized markers of these transitions.

  18. GATA binding protein 3 is correlated with leptin regulation of PPARγ1 in hepatic stellate cells.

    Science.gov (United States)

    Guan, Wei; Cheng, Fangyun; Wu, Hao; Cao, Qing; Zhu, Xiaofei; Fan, Yan; Zhu, Huixia; Zhou, Yajun

    2017-03-01

    Accumulating evidence reveals that hormone leptin, mainly produced by adipocyte, plays a unique role in promotion of liver fibrosis. Hepatic stellate cell (HSC) activation is a key step in liver fibrosis and peroxisome-proliferator activated receptor γ (PPARγ) exerts a crucial role in inhibition of HSC activation. Our previous researches demonstrated that leptin reduced PPARγ1 (a major subtype of PPARγ in HSCs) expression through GATA binding protein 2 (GATA2) binding to a site around -2323 in PPARγ1 promoter. The present researches aimed to examine the effect of GATA3 on leptin-induced inhibition of PPARγ1 and elucidate the relationship between GATA3 and GATA2. Gene expressions were analysed by real-time PCR, western blot, luciferase assay and immunostaining. C57BL/6J ob/ob mouse model of thioacetamide-induced liver injury was used in vivo. Results demonstrate that leptin significantly induces GATA3 expression in HSCs by multiple signalling pathways including NADPH oxidase pathway. There exist crosstalks between NADPH oxidase pathway and the other pathways. GATA3 can bind to GATA2-binding site in PPARγ1 promoter and interacts with GATA2, contributing to leptin inhibition of PPARγ1 expression in HSCs. These data demonstrated novel molecular events for leptin inhibition of PPARγ1 expression in HSCs and thus might have potential implications for clarifying the detailed mechanisms underlying liver fibrosis in diseases in which circulating leptin levels are elevated such as non-alcoholic steatohepatitis in obese patients. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  19. Gata3 drives development of RORγt+ group 3 innate lymphoid cells

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    Serafini, Nicolas; Klein Wolterink, Roel G.J.; Satoh-Takayama, Naoko; Xu, Wei; Vosshenrich, Christian A.J.; Hendriks, Rudi W.

    2014-01-01

    Group 3 innate lymphoid cells (ILC3) include IL-22–producing NKp46+ cells and IL-17A/IL-22–producing CD4+ lymphoid tissue inducerlike cells that express RORγt and are implicated in protective immunity at mucosal surfaces. Whereas the transcription factor Gata3 is essential for T cell and ILC2 development from hematopoietic stem cells (HSCs) and for IL-5 and IL-13 production by T cells and ILC2, the role for Gata3 in the generation or function of other ILC subsets is not known. We found that abundant GATA-3 protein is expressed in mucosa-associated ILC3 subsets with levels intermediate between mature B cells and ILC2. Chimeric mice generated with Gata3-deficient fetal liver hematopoietic precursors lack all intestinal RORγt+ ILC3 subsets, and these mice show defective production of IL-22 early after infection with the intestinal pathogen Citrobacter rodentium, leading to impaired survival. Further analyses demonstrated that ILC3 development requires cell-intrinsic Gata3 expression in fetal liver hematopoietic precursors. Our results demonstrate that Gata3 plays a generalized role in ILC lineage determination and is critical for the development of gut RORγt+ ILC3 subsets that maintain mucosal barrier homeostasis. These results further extend the paradigm of Gata3-dependent regulation of diversified innate ILC and adaptive T cell subsets. PMID:24419270

  20. Regulation of an Autoimmune Model for Multiple Sclerosis in Th2-Biased GATA3 Transgenic Mice

    Directory of Open Access Journals (Sweden)

    Viromi Fernando

    2014-01-01

    Full Text Available T helper (Th2 cells have been proposed to play a neuroprotective role in multiple sclerosis (MS. This is mainly based on “loss-of-function” studies in an animal model for MS, experimental autoimmune encephalomyelitis (EAE, using blocking antibodies against Th2 related cytokines, and knockout mice lacking Th2-related molecules. We tested whether an increase of Th2 responses (“gain-of-function” approach could alter EAE, the approach of novel GATA binding protein 3 (GATA3-transgenic (tg mice that overexpress GATA3, a transcription factor required for Th2 differentiation. In EAE induced with myelin oligodendrocyte glycoprotein (MOG35−55 peptide, GATA3-tg mice had a significantly delayed onset of disease and a less severe maximum clinical score, compared with wild-type C57BL/6 mice. Histologically, GATA3-tg mice had decreased levels of meningitis and demyelination in the spinal cord, and anti-inflammatory cytokine profiles immunologically, however both groups developed similar levels of MOG-specific lymphoproliferative responses. During the early stage, we detected higher levels of interleukin (IL-4 and IL-10, with MOG and mitogen stimulation of regional lymph node cells in GATA3-tg mice. During the late stage, only mitogen stimulation induced higher IL-4 and lower interferon-γ and IL-17 production in GATA3-tg mice. These results suggest that a preexisting bias toward a Th2 immune response may reduce the severity of inflammatory demyelinating diseases, including MS.

  1. Disease Model of GATA4 Mutation Reveals Transcription Factor Cooperativity in Human Cardiogenesis.

    Science.gov (United States)

    Ang, Yen-Sin; Rivas, Renee N; Ribeiro, Alexandre J S; Srivas, Rohith; Rivera, Janell; Stone, Nicole R; Pratt, Karishma; Mohamed, Tamer M A; Fu, Ji-Dong; Spencer, C Ian; Tippens, Nathaniel D; Li, Molong; Narasimha, Anil; Radzinsky, Ethan; Moon-Grady, Anita J; Yu, Haiyuan; Pruitt, Beth L; Snyder, Michael P; Srivastava, Deepak

    2016-12-15

    Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions, leading to gene network dysregulation and human disease. Human mutations in GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling, and metabolic activity. In human cardiomyocytes, GATA4 broadly co-occupied cardiac enhancers with TBX5, another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment, particularly to cardiac super-enhancers, concomitant with dysregulation of genes related to the phenotypic abnormalities, including cardiac septation. Conversely, the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity, leading to aberrant chromatin states and cellular dysfunction, including those related to morphogenetic defects. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. GATA6 mutations cause human cardiac outflow tract defects by disrupting semaphorin-plexin signaling

    Science.gov (United States)

    Kodo, Kazuki; Nishizawa, Tsutomu; Furutani, Michiko; Arai, Shoichi; Yamamura, Eiji; Joo, Kunitaka; Takahashi, Takao; Matsuoka, Rumiko; Yamagishi, Hiroyuki

    2009-01-01

    Congenital heart diseases (CHD) occur in nearly 1% of all live births and are the major cause of infant mortality and morbidity. Although an improved understanding of the genetic causes of CHD would provide insight into the underlying pathobiology, the genetic etiology of most CHD remains unknown. Here we show that mutations in the gene encoding the transcription factor GATA6 cause CHD characteristic of a severe form of cardiac outflow tract (OFT) defect, namely persistent truncus arteriosus (PTA). Two different GATA6 mutations were identified by systematic genetic analysis using DNA from patients with PTA. Genes encoding the neurovascular guiding molecule semaphorin 3C (SEMA3C) and its receptor plexin A2 (PLXNA2) appear to be regulated directly by GATA6, and both GATA6 mutant proteins failed to transactivate these genes. Transgenic analysis further suggests that, in the developing heart, the expression of SEMA3C in the OFT/subpulmonary myocardium and PLXNA2 in the cardiac neural crest contributing to the OFT is dependent on GATA transcription factors. Together, our data implicate mutations in GATA6 as genetic causes of CHD involving OFT development, as a result of the disruption of the direct regulation of semaphorin-plexin signaling. PMID:19666519

  3. DNA methylation restricts lineage-specific functions of transcription factor Gata4 during embryonic stem cell differentiation.

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    Masaaki Oda

    2013-06-01

    Full Text Available DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Gata4-binding-site profiles and DNA methylation analyses suggested that DNA methylation modulates the Gata4 response through diverse mechanisms. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells.

  4. Serine 105 phosphorylation of transcription factor GATA4 is necessary for stress-induced cardiac hypertrophy in vivo.

    Science.gov (United States)

    van Berlo, Jop H; Elrod, John W; Aronow, Bruce J; Pu, William T; Molkentin, Jeffery D

    2011-07-26

    Cardiac hypertrophy is an adaptive growth process that occurs in response to stress stimulation or injury wherein multiple signal transduction pathways are induced, culminating in transcription factor activation and the reprogramming of gene expression. GATA4 is a critical transcription factor in the heart that is known to induce/regulate the hypertrophic program, in part, by receiving signals from MAPKs. Here we generated knock-in mice in which a known MAPK phosphorylation site at serine 105 (S105) in Gata4 that augments activity was mutated to alanine. Homozygous Gata4-S105A mutant mice were viable as adults, although they showed a compromised stress response of the myocardium. For example, cardiac hypertrophy in response to phenylephrine agonist infusion for 2 wk was largely blunted in Gata4-S105A mice, as was the hypertrophic response to pressure overload at 1 and 2 wk of applied stimulation. Gata4-S105A mice were also more susceptible to heart failure and cardiac dilation after 2 wk of pressure overload. With respect to the upstream pathway, hearts from Gata4-S105A mice did not efficiently hypertrophy following direct ERK1/2 activation using an activated MEK1 transgene in vivo. Mechanistically, GATA4 mutant protein from these hearts failed to show enhanced DNA binding in response to hypertrophic stimulation. Moreover, hearts from Gata4-S105A mice had significant changes in the expression of hypertrophy-inducible, fetal, and remodeling-related genes.

  5. Myocardial Gene Expression of T-bet, GATA-3, Ror-γt, FoxP3, and Hallmark Cytokines in Chronic Chagas Disease Cardiomyopathy: An Essentially Unopposed TH1-Type Response

    Directory of Open Access Journals (Sweden)

    Luciana Gabriel Nogueira

    2014-01-01

    Full Text Available Background. Chronic Chagas disease cardiomyopathy (CCC, a late consequence of Trypanosoma cruzi infection, is an inflammatory cardiomyopathy with prognosis worse than those of noninflammatory etiology (NIC. Although the T cell-rich myocarditis is known to play a pathogenetic role, the relative contribution of each of the functional T cell subsets has never been thoroughly investigated. We therefore assessed gene expression of cytokines and transcription factors involved in differentiation and effector function of each functional T cell subset (TH1/TH2/TH17/Treg in CCC, NIC, and heart donor myocardial samples. Methods and Results. Quantitative PCR showed markedly upregulated expression of IFN-γ and transcription factor T-bet, and minor increases of GATA-3; FoxP3 and CTLA-4; IL-17 and IL-18 in CCC as compared with NIC samples. Conversely, cytokines expressed by TH2 cells (IL-4, IL-5, and IL-13 or associated with Treg (TGF-β and IL-10 were not upregulated in CCC myocardium. Expression of TH1-related genes such as T-bet, IFN-γ, and IL-18 correlated with ventricular dilation, FoxP3, and CTLA-4. Conclusions. Results are consistent with a strong local TH1-mediated response in most samples, possibly associated with pathological myocardial remodeling, and a proportionally smaller FoxP3+CTLA4+ Treg cell population, which is unable to completely curb IFN-γ production in CCC myocardium, therefore fueling inflammation.

  6. Genome-wide DNA methylation analysis predicts an epigenetic switch for GATA factor expression in endometriosis.

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    Matthew T Dyson

    2014-03-01

    Full Text Available Endometriosis is a gynecological disease defined by the extrauterine growth of endometrial-like cells that cause chronic pain and infertility. The disease is limited to primates that exhibit spontaneous decidualization, and diseased cells are characterized by significant defects in the steroid-dependent genetic pathways that typify this process. Altered DNA methylation may underlie these defects, but few regions with differential methylation have been implicated in the disease. We mapped genome-wide differences in DNA methylation between healthy human endometrial and endometriotic stromal cells and correlated this with gene expression using an interaction analysis strategy. We identified 42,248 differentially methylated CpGs in endometriosis compared to healthy cells. These extensive differences were not unidirectional, but were focused intragenically and at sites distal to classic CpG islands where methylation status was typically negatively correlated with gene expression. Significant differences in methylation were mapped to 403 genes, which included a disproportionally large number of transcription factors. Furthermore, many of these genes are implicated in the pathology of endometriosis and decidualization. Our results tremendously improve the scope and resolution of differential methylation affecting the HOX gene clusters, nuclear receptor genes, and intriguingly the GATA family of transcription factors. Functional analysis of the GATA family revealed that GATA2 regulates key genes necessary for the hormone-driven differentiation of healthy stromal cells, but is hypermethylated and repressed in endometriotic cells. GATA6, which is hypomethylated and abundant in endometriotic cells, potently blocked hormone sensitivity, repressed GATA2, and induced markers of endometriosis when expressed in healthy endometrial cells. The unique epigenetic fingerprint in endometriosis suggests DNA methylation is an integral component of the disease, and

  7. A rare case of GATA1 negative chemoresistant acute megakaryocytic leukemia in an 8-month-old infant with trisomy 21.

    Science.gov (United States)

    Stepensky, Polina; Brooks, Rebecca; Waldman, Elisha; Revel-Vilk, Shoshana; Izraeli, Shai; Resnick, Igor; Weintraub, Michael

    2010-07-01

    Children with Down syndrome (DS) have a unique form of acute megakaryocytic leukemia (AMKL) characterized by the presence of mutations in the GATA1 gene leading to increased chemosensitivity and a favorable outcome. We describe an 8-month-old male with DS who was diagnosed with AMKL without a mutation in the GATA1 gene. The patient was treated according to the DS-AML-regimen but his disease progressed and he succumbed 9 months later. This rare case of DS AMKL without a GATA1 mutation with an unfavorable outcome suggests that GATA1 testing may play a useful role in initial stratification. Copyright 2010 Wiley-Liss, Inc.

  8. Nucleocytoplasmic shuttling of a GATA transcription factor functions as a development timer.

    Science.gov (United States)

    Cai, Huaqing; Katoh-Kurasawa, Mariko; Muramoto, Tetsuya; Santhanam, Balaji; Long, Yu; Li, Lei; Ueda, Masahiro; Iglesias, Pablo A; Shaulsky, Gad; Devreotes, Peter N

    2014-03-21

    Biological oscillations are observed at many levels of cellular organization. In the social amoeba Dictyostelium discoideum, starvation-triggered multicellular development is organized by periodic cyclic adenosine 3',5'-monophosphate (cAMP) waves, which provide both chemoattractant gradients and developmental signals. We report that GtaC, a GATA transcription factor, exhibits rapid nucleocytoplasmic shuttling in response to cAMP waves. This behavior requires coordinated action of a nuclear localization signal and reversible G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor-mediated phosphorylation. Although both are required for developmental gene expression, receptor occupancy promotes nuclear exit of GtaC, which leads to a transient burst of transcription at each cAMP cycle. We demonstrate that this biological circuit filters out high-frequency signals and counts those admitted, thereby enabling cells to modulate gene expression according to the dynamic pattern of the external stimuli.

  9. Electrostatic study of Alanine mutational effects on transcription: application to GATA-3:DNA interaction complex.

    Science.gov (United States)

    El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges

    2015-01-01

    Protein-DNA interaction is of fundamental importance in molecular biology, playing roles in functions as diverse as DNA transcription, DNA structure formation, and DNA repair. Protein-DNA association is also important in medicine; understanding Protein-DNA binding kinetics can assist in identifying disease root causes which can contribute to drug development. In this perspective, this work focuses on the transcription process by the GATA Transcription Factor (TF). GATA TF binds to DNA promoter region represented by `G,A,T,A' nucleotides sequence, and initiates transcription of target genes. When proper regulation fails due to some mutations on the GATA TF protein sequence or on the DNA promoter sequence (weak promoter), deregulation of the target genes might lead to various disorders. In this study, we aim to understand the electrostatic mechanism behind GATA TF and DNA promoter interactions, in order to predict Protein-DNA binding in the presence of mutations, while elaborating on non-covalent binding kinetics. To generate a family of mutants for the GATA:DNA complex, we replaced every charged amino acid, one at a time, with a neutral amino acid like Alanine (Ala). We then applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations, for each mutation. These calculations delineate the contribution to binding from each Ala-replaced amino acid in the GATA:DNA interaction. After analyzing the obtained data in view of a two-step model, we are able to identify potential key amino acids in binding. Finally, we applied the model to GATA-3:DNA (crystal structure with PDB-ID: 3DFV) binding complex and validated it against experimental results from the literature.

  10. The evolutionary duplication and probable demise of an endodermal GATA factor in Caenorhabditis elegans.

    OpenAIRE

    Fukushige, Tetsunari; Goszczynski, Barbara; Tian, Helen; McGhee, James D

    2003-01-01

    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 intes...

  11. High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells.

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    Yo Saito

    Full Text Available Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in both hematopoietic stem and progenitor cells and is essential for cell proliferation, survival, and differentiation. Recently, evidence from studies of aplastic anemia, MonoMAC syndrome, and lung cancer has demonstrated a mechanistic link between GATA-2 and human pathophysiology. GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood. Here, we conducted high-throughput small-interfering-RNA (siRNA library screening and showed that YN-1, a human erythroleukemia cell line, expressed high levels of GATA-2 following the activation of the hematopoietic-specific 1S promoter. As transient luciferase reporter assay in YN-1 cells revealed the highest promoter activity in the 1S promoter fused with GATA-2 intronic enhancer (+9.9 kb/1S; therefore, we established a cell line capable of stably expressing +9.9 kb/1S-Luciferase. Subsequently, we screened 995 transcription factor genes and revealed that CITED2 acts as a GATA-2 activator in human hematopoietic cells. These results provide novel insights into and further identify the regulatory mechanism of GATA-2.

  12. 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.; Mitchell, Aaron P.

    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

  13. GATA3 is a master regulator of the transcriptional response to low-dose ionizing radiation in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bonin, F.; Molina, M.; Berthier-Vergnes, O.; Lamartine, J. [Universite de Lyon, Lyon, F-69003 (France); Universite Lyon 1, Lyon, F-69003 (France); CNRS, UMR5534, Centre de Genetique Moleculaire et Cellulaire, Villeurbanne, F-69622 (France); Malet, C.; Ginestet, C. [Centre Leon Berard, Service de Radiotherapie, Lyon F-69008 (France); Martin, M.T. [Laboratoire de Genomique et Radiobiologie de la Keratinopoiese, CEA, IRCM, Evry F-91000 (France)

    2009-07-01

    Background: The general population is constantly exposed to low levels of radiation through natural, occupational or medical irradiation. Even if the biological effects of low-level radiation have been intensely debated and investigated, the molecular mechanisms underlying the cellular response to low doses remain largely unknown. Results: The present study investigated the role of GATA3 protein in the control of the cellular and molecular response of human keratinocytes exposed to a 1 cGy dose of X-rays. Chromatin immunoprecipitation showed GATA3 to be able to bind the promoter of 4 genes responding to a 1 cGy exposure. To go further into the role of GATA3 after ionizing radiation exposure, we studied the cellular and molecular consequences of radiation in GATA3 knock-down cells. Knockdown was obtained by lentiviral-mediated expression of an shRNA targeting the GATA3 transcript in differentiated keratinocytes. First, radiosensitivity was assessed: the toxicity, in terms of immediate survival (with XTT test), associated with 1 cGy radiation was found to be increased in GATA3 knock-down cells. The impact of GATA3 knock-down on the transcriptome of X-ray irradiated cells was also investigated, using oligonucleotide micro-arrays to assess changes between 3 h and 72 h post-irradiation in normal vs GATA3 knock-down backgrounds; transcriptome response was found to be completely altered in GATA3 knock-down cells, with a strong induction/repression peak 48 h after irradiation. Functional annotation revealed enrichment in genes known to be involved in chaperone activity, TGF{beta} signalling and stress response. Conclusion: Taken together, these data indicate that GATA3 is an important regulator of the cellular and molecular response of epidermal cells to very low doses of radiation. (authors)

  14. GATA2 mutations in patients with acute myeloid leukemia-paired samples analyses show that the mutation is unstable during disease evolution.

    Science.gov (United States)

    Hou, Hsin-An; Lin, Yun-Chu; Kuo, Yuan-Yeh; Chou, Wen-Chien; Lin, Chien-Chin; Liu, Chieh-Yu; Chen, Chien-Yuan; Lin, Liang-In; Tseng, Mei-Hsuan; Huang, Chi-Fei; Chiang, Ying-Chieh; Liu, Ming-Chih; Liu, Chia-Wen; Tang, Jih-Luh; Yao, Ming; Huang, Shang-Yi; Ko, Bor-Sheng; Hsu, Szu-Chun; Wu, Shang-Ju; Tsay, Woei; Chen, Yao-Chang; Tien, Hwei-Fang

    2015-02-01

    Recently, mutations of the GATA binding protein 2 (GATA2) gene were identified in acute myeloid leukemia (AML) patients with CEBPA double mutations (CEBPA (double-mut)), but the interaction of this mutation with other genetic alterations and its dynamic changes during disease progression remain to be determined. In this study, 14 different missense GATA2 mutations, which were all clustered in the highly conserved N-terminal zinc finger 1 domain, were identified in 27.4, 6.7, and 1 % of patients with CEBPA (double-mut), CEBPA (single-mut), and CEBPA wild type, respectively. All but one patient with GATA2 mutation had concurrent CEBPA mutation. GATA2 mutations were closely associated with younger age, FAB M1 subtype, intermediate-risk cytogenetics, expression of HLA-DR, CD7, CD15, or CD34 on leukemic cells, and CEBPA mutation, but negatively associated with FAB M4 subtype, favorable-risk cytogenetics, and NPM1 mutation. Patients with GATA2 mutation had significantly better overall survival and relapse-free survival than those without GATA2 mutation. Sequential analysis showed that the original GATA2 mutations might be lost during disease progression in GATA2-mutated patients, while novel GATA2 mutations might be acquired at relapse in GATA2-wild patients. In conclusion, AML patients with GATA2 mutations had distinct clinic-biological features and a favorable prognosis. GATA2 mutations might be lost or acquired at disease progression, implying that it was a second hit in the leukemogenesis of AML, especially those with CEBPA mutation.

  15. Protective Effects of Folic Acid on Cardiac Development Related Genes GATA-4 and NKx2.5 Expression of Offspring Rats with Maternal Coxsackievirus B3 Infection%柯萨奇B3病毒对心脏发育因子GATA-4、NKx2.5表达的影响及叶酸对其保护作用

    Institute of Scientific and Technical Information of China (English)

    薛雅馨; 仇小强; 余红平; 谭盛葵

    2013-01-01

    Objective To investigate the effects of oral folic acid on cardiac development related gene expression of offspring in an experimental model of coxsackievirus B3(CVB3) infection of pregnant rats. Methods SD female rats were randomized into control group, folic acid group, CVB3 group and CVB3+folic acid group. The female rats were given folic acid by gavage for 2 weeks before pregnancy in folic acid group and CVB3+folic acid group. After conception for 7 days, rat model was established by intraperitoneal injection of CVB3 for 5 days in CVB3 group and CVB3+folic acid group. After nat-ural childbirth neonatal heart was taken and stored in liquid nitrogen. The morphological changes of neonatal rat myocardial tissues were observed by HE staining. The expressions of GATA-4 and NKx2.5 mRNA were detected by RT-PCR and West-ern blot assay. Results There was significant myocardial injury, such as myocardial fiber disarray and myocardial fiber breakage, in neonatal rats in CVB3 group. These damages were improved in CVB3+folic acid group. The expression levels of GATA-4 and NKx2.5 genes in myocardial tissues were significantly lower in CVB3 group than those of control group (P<0.05). The expression levels of GATA-4 and NKx2.5 proteins in myocardial tissues were significantly higher in CVB3+folic acid group than those of CVB3 group (P<0.05). Conclusion CVB3 infection in the early pregnancy inhibited the expres-sion of neonatal rat cardiac development factor. Folic acid supplementation has obvious protective effects on the neonatal rat cardiac development.%目的:探讨叶酸对孕期感染柯萨奇B3病毒(CVB3)的新生鼠心脏发育因子GATA-4、NKx2.5的基因与蛋白表达的影响。方法 SD雌鼠分为对照组、叶酸组、CVB3组、CVB3+叶酸组。叶酸组及CVB3+叶酸组孕前给予叶酸灌胃2周;CVB3组及CVB3+叶酸组孕第7天腹腔注射CVB3,连续5 d,自然分娩后取新生鼠心脏组织,病理切片HE染色观察新生鼠心脏组织形态

  16. hHO-1 combined with GATA-4 transduction promotes myocardial transdifferentiation and anti- apoptosis of rat mesenchymal stem cells

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    Ning-bo DENG

    2017-06-01

    Full Text Available Objectives To explore if the rat bone marrow mesenchymal stem cells (BMSCs modified by human heme oxygenase 1 (hHO-1 gene combined with GATA-4 gene may promote the ability of anti-apoptosis and myocardial transdifferentiation in vitro in hypoxia ischemic environment. Methods The rat BMSCs were isolated and cultured by whole bone marrow adherence and identified in vitro, and then were transfected with recombinant adenovirus; Western blotting was used to determinate the optimal time of gene expression; the genetically modified BMSCs were taken to hypoxia serum-free conditions simulating ischemia hypoxia microenvironment in vivo; CCK-8 kit and trypan blue staining were performed to detect the 12, 24, 48 and 72h survival rates in hypoxia ischemia respectively; flow cytometry was used to detect the apoptosis of BMSCs in hypoxia ischemia for 24h. The cardiomyocyte-specific cardiac troponin I (cTnI was detected by Western blotting and cellular immunofluorescence. Results The 12, 24, 48 and 72h survival rates were higher in hHO-1+GATA-4 group cultured in ischemia and hypoxia condition than in hHO-1 group (P<0.05 and GATA-4 group (P<0.05. After 24h cultivation in ischemia hypoxia condition, the apoptotic rates were lower in hHO-1+GATA-4 group than in hHO-1 group (P<0.05 and GATA-4 group (P<0.05. No significant difference existed in cTnI expressions between GATA-4 group and hHO-1+GATA-4 group. Conclusion Compared with transfection of hHO-1 or GATA-4 single gene, hHO-1 combined with GATA-4 transduction can significantly increase the survival rate of BMSCs in hypoxia ischemic condition, but myocardial transdifferentiation does not increase significantly. DOI: 10.11855/j.issn.0577-7402.2017.04.08

  17. A +220 GATA motif mediates basal but not endotoxin-repressible expression of the von Willebrand factor promoter in Hprt-targeted transgenic mice

    Science.gov (United States)

    LIU, J.; KANKI, Y.; OKADA, Y.; JIN, E.; YANO, K.; SHIH, S.-C.; MINAMI, T.; AIRD, W. C.

    2012-01-01

    Summary Background The von Willebrand factor (VWF) gene is a marker for spatial and temporal heterogeneity of the endothelium. A GATA motif at +220 has been implicated in basal VWF expression in vitro. Other studies have shown that GATA3 and VWF are transcriptionally downregulated in response to inflammatory mediators. Objectives Our goal was to determine the importance of the +220 GATA motif in mediating expression of VWF promoter in vivo, and to elucidate whether the GATA element plays a role in spatial and/or temporal regulation of VWF expression. Methods ChIP and electrophoretic mobility shift assays were carried out in human umbilical vein endothelial cells (HUVEC). Reporter gene constructs containing 3.6 kb of the human VWF promoter with and without amutation of the +220 GATA element were transfected into cultured endothelial cells or targeted to the Hprt locus of mice. The Hprt-targeted mice were subjected to endotoxemia. Results In protein-DNA binding assays, the +220 GATA motif bound GATA-2, -3 and -6. Mutation of the GATA site resulted in reduced basal promoter activity in HUVEC. When targeted to the Hprt locus of mice, the GATA mutation resulted in a significant, proportionate reduction of promoter activity in LacZ expressing vascular beds. Systemic administration of lipopolysaccharide (LPS) resulted in a widespread reduction in VWF mRNA expression and promoter activity. LPS-mediated repression of the VWF promoter was unaffected by the GATA mutation. Conclusions A region of the VWF promoter between −2182 and the end of the first intron contains information for LPS-mediated gene repression. The +220 GATA motif is important for basal, but not LPS-repressible expression of the VWF gene. PMID:19496923

  18. The GATA1s isoform is normally down-regulated during terminal haematopoietic differentiation and over-expression leads to failure to repress MYB, CCND2 and SKI during erythroid differentiation of K562 cells

    Directory of Open Access Journals (Sweden)

    Halsey Christina

    2012-08-01

    Full Text Available Abstract Background Although GATA1 is one of the most extensively studied haematopoietic transcription factors little is currently known about the physiological functions of its naturally occurring isoforms GATA1s and GATA1FL in humans—particularly whether the isoforms have distinct roles in different lineages and whether they have non-redundant roles in haematopoietic differentiation. As well as being of general interest to understanding of haematopoiesis, GATA1 isoform biology is important for children with Down syndrome associated acute megakaryoblastic leukaemia (DS-AMKL where GATA1FL mutations are an essential driver for disease pathogenesis. Methods Human primary cells and cell lines were analyzed using GATA1 isoform specific PCR. K562 cells expressing GATA1s or GATA1FL transgenes were used to model the effects of the two isoforms on in vitro haematopoietic differentiation. Results We found no evidence for lineage specific use of GATA1 isoforms; however GATA1s transcripts, but not GATA1FL transcripts, are down-regulated during in vitro induction of terminal megakaryocytic and erythroid differentiation in the cell line K562. In addition, transgenic K562-GATA1s and K562-GATA1FL cells have distinct gene expression profiles both in steady state and during terminal erythroid differentiation, with GATA1s expression characterised by lack of repression of MYB, CCND2 and SKI. Conclusions These findings support the theory that the GATA1s isoform plays a role in the maintenance of proliferative multipotent megakaryocyte-erythroid precursor cells and must be down-regulated prior to terminal differentiation. In addition our data suggest that SKI may be a potential therapeutic target for the treatment of children with DS-AMKL.

  19. An elt-3/elt-5/elt-6 GATA Transcription Circuit Guides Aging in C. elegans

    OpenAIRE

    Budovskaya, Yelena V.; Wu, Kendall; Southworth, Lucinda K; Jiang, Min; Tedesco, Patricia; Johnson, Thomas E.; Kim, Stuart K.

    2008-01-01

    To define the C. elegans aging process at the molecular level, we used DNA microarray experiments to identify a set of 1294 age-regulated genes and found that the GATA transcription factors ELT-3, ELT-5, and ELT-6 are responsible for age regulation of a large fraction of these genes. Expression of elt-5 and elt-6 increases during normal aging, and both of these GATA factors repress expression of elt-3, which shows a corresponding decrease in expression in old worms. elt-3 regulates a large nu...

  20. GATA2 is associated with familial early-onset coronary artery disease.

    Directory of Open Access Journals (Sweden)

    Jessica J Connelly

    2006-08-01

    Full Text Available The transcription factor GATA2 plays an essential role in the establishment and maintenance of adult hematopoiesis. It is expressed in hematopoietic stem cells, as well as the cells that make up the aortic vasculature, namely aortic endothelial cells and smooth muscle cells. We have shown that GATA2 expression is predictive of location within the thoracic aorta; location is suggested to be a surrogate for disease susceptibility. The GATA2 gene maps beneath the Chromosome 3q linkage peak from our family-based sample set (GENECARD study of early-onset coronary artery disease. Given these observations, we investigated the relationship of several known and novel polymorphisms within GATA2 to coronary artery disease. We identified five single nucleotide polymorphisms that were significantly associated with early-onset coronary artery disease in GENECARD. These results were validated by identifying significant association of two of these single nucleotide polymorphisms in an independent case-control sample set that was phenotypically similar to the GENECARD families. These observations identify GATA2 as a novel susceptibility gene for coronary artery disease and suggest that the study of this transcription factor and its downstream targets may uncover a regulatory network important for coronary artery disease inheritance.

  1. GATA3 induces human T-cell commitment by restraining Notch activity and repressing NK-cell fate

    Science.gov (United States)

    Van de Walle, Inge; Dolens, Anne-Catherine; Durinck, Kaat; De Mulder, Katrien; Van Loocke, Wouter; Damle, Sagar; Waegemans, Els; De Medts, Jelle; Velghe, Imke; De Smedt, Magda; Vandekerckhove, Bart; Kerre, Tessa; Plum, Jean; Leclercq, Georges; Rothenberg, Ellen V.; Van Vlierberghe, Pieter; Speleman, Frank; Taghon, Tom

    2016-01-01

    The gradual reprogramming of haematopoietic precursors into the T-cell fate is characterized by at least two sequential developmental stages. Following Notch1-dependent T-cell lineage specification during which the first T-cell lineage genes are expressed and myeloid and dendritic cell potential is lost, T-cell specific transcription factors subsequently induce T-cell commitment by repressing residual natural killer (NK)-cell potential. How these processes are regulated in human is poorly understood, especially since efficient T-cell lineage commitment requires a reduction in Notch signalling activity following T-cell specification. Here, we show that GATA3, in contrast to TCF1, controls human T-cell lineage commitment through direct regulation of three distinct processes: repression of NK-cell fate, upregulation of T-cell lineage genes to promote further differentiation and restraint of Notch activity. Repression of the Notch1 target gene DTX1 hereby is essential to prevent NK-cell differentiation. Thus, GATA3-mediated positive and negative feedback mechanisms control human T-cell lineage commitment. PMID:27048872

  2. Forced FOG1 expression in erythroleukemia cells: Induction of erythroid genes and repression of myelo-lymphoid transcription factor PU.1.

    Science.gov (United States)

    Fujiwara, Tohru; Sasaki, Katsuyuki; Saito, Kei; Hatta, Shunsuke; Ichikawa, Satoshi; Kobayashi, Masahiro; Okitsu, Yoko; Fukuhara, Noriko; Onishi, Yasushi; Harigae, Hideo

    2017-02-16

    The transcription factor GATA-1-interacting protein Friend of GATA-1 (FOG1) is essential for proper transcriptional activation and repression of GATA-1 target genes; yet, the mechanisms by which FOG1 exerts its activating and repressing functions remain unknown. Forced FOG1 expression in human K562 erythroleukemia cells induced the expression of erythroid genes (SLC4A1, globins) but repressed that of GATA-2 and PU.1. A quantitative chromatin immunoprecipitation (ChIP) analysis demonstrated increased GATA-1 chromatin occupancy at both FOG1-activated as well as FOG1-repressed gene loci. However, while TAL1 chromatin occupancy was significantly increased at FOG1-activated gene loci, it was significantly decreased at FOG1-repressed gene loci. When FOG1 was overexpressed in TAL1-knocked down K562 cells, FOG1-mediated activation of HBA, HBG, and SLC4A1 was significantly compromised by TAL1 knockdown, suggesting that FOG1 may require TAL1 to activate GATA-1 target genes. Promoter analysis and quantitative ChIP analysis demonstrated that FOG1-mediated transcriptional repression of PU.1 would be mediated through a GATA-binding element located at its promoter, accompanied by significantly decreased H3 acetylation at lysine 4 and 9 (K4 and K9) as well as H3K4 trimethylation. Our results provide important mechanistic insight into the role of FOG1 in the regulation of GATA-1-regulated genes and suggest that FOG1 has an important role in inducing cells to differentiate toward the erythroid lineage rather than the myelo-lymphoid one by repressing the expression of PU.1.

  3. GATA4 regulates Fgf16 to promote heart repair after injury.

    Science.gov (United States)

    Yu, Wei; Huang, Xiuzhen; Tian, Xueying; Zhang, Hui; He, Lingjuan; Wang, Yue; Nie, Yu; Hu, Shengshou; Lin, Zhiqiang; Zhou, Bin; Pu, William; Lui, Kathy O; Zhou, Bin

    2016-03-15

    Although the mammalian heart can regenerate during the neonatal stage, this endogenous regenerative capacity is lost with age. Importantly, replication of cardiomyocytes has been found to be the key mechanism responsible for neonatal cardiac regeneration. Unraveling the transcriptional regulatory network for inducing cardiomyocyte replication will, therefore, be crucial for the development of novel therapies to drive cardiac repair after injury. Here, we investigated whether the key cardiac transcription factor GATA4 is required for neonatal mouse heart regeneration. Using the neonatal mouse heart cryoinjury and apical resection models with an inducible loss of GATA4 specifically in cardiomyocytes, we found severely depressed ventricular function in the Gata4-ablated mice (mutant) after injury. This was accompanied by reduced cardiomyocyte replication. In addition, the mutant hearts displayed impaired coronary angiogenesis and increased hypertrophy and fibrosis after injury. Mechanistically, we found that the paracrine factor FGF16 was significantly reduced in the mutant hearts after injury compared with littermate controls and was directly regulated by GATA4. Cardiac-specific overexpression of FGF16 via adeno-associated virus subtype 9 (AAV9) in the mutant hearts partially rescued the cryoinjury-induced cardiac hypertrophy, promoted cardiomyocyte replication and improved heart function after injury. Altogether, our data demonstrate that GATA4 is required for neonatal heart regeneration through regulation of Fgf16, suggesting that paracrine factors could be of potential use in promoting myocardial repair.

  4. Sphingosine 1-Phosphate Induces Differentiation of Mesoangioblasts towards Smooth Muscle. A Role for GATA6

    Science.gov (United States)

    Donati, Chiara; Marseglia, Giuseppina; Magi, Alberto; Serratì, Simona; Cencetti, Francesca; Bernacchioni, Caterina; Nannetti, Genni; Benelli, Matteo; Brunelli, Silvia; Torricelli, Francesca; Cossu, Giulio; Bruni, Paola

    2011-01-01

    Different cells can contribute to repair following vascular injury by differentiating into smooth muscle (SM) cells; however the extracellular signals involved are presently poorly characterized. Mesoangioblasts are progenitor cells capable of differentiating into various mesoderm cell types including SM cells. In this study the biological action exerted by the pleiotropic sphingolipid sphingosine 1-phosphate (S1P) in human mesoangioblasts has been initially investigated by cDNA microarray analysis. Obtained data confirmed the anti-apoptotic action of this sphingolipid and identified for the first time a strong differentiating action toward SM cells. Quantitative mRNA and protein analysis corroborated the microarray results demonstrating enhanced expression of myogenic marker proteins and regulation of the expression of transcription factor GATA6 and its co-regulator, LMCD1. Importantly, GATA6 up-regulation induced by S1P was responsible for the enhanced expression of SM-specific contractile proteins. Moreover, by specific gene silencing experiments GATA6 was critical in the pro-differentiating activity of the cytokine TGFβ. Finally, the pharmacological inhibition of endogenous S1P formation in response to TGFβ abrogated GATA6 up-regulation, supporting the view that the S1P pathway plays a physiological role in mediating the pro-myogenic effect of TGFβ. This study individuates GATA6 as novel player in the complex transcriptional regulation of mesoangioblast differentiation into SM cells and highlights a role for S1P to favour vascular regeneration. PMID:21629665

  5. A GATA-1-regulated microRNA locus essential for erythropoiesis

    OpenAIRE

    Dore, L. C.; Amigo, J. D.; Dos Santos, Carolina; Zhang, Z; Gai, Xiaowu; Tobias, J W; Yu, D.; Klein, A.M.; Dorman, C.; Wu, W; Hardison, R. C.; Paw, Barry Htin; Weiss, M. J.

    2008-01-01

    MicroRNAs (miRNAs) control tissue development, but their mechanism of regulation is not well understood. We used a gene complementation strategy combined with microarray screening to identify miRNAs involved in the formation of erythroid (red blood) cells. Two conserved miRNAs, miR 144 and miR 451, emerged as direct targets of the critical hematopoietic transcription factor GATA-1. In vivo, GATA-1 binds a distal upstream regulatory element to activate RNA polymerase II-mediated transcription ...

  6. Screening of 99 Danish patients with congenital heart disease for GATA4 mutations

    DEFF Research Database (Denmark)

    Zhang, Litu; Tümer, Zeynep; Jacobsen, Joes Ramsøe;

    2006-01-01

    Congenital heart disease (CHD) affects nearly 1% of the population, but only few genes involved in human CHD are presently known. Germ-line mutations in the zinc finger transcription factor GATA4 have been associated with familial cases of atrial and ventricular septal defects and pulmonary...

  7. 间充质干细胞治疗提升免疫性血小板减少症小鼠的血小板数并影响其外周血单核细胞中 T-bet 和 GATA-3基因表达%Mesenchymal stem cells increase platelet counts in mice with immune-mediated thrombocytopenia and affect T-bet and GATA-3 gene expression

    Institute of Scientific and Technical Information of China (English)

    徐敏; 王椋; 刘国强; 鲁光; 丁慧芳; 邢健; 赵霞; 韩芳; 尚应辉

    2013-01-01

    BALB/c mice.The mice were then divided into experiment and control groups with 20 mice in each.Each mouse in experimental group was injected with 2×107 mesenchymal stem cells (MSC) through the tail vein .The numbers of blood platelets in mice from two groups were counted on days 5, 7 and 14 after MSC injection .Reverse transcriptase polymerase chain reaction ( RT-PCR) was performed to meas-ure T-bet and GATA-3 gene expression in peripheral blood mononuclear cells ( PBMCs ) at mRNA level on day 14.The levels of IFN-γ, IL-2, IL-4 and IL-10 in serum were detected by ELISA .Results The platelet counts in experimental group were significantly higher than those in control group on days 7 and 14 after MSC injection [(588.0±81.6)×109/L and (623.0±78.9) ×109/L vs.(317.0±90.1) ×109/L and (288.0± 87.8)×109/L ] (P<0.05).On day 14 after MSC injection, the T-bet expression at mRNA level in PBMCs from mice in experimental group was significantly lower than that in control group [(0.04±0.03) vs.(0.27 ±0.05)] (P<0.05), while the GATA-3 expression at mRNA level was higher than those in control group [ (0.14±0.04) vs.(0.07±0.05)] (P<0.05).Compared with control group, the concentrations of Th1 type cytokines such as IFN-γand IL-2 were remarkably down-regulated in experimental group [(3.1±1.7) pg/ml and (3.2±2.1) pg/ml vs.(10.3±4.8) pg/ml and (16.3±5.7) pg/ml](P<0.05), while the con-centrations of Th2 type cytokines such as IL-4 and IL-10 were up-regulated in experimental group [(88.6± 15.2) pg/ml and (38.3±11.8) pg/ml vs.(32.7±5.7) pg/ml and (22.1±3.4) pg/ml ] (P<0.05). Conclusion MSC treatment can effectively increase platelet counts in mice with immune-mediated thrombo-cytopenia, which may be associated with the suppression of Th 1-dominant response mediated by abnormal ex-pression of T-bet and GATA-3.

  8. GATA: a graphic alignment tool for comparative sequence analysis

    Directory of Open Access Journals (Sweden)

    Nix David A

    2005-01-01

    Full Text Available Abstract Background Several problems exist with current methods used to align DNA sequences for comparative sequence analysis. Most dynamic programming algorithms assume that conserved sequence elements are collinear. This assumption appears valid when comparing orthologous protein coding sequences. Functional constraints on proteins provide strong selective pressure against sequence inversions, and minimize sequence duplications and feature shuffling. For non-coding sequences this collinearity assumption is often invalid. For example, enhancers contain clusters of transcription factor binding sites that change in number, orientation, and spacing during evolution yet the enhancer retains its activity. Dot plot analysis is often used to estimate non-coding sequence relatedness. Yet dot plots do not actually align sequences and thus cannot account well for base insertions or deletions. Moreover, they lack an adequate statistical framework for comparing sequence relatedness and are limited to pairwise comparisons. Lastly, dot plots and dynamic programming text outputs fail to provide an intuitive means for visualizing DNA alignments. Results To address some of these issues, we created a stand alone, platform independent, graphic alignment tool for comparative sequence analysis (GATA http://gata.sourceforge.net/. GATA uses the NCBI-BLASTN program and extensive post-processing to identify all small sub-alignments above a low cut-off score. These are graphed as two shaded boxes, one for each sequence, connected by a line using the coordinate system of their parent sequence. Shading and colour are used to indicate score and orientation. A variety of options exist for querying, modifying and retrieving conserved sequence elements. Extensive gene annotation can be added to both sequences using a standardized General Feature Format (GFF file. Conclusions GATA uses the NCBI-BLASTN program in conjunction with post-processing to exhaustively align two DNA

  9. Analysis list: Gata3 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Gata3 Blood + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Gata3.1.tsv... http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Gata3.5.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Gata3....10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Gata3.Blood.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Blood.gml ...

  10. Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo

    National Research Council Canada - National Science Library

    Yu, Channing; Cantor, Alan B; Yang, Haidi; Browne, Carol; Wells, Richard A; Fujiwara, Yuko; Orkin, Stuart H

    2002-01-01

    .... Here we demonstrate that deletion of a high-affinity GATA-binding site in the GATA-1 promoter, an element presumed to mediate positive autoregulation of GATA-1 expression, leads to selective loss...

  11. Organization and differential expression of the GACA/GATA tagged somatic and spermatozoal transcriptomes in Buffalo Bubalus bubalis

    Directory of Open Access Journals (Sweden)

    Srivastava Jyoti

    2008-03-01

    Full Text Available Abstract Background Simple sequence repeats (SSRs of GACA/GATA have been implicated with differentiation of sex-chromosomes and speciation. However, the organization of these repeats within genomes and transcriptomes, even in the best characterized organisms including human, remains unclear. The main objective of this study was to explore the buffalo transcriptome for its association with GACA/GATA repeats, and study the structural organization and differential expression of the GACA/GATA repeat tagged transcripts. Moreover, the distribution of GACA and GATA repeats in the prokaryotic and eukaryotic genomes was studied to highlight their significance in genome evolution. Results We explored several genomes and transcriptomes, and observed total absence of these repeats in the prokaryotes, with their gradual accumulation in higher eukaryotes. Further, employing novel microsatellite associated sequence amplification (MASA approach using varying length oligos based on GACA and GATA repeats; we identified and characterized 44 types of known and novel mRNA transcripts tagged with these repeats from different somatic tissues, gonads and spermatozoa of water buffalo Bubalus bubalis. GACA was found to be associated with higher number of transcripts compared to that with GATA. Exclusive presence of several GACA-tagged transcripts in a tissue or spermatozoa, and absence of the GATA-tagged ones in lung/heart highlights their tissue-specific significance. Of all the GACA/GATA tagged transcripts, ~30% demonstrated inter-tissue and/or tissue-spermatozoal sequence polymorphisms. Significantly, ~60% of the GACA-tagged and all the GATA-tagged transcripts showed highest or unique expression in the testis and/or spermatozoa. Moreover, ~75% GACA-tagged and all the GATA-tagged transcripts were found to be conserved across the species. Conclusion Present study is a pioneer attempt exploring GACA/GATA tagged transcriptome in any mammalian species highlighting their

  12. Analysis list: GATA3 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available GATA3 Blood,Breast,Neural + hg19 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/GATA3.1.tsv http:...//dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/GATA3.5.tsv http://dbarchive.bioscie...ncedbc.jp/kyushu-u/hg19/target/GATA3.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/GATA3.Blood.tsv,http:...//dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/GATA3.Breast.tsv,http:/.../dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/GATA3.Neural.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/Blood.gml,http:

  13. GATA-1 directly regulates Nanog in mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Wen-Zhong; Ai, Zhi-Ying [College of Life Sciences, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Wang, Zhi-Wei [School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui 230027 (China); Chen, Lin-Lin [College of Life Sciences, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Guo, Ze-Kun, E-mail: gzknwaf@126.com [College of Veterinary Medicine, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Zhang, Yong, E-mail: zylabnwaf@126.com [College of Veterinary Medicine, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China)

    2015-09-25

    Nanog safeguards pluripotency in mouse embryonic stem cells (mESCs). Insight into the regulation of Nanog is important for a better understanding of the molecular mechanisms that control pluripotency of mESCs. In a silico analysis, we identify four GATA-1 putative binding sites in Nanog proximal promoter. The Nanog promoter activity can be significantly repressed by ectopic expression of GATA-1 evidenced by a promoter reporter assay. Mutation studies reveal that one of the four putative binding sites counts for GATA-1 repressing Nanog promoter activity. Direct binding of GATA-1 on Nanog proximal promoter is confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Our data provide new insights into the expanded regulatory circuitry that coordinates Nanog expression. - Highlights: • The Nanog proximal promoter conceives functional element for GATA-1. • GATA-1 occupies the Nanog proximal promoter in vitro and in vivo. • GATA-1 transcriptionally suppresses Nanog.

  14. Runx1-mediated hematopoietic stem-cell emergence is controlled by a Gata/Ets/SCL-regulated enhancer

    Science.gov (United States)

    Nottingham, Wade T.; Jarratt, Andrew; Burgess, Matthew; Speck, Caroline L.; Cheng, Jan-Fang; Prabhakar, Shyam; Rubin, Eddy M.; Li, Pik-Shan; Sloane-Stanley, Jackie; Kong-a-San, John

    2007-01-01

    The transcription factor Runx1/AML1 is an important regulator of hematopoiesis and is critically required for the generation of the first definitive hematopoietic stem cells (HSCs) in the major vasculature of the mouse embryo. As a pivotal factor in HSC ontogeny, its transcriptional regulation is of high interest but is largely undefined. In this study, we used a combination of comparative genomics and chromatin analysis to identify a highly conserved 531-bp enhancer located at position + 23.5 in the first intron of the 224-kb mouse Runx1 gene. We show that this enhancer contributes to the early hematopoietic expression of Runx1. Transcription factor binding in vivo and analysis of the mutated enhancer in transient transgenic mouse embryos implicate Gata2 and Ets proteins as critical factors for its function. We also show that the SCL/Lmo2/Ldb-1 complex is recruited to the enhancer in vivo. Importantly, transplantation experiments demonstrate that the intronic Runx1 enhancer targets all definitive HSCs in the mouse embryo, suggesting that it functions as a crucial cis-regulatory element that integrates the Gata, Ets, and SCL transcriptional networks to initiate HSC generation. PMID:17823307

  15. GATA-1 regulates the generation and function of basophils.

    Science.gov (United States)

    Nei, Yuichiro; Obata-Ninomiya, Kazushige; Tsutsui, Hidemitsu; Ishiwata, Kenji; Miyasaka, Masayuki; Matsumoto, Kenji; Nakae, Susumu; Kanuka, Hirotaka; Inase, Naohiko; Karasuyama, Hajime

    2013-11-12

    Developmental processes of hematopoietic cells are orchestrated by transcriptional networks. GATA-1, the founding member of the GATA family of transcription factors, has been demonstrated to play crucial roles in the differentiation of erythroid cells, magakaryocytes, eosinophils, and mast cells. However, the role of GATA-1 in basophils remains elusive. Here we show that basophils abundantly express Gata1 mRNAs, and that siRNA-mediated knockdown of Gata1 resulted in impaired production of IL-4 by basophils in response to the stimulation with IgE plus antigens. ΔdblGATA mice that carry the mutated Gata1 promoter and are widely used for functional analysis of eosinophils owing to their selective loss of eosinophils showed a decreased number of basophils with reduced expression of Gata1 mRNAs. The number of basophil progenitors in bone marrow was reduced in these mice, and the generation of basophils from their bone marrow cells in culture with IL-3 or thymic stromal lymphopoietin was impaired. ΔdblGATA basophils responded poorly ex vivo to stimulation with IgE plus antigens compared with wild-type basophils as assessed by degranulation and production of IL-4 and IL-6. Moreover, ΔdblGATA mice showed impaired responses in basophil-mediated protective immunity against intestinal helminth infection. Thus, ΔdblGATA mice showed numerical and functional aberrancy in basophils in addition to the known deficiency of eosinophils. Our findings demonstrate that GATA-1 plays a key role in the generation and function of basophils and underscore the need for careful distinction of the cell lineage responsible for each phenotype observed in ΔdblGATA mice.

  16. Analysis list: Gata2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Gata2 Blood,Uterus + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Gata...2.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Gata2.5.tsv http://dbarchive.biosciencedbc.jp/...kyushu-u/mm9/target/Gata2.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Gata2.Blood.tsv,http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/colo/Gata2.Uterus.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/colo/Blood.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Uterus.gml ...

  17. A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in leukemia.

    Science.gov (United States)

    Gröschel, Stefan; Sanders, Mathijs A; Hoogenboezem, Remco; de Wit, Elzo; Bouwman, Britta A M; Erpelinck, Claudia; van der Velden, Vincent H J; Havermans, Marije; Avellino, Roberto; van Lom, Kirsten; Rombouts, Elwin J; van Duin, Mark; Döhner, Konstanze; Beverloo, H Berna; Bradner, James E; Döhner, Hartmut; Löwenberg, Bob; Valk, Peter J M; Bindels, Eric M J; de Laat, Wouter; Delwel, Ruud

    2014-04-10

    Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.

  18. Acquired Senescent T-Cell Phenotype Correlates with Clinical Severity in GATA Binding Protein 2-Deficient Patients

    Directory of Open Access Journals (Sweden)

    Raquel Ruiz-García

    2017-07-01

    Full Text Available GATA binding protein 2 (GATA2 deficiency is a rare disorder of hematopoiesis, lymphatics, and immunity caused by spontaneous or autosomal dominant mutations in the GATA2 gene. Clinical manifestations range from neutropenia, lymphedema, deafness, to severe viral and mycobacterial infections, bone marrow failure, and acute myeloid leukemia. Patients also present with monocytopenia, dendritic cell, B- and natural killer (NK-cell deficiency. We studied the T-cell and NK-cell compartments of four GATA2-deficient patients to assess if changes in these lymphocyte populations could be correlated with clinical phenotype. Patients with more severe clinical complications demonstrated a senescent T-cell phenotype whereas patients with lower clinical score had undetectable changes relative to controls. In contrast, patients’ NK-cells demonstrated an immature/activated phenotype that did not correlate with clinical score, suggesting an intrinsic NK-cell defect. These studies will help us to determine the contribution of T- and NK-cell dysregulation to the clinical phenotype of GATA2 patients, and may help to establish the most accurate therapeutic options for these patients. Asymptomatic patients may be taken into consideration for hematopoietic stem cell transplantation when dysregulation of T-cell and NK-cell compartment is present.

  19. Systematic Cellular Disease Models Reveal Synergistic Interaction of Trisomy 21 and GATA1 Mutations in Hematopoietic Abnormalities

    Directory of Open Access Journals (Sweden)

    Kimihiko Banno

    2016-05-01

    Full Text Available Chromosomal aneuploidy and specific gene mutations are recognized early hallmarks of many oncogenic processes. However, the net effect of these abnormalities has generally not been explored. We focused on transient myeloproliferative disorder (TMD in Down syndrome, which is characteristically associated with somatic mutations in GATA1. To better understand functional interplay between trisomy 21 and GATA1 mutations in hematopoiesis, we constructed cellular disease models using human induced pluripotent stem cells (iPSCs and genome-editing technologies. Comparative analysis of these engineered iPSCs demonstrated that trisomy 21 perturbed hematopoietic development through the enhanced production of early hematopoietic progenitors and the upregulation of mutated GATA1, resulting in the accelerated production of aberrantly differentiated cells. These effects were mediated by dosage alterations of RUNX1, ETS2, and ERG, which are located in a critical 4-Mb region of chromosome 21. Our study provides insight into the genetic synergy that contributes to multi-step leukemogenesis.

  20. GATA6 expression in Barrett's oesophagus and oesophageal adenocarcinoma

    NARCIS (Netherlands)

    Pavlov, Kirill; Honing, Judith; Meijer, Coby; Boersma-van Ek, Wytske; Peters, Frans T. M.; van den Berg, Anke; Karrenbeld, Arend; Plukker, John T. M.; Kruyt, Frank A. E.; Kleibeuker, Jan H.

    2015-01-01

    Background: Barrett's oesophagus can progress towards oesophageal adenocarcinoma through a metaplasia-dysplasia-carcinoma sequence, but the underlying mechanisms are poorly understood. The transcription factor GATA6 is known to be involved in columnar differentiation and proliferation, and GATA6 gen

  1. GATA6 expression in Barrett's oesophagus and oesophageal adenocarcinoma

    NARCIS (Netherlands)

    Pavlov, Kirill; Honing, Judith; Meijer, Coby; Boersma-van Ek, Wytske; Peters, Frans T.M.; van den Berg, Anke; Karrenbeld, Arend; Plukker, John T.M.; Kruyt, Frank A.E.; Kleibeuker, Jan H.

    2015-01-01

    BACKGROUND: Barrett's oesophagus can progress towards oesophageal adenocarcinoma through a metaplasia-dysplasia-carcinoma sequence, but the underlying mechanisms are poorly understood. The transcription factor GATA6 is known to be involved in columnar differentiation and proliferation, and GATA6 gen

  2. Fungal Morphology, Iron Homeostasis, and Lipid Metabolism Regulated by a GATA Transcription Factor in Blastomyces dermatitidis.

    Directory of Open Access Journals (Sweden)

    Amber J Marty

    2015-06-01

    Full Text Available In response to temperature, Blastomyces dermatitidis converts between yeast and mold forms. Knowledge of the mechanism(s underlying this response to temperature remains limited. In B. dermatitidis, we identified a GATA transcription factor, SREB, important for the transition to mold. Null mutants (SREBΔ fail to fully complete the conversion to mold and cannot properly regulate siderophore biosynthesis. To capture the transcriptional response regulated by SREB early in the phase transition (0-48 hours, gene expression microarrays were used to compare SREB∆ to an isogenic wild type isolate. Analysis of the time course microarray data demonstrated SREB functioned as a transcriptional regulator at 37°C and 22°C. Bioinformatic and biochemical analyses indicated SREB was involved in diverse biological processes including iron homeostasis, biosynthesis of triacylglycerol and ergosterol, and lipid droplet formation. Integration of microarray data, bioinformatics, and chromatin immunoprecipitation identified a subset of genes directly bound and regulated by SREB in vivo in yeast (37°C and during the phase transition to mold (22°C. This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation. Functional analysis suggested that lipid droplets were actively metabolized during the phase transition and lipid metabolism may contribute to filamentous growth at 22°C. Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX. Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition.

  3. Generalized verrucosis in a patient with GATA2 deficiency.

    Science.gov (United States)

    West, E S; Kingsbery, M Y; Mintz, E M; Hsu, A P; Holland, S M; Rady, P L; Tyring, S K; Grossman, M E

    2014-05-01

    Generalized verrucosis is a characteristic of several genetic and immunodeficiency disorders including epidermodysplasia verruciformis; warts, hypogammaglobulinaemia, infections and myelokathexis (WHIM) syndrome; warts, immunodeficiency, lymphoedema and anogenital dysplasia (WILD) syndrome; severe combined immune deficiency and HIV, among others. In recent years, it has been consistently recognized in patients with GATA2 deficiency, a novel immunodeficiency syndrome characterized by monocytopenia, B-cell and natural killer-cell lymphopenia, and a tendency to develop myeloid leukaemias and disseminated mycobacterial, human papillomavirus (HPV) and opportunistic fungal infections. Mutations in GATA2 cause haploinsufficiency and track in families as an autosomal dominant immunodeficiency. GATA2 is a transcription factor involved in early haematopoietic differentiation and lymphatic and vascular development. We describe a case of generalized verrucosis with HPV type 57 presenting in a young man with GATA2 deficiency. GATA2 deficiency is a novel dominant immunodeficiency that is often recognized later in life and should be considered in the differential diagnosis of patients with generalized verrucosis.

  4. Association of autoimmune Addison's disease with alleles of STAT4 and GATA3 in European cohorts.

    Directory of Open Access Journals (Sweden)

    Anna L Mitchell

    Full Text Available Gene variants known to contribute to Autoimmune Addison's disease (AAD susceptibility include those at the MHC, MICA, CIITA, CTLA4, PTPN22, CYP27B1, NLRP-1 and CD274 loci. The majority of the genetic component to disease susceptibility has yet to be accounted for.To investigate the role of 19 candidate genes in AAD susceptibility in six European case-control cohorts.A sequential association study design was employed with genotyping using Sequenom iPlex technology. In phase one, 85 SNPs in 19 genes were genotyped in UK and Norwegian AAD cohorts (691 AAD, 715 controls. In phase two, 21 SNPs in 11 genes were genotyped in German, Swedish, Italian and Polish cohorts (1264 AAD, 1221 controls. In phase three, to explore association of GATA3 polymorphisms with AAD and to determine if this association extended to other autoimmune conditions, 15 SNPs in GATA3 were studied in UK and Norwegian AAD cohorts, 1195 type 1 diabetes patients from Norway, 650 rheumatoid arthritis patients from New Zealand and in 283 UK Graves' disease patients. Meta-analysis was used to compare genotype frequencies between the participating centres, allowing for heterogeneity.We report significant association with alleles of two STAT4 markers in AAD cohorts (rs4274624: P = 0.00016; rs10931481: P = 0.0007. In addition, nominal association of AAD with alleles at GATA3 was found in 3 patient cohorts and supported by meta-analysis. Association of AAD with CYP27B1 alleles was also confirmed, which replicates previous published data. Finally, nominal association was found at SNPs in both the NF-κB1 and IL23A genes in the UK and Italian cohorts respectively.Variants in the STAT4 gene, previously associated with other autoimmune conditions, confer susceptibility to AAD. Additionally, we report association of GATA3 variants with AAD: this adds to the recent report of association of GATA3 variants with rheumatoid arthritis.

  5. Activation of the control reporter plasmids pRL-TK and pRL-SV40 by multiple GATA transcription factors can lead to aberrant normalization of transfection efficiency

    Directory of Open Access Journals (Sweden)

    Strauss Jerome F

    2004-04-01

    Full Text Available Abstract Background Members of the GATA transcription factor family have been used in many transfection studies to investigate their roles in the regulation of gene expression. To correct for variations in transfection efficiency, the Renilla luciferase encoding plasmids pRL-TK and pRL-SV40 are commonly used as normalization controls. Results We report here that plasmids expressing GATA-4 or GATA-6 transcription factor increased Renilla luciferase gene expression by 2 to 8 fold when co-transfected with pRL-TK or pRL-SV40. This alteration of the control reporter gene activity was shown to cause erroneous normalization of transfection efficiency and thus misinterpretation of results in a transactivation assay. To circumvent the problem, we generated two mutant control plasmids from which putative GATA response elements were deleted. These deletions rendered pRL-SV40 unresponsive to GATA transcription factor stimulation and reduced the response of pRL-TK. A database search also indicates that consensus GATA binding sequences are present in other commercially available Renilla luciferase encoding plasmids; therefore, the latter can potentially be transactivated by GATA transcription factors. Conclusion Taken together, these findings highlight the importance of the selection of an appropriate control reporter plasmid for the normalization of transfection efficiency.

  6. Retinoid X receptor alpha represses GATA-4-mediated transcription via a retinoid-dependent interaction with the cardiac-enriched repressor FOG-2.

    Science.gov (United States)

    Clabby, Martha L; Robison, Trevor A; Quigley, Heather F; Wilson, David B; Kelly, Daniel P

    2003-02-21

    Dietary vitamin A and its derivatives, retinoids, regulate cardiac growth and development. To delineate mechanisms involved in retinoid-mediated control of cardiac gene expression, the regulatory effects of the retinoid X receptor alpha (RXR alpha) on atrial naturietic factor (ANF) gene transcription was investigated. The transcriptional activity of an ANF promoter-reporter in rat neonatal ventricular myocytes was repressed by RXR alpha in the presence of 9-cis-RA and by the constitutively active mutant RXR alpha F318A indicating that liganded RXR confers the regulatory effect. The RXR alpha-mediated repression mapped to the proximal 147 bp of the rat ANF promoter, a region lacking a consensus retinoid response element but containing several known cardiogenic cis elements including a well characterized GATA response element. Glutathione S-transferase "pull-down" assays revealed that RXR alpha interacts directly with GATA-4, in a ligand-independent manner, via the DNA binding domain of RXR alpha and the second zinc finger of GATA-4. Liganded RXR alpha repressed the activity of a heterologous promoter-reporter construct containing GATA-response element recognition sites in cardiac myocytes but not in several other cell types, suggesting that additional cardiac-enriched factors participate in the repression complex. Co-transfection of liganded RXR alpha and the known cardiac-enriched GATA-4 repressor, FOG-2, resulted in additive repression of GATA-4 activity in ventricular myocytes. In addition, RXR alpha was found to bind FOG-2, in a 9-cis-RA-dependent manner. These data reveal a novel mechanism by which retinoids regulate cardiogenic gene expression through direct interaction with GATA-4 and its co-repressor, FOG-2.

  7. GATA Transcription Factors in Tissue Homeostasis and Pathology of the Gastrointestinal Tract and Liver

    OpenAIRE

    Haveri, Hanna

    2008-01-01

    Mammalian gastrointestinal tract and liver are self-renewing organs that are able to sustain themselves due to stem cells present in their tissues. In constant, inflammation-related epithelial damage, vigorous activation of stem cells may lead to their uncontrolled proliferation, and further, to cancer. GATA-4, GATA-5, and GATA-6 regulate cell proliferation and differentiation in many mammalian organs. Lack of GATA-4 or GATA-6 leads to defective endodermal development and cell differentiation...

  8. Expression of FoxA and GATA transcription factors correlates with regionalized gut development in two lophotrochozoan marine worms: Chaetopterus (Annelida and Themiste lageniformis (Sipuncula

    Directory of Open Access Journals (Sweden)

    Boyle Michael J

    2010-07-01

    Full Text Available Abstract Background A through gut is present in almost all metazoans, and most likely represents an ancient innovation that enabled bilaterian animals to exploit a wide range of habitats. Molecular developmental studies indicate that Fox and GATA regulatory genes specify tissue regions along the gut tube in a broad diversity of taxa, although little is known about gut regionalization within the Lophotrochozoa. In this study, we isolated FoxA and GATA456 orthologs and used whole mount in situ hybridization during larval gut formation in two marine worms: the segmented, polychaete annelid Chaetopterus, which develops a planktotrophic larva with a tripartite gut, and the non-segmented sipunculan Themiste lageniformis, which develops a lecithotrophic larva with a U-shaped gut. Results FoxA and GATA456 transcripts are predominantly restricted to gut tissue, and together show regional expression spanning most of the alimentary canal in each of these lophotrochozoans, although neither FoxA nor GATA456 is expressed in the posterior intestine of Chaetopterus. In both species, FoxA is expressed at the blastula stage, transiently in presumptive endoderm before formation of a definitive gut tube, and throughout early larval development in discrete foregut and hindgut domains. GATA456 genes are expressed during endoderm formation, and in endoderm and mesoderm associated with the midgut in each species. Several species-specific differences were detected, including an overlap of FoxA and GATA456 expression in the intestinal system of Themiste, which is instead complimentary in Chaetopterus. Other differences include additional discrete expression domains of FoxA in ectodermal trunk cells in Themiste but not Chaetopterus, and expression of GATA456 in anterior ectoderm and midgut cells unique to Chaetopterus. Conclusions This study of gene expression in a sipunculan contributes new comparative developmental insights from lophotrochozoans, and shows that FoxA and

  9. Identification of Drosophila Gene Products Required for Phagocytosis of Candidaalbicans.

    Directory of Open Access Journals (Sweden)

    2005-12-01

    Full Text Available Phagocytosis is a highly conserved aspect of innate immunity. We used Drosophila melanogaster S2 cells as a model system to study the phagocytosis of Candida albicans, the major fungal pathogen of humans, by screening an RNAi library representing 7,216 fly genes conserved among metazoans. After rescreening the initial genes identified and eliminating certain classes of housekeeping genes, we identified 184 genes required for efficient phagocytosis of C. albicans. Diverse biological processes are represented, with actin cytoskeleton regulation, vesicle transport, signaling, and transcriptional regulation being prominent. Secondary screens using Escherichia coli and latex beads revealed several genes specific for C. albicans phagocytosis. Characterization of one of those gene products, Macroglobulin complement related (Mcr, shows that it is secreted, that it binds specifically to the surface of C. albicans, and that it promotes its subsequent phagocytosis. Mcr is closely related to the four Drosophila thioester proteins (Teps, and we show that TepII is required for efficient phagocytosis of E. coli (but not C. albicans or Staphylococcus aureus and that TepIII is required for the efficient phagocytosis of S. aureus (but not C. albicans or E. coli. Thus, this family of fly proteins distinguishes different pathogens for subsequent phagocytosis.

  10. Deactivation of the GATA Transcription Factor ELT-2 Is a Major Driver of Normal Aging in C. elegans

    OpenAIRE

    Mann, Frederick G.; Van Nostrand, Eric L.; Friedland, Ari E.; Xiao Liu; Kim, Stuart K.

    2016-01-01

    Author Summary A central question in aging is to uncover the molecular drivers of the normal aging process. Using the roundworm C. elegans as a model organism, we found that the ELT-2 GATA transcription factor regulates many gene expression changes that occur during aging. During development, elt-2 functions as a key regulator of intestinal development. During aging, the levels of ELT-2 transcription factor decline, which causes expression of its target genes to decline and this limits lifesp...

  11. CREB-binding proteins (CBP) as a transcriptional coactivator of GATA-2

    Institute of Scientific and Technical Information of China (English)

    JIANG HuiJie; LIU LinDe; YANG ShuDe; TOMOMI Takahashi; TORU Nakano

    2008-01-01

    The GATA family consists of six members, GATA 1-6. In this study, we focused on GATA-2, which is expressed predominantly in hematopoietic progenitor cells and plays the key role in keeping these cells in the undifferentiated status. CREB-binding proteins (CBP) are essential transcriptional coactivators for a large number of regulated DNA-binding transcription factors, including GATA-1. But there have been no reports on whether CBP is still a co-activator of GATA-2. Here, we used the immunoprecipitation and pull-down experiments to show that the GATA-2 and CBP were physically binding together, and clarified the binding sites CH1, CH3, CH452 and CT1430 in CBP and N-finger, C-finger and N-C-finger in GATA-2. Luciferase assay results in our experiment indicated that CBP could increase GATA-2 transcriptional activity in the dose-dependent manner. GATA-1 is mainly expressed in differentiated hematopoietic cells, but still has overlap expression with GATA-2. CBP is a coactivator of GATA-2 and GATA-1. The investigation on the mechanism that could decide whether CBP binds to GATA-2 to keep hematopoietic cells in the progenitor status or to GATA-1 to start differentiation will be a very interesting and very meaningful project in the near future.

  12. CREB-binding proteins (CBP) as a transcriptional coactivator of GATA-2

    Institute of Scientific and Technical Information of China (English)

    TOMOMI; Takahashi; TORU; Nakano

    2008-01-01

    The GATA family consists of six members, GATA 1-6. In this study, we focused on GATA-2, which is expressed predominantly in hematopoietic progenitor cells and plays the key role in keeping these cells in the undifferentiated status. CREB-binding proteins (CBP) are essential transcriptional coacti- vators for a large number of regulated DNA-binding transcription factors, including GATA-1. But there have been no reports on whether CBP is still a co-activator of GATA-2. Here, we used the immunopre- cipitation and pull-down experiments to show that the GATA-2 and CBP were physically binding to- gether, and clarified the binding sites CH1, CH3 , CH452 and CT1430 in CBP and N-finger, C-finger and N-C-finger in GATA-2. Luciferase assay results in our experiment indicated that CBP could increase GATA-2 transcriptional activity in the dose-dependent manner. GATA-1 is mainly expressed in differen- tiated hematopoietic cells, but still has overlap expression with GATA-2. CBP is a coactivator of GATA-2 and GATA-1. The investigation on the mechanism that could decide whether CBP binds to GATA-2 to keep hematopoietic cells in the progenitor status or to GATA-1 to start differentiation will be a very in- teresting and very meaningful project in the near future.

  13. GATA3 Expression Is a Poor Prognostic Factor in Soft Tissue Sarcomas.

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    Toshiaki Haraguchi

    Full Text Available Recent studies have investigated the significance of GATA3 expression in patients with various malignant tumors. However, no previous studies have evaluated the clinicopathological importance of GATA3 expression in soft tissue sarcomas (STS patients.We evaluated GATA3 expression in 76 STS cases using immunohistochemical analysis, and statistically compared clinicopathological characteristics between GATA3-positive and GATA3-negative cases.GATA3-positive expression was significantly associated with a higher mitotic count (P < 0.0001. Disease-free survival (DFS of GATA3-positive cases was significantly shorter than that of cases without GATA3 expression (P = 0.0104. Overall survival (OS of GATA3-positive cases was significantly shorter than that of cases without GATA3 expression (P = 0.0006. GATA3-positive expression was significantly associated with shorter DFS in both univariate analysis (hazard ratio [HR], 2.719; P = 0.012 and multivariate analysis (HR, 2.711; P = 0.014. GATA3-positive expression was also significantly associated with worse OS in both univariate analysis (HR, 5.730; P = 0.0007 and multivariate analysis (HR, 5.789; P = 0.0008.These results indicate that GATA3 is an independent prognostic factor and suggest that evaluation of GATA3 expression might enable more effective clinical follow-up using prognostic stratification of STS patients.

  14. Characterization of the conserved interaction between GATA and FOG family proteins.

    Science.gov (United States)

    Kowalski, Kasper; Liew, Chu Kong; Matthews, Jacqueline M; Gell, David A; Crossley, Merlin; Mackay, Joel P

    2002-09-20

    The N-terminal zinc finger (ZnF) from GATA transcription factors mediates interactions with FOG family proteins. In FOG proteins, the interacting domains are also ZnFs; these domains are related to classical CCHH fingers but have an His --> Cys substitution at the final zinc-ligating position. Here we demonstrate that different CCHC fingers in the FOG family protein U-shaped contact the N-terminal ZnF of GATA-1 in the same fashion although with different affinities. We also show that these interactions are of moderate affinity, which is interesting given the presumed low concentrations of these proteins in the nucleus. Furthermore, we demonstrate that the variant CCHC topology enhances binding affinity, although the His --> Cys change is not essential for the formation of a stably folded domain. To ascertain the structural basis for the contribution of the CCHC arrangement, we have determined the structure of a CCHH mutant of finger nine from U-shaped. The structure is very similar overall to the wild-type domain, with subtle differences at the C terminus that result in loss of the interaction in vivo. Taken together, these results suggest that the CCHC zinc binding topology is required for the integrity of GATA-FOG interactions and that weak interactions can play important roles in vivo.

  15. A core transcriptional network composed of Pax2/8, Gata3 and Lim1 regulates key players of pro/mesonephros morphogenesis.

    Science.gov (United States)

    Boualia, Sami Kamel; Gaitan, Yaned; Tremblay, Mathieu; Sharma, Richa; Cardin, Julie; Kania, Artur; Bouchard, Maxime

    2013-10-15

    Translating the developmental program encoded in the genome into cellular and morphogenetic functions requires the deployment of elaborate gene regulatory networks (GRNs). GRNs are especially crucial at the onset of organ development where a few regulatory signals establish the different programs required for tissue organization. In the renal system primordium (the pro/mesonephros), important regulators have been identified but their hierarchical and regulatory organization is still elusive. Here, we have performed a detailed analysis of the GRN underlying mouse pro/mesonephros development. We find that a core regulatory subcircuit composed of Pax2/8, Gata3 and Lim1 turns on a deeper layer of transcriptional regulators while activating effector genes responsible for cell signaling and tissue organization. Among the genes directly affected by the core components are the key developmental molecules Nephronectin (Npnt) and Plac8. Hence, the pro/mesonephros GRN links together several essential genes regulating tissue morphogenesis. This renal GRN sheds new light on the disease group Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) in that gene mutations are expected to generate different phenotypic outcomes as a consequence of regulatory network deficiencies rather than threshold effects from single genes.

  16. Quantitative gene expression of transcription factors T-bet and GATA-3 in rat lung allograft rejection%转录因子T-bet与GATA结合蛋白3的比值在大鼠肺移植急性排斥反应中的mRNA表达及其意义

    Institute of Scientific and Technical Information of China (English)

    纪玲; 武延格; 孙学峰; 王正; 杨林

    2009-01-01

    目的 探讨T-bet(T-box expression in T cells)和GATA结合蛋白3(GATA-3)在大鼠肺移植急性排斥反应中的mRNA表达及其意义.方法 建立同种异体大鼠肺移植模型,实验分为正常对照组(n=5只)、同种异体移植3 d组(n=4只)和5 d组(n=6只),用实时荧光定量逆转录-聚合酶链反应(RT-PCR)法,检测移植后肺组织中T-bet和GATA-3的mRNA表达.结果 实时荧光定量RT-PCR检测T-bet和GATA-3的扩增效率为78%~85%,批内和批间实验循环阈值(Ct)变化均<0.15.与止常对照和右侧非移植肺比较,同种异体移植术后3 d组,T-bet和GATA-3无显著性改变,但其比值明显高于右侧肺;同种异体移植术后5 d组,T-bet表达增高(9.31拷贝/106 18 S拷贝),但差异无统计学意义(P>0.05),GATA-3表达下降(0.88拷贝/105 18 S拷贝),T-bet/GATA3的比值增高(1.12),差异均有统计学意义(P<0.05).结论 实时荧光定量RT-PCR检测T-bet和GATA-3结果准确可靠.在急性排斥反应中,T-bet表达升高,GATA-3表达降低,其中GATA-3的表达起主导作用.T-bet/GATA-3的比值比单独检测T-bet或GATA-3更能客观地评价大鼠肺移植急性排斥反应.%Objective To investigate the mRNA expression and significance of transcription factots T-bet and GATA-3 in rat lung acute allograft rejection.Methods Experimental animals were established and divided into normal group(n=5),allogeneic 3-day group(n=4)and allogeneic 5-day group (n=6).The expression of transcription factors T-bet and GATA-3 mRNA in rat lung allografts was detected by real-time PCR.Results The amplification efficiency of quantification of T-bet and GATA-3 with real-time PCR was 78%-85%.The Ct in intra-and interassay was<0.15.Compared with normal controls (n=5),the expression had no significant change in allogeneic 3-day group:the expression of T-bet was increased(9.60±4.07 copies/106 copies),the expression of GATA-3 was decreased(0.82±0.04 copies/106 copies),and the ratio of T-bet and GATA-3 was

  17. Genetic dissection of Gata2 selective functions during specification of V2 interneurons in the developing spinal cord.

    Science.gov (United States)

    Francius, Cédric; Ravassard, Philippe; Hidalgo-Figueroa, María; Mallet, Jacques; Clotman, Frédéric; Nardelli, Jeannette

    2015-07-01

    Motor activities are controlled by neural networks in the ventral spinal cord and consist in motor neurons and a set of distinct cardinal classes of spinal interneurons. These interneurons arise from distinct progenitor domains (p0-p3) delineated according to a transcriptional code. Neural progenitors of each domain express a unique combination of transcription factors (TFs) that largely contribute to determine the fate of four classes of interneurons (V0-V3) and motor neurons. In p2 domain, at least four subtypes of interneurons namely V2a, V2b, V2c, and Pax6(+) V2 are generated. Although genetic and molecular mechanisms that specify V2a and V2b are dependent on complex interplay between several TFs including Nkx6.1, Irx3, Gata2, Foxn4, and Ascl1, and signaling pathways such as Notch and TGF-β, the sequence order of the activation of these regulators and their respective contribution are not completely elucidated yet. Here, we provide evidence by loss- or gain-of-function experiments that Gata2 is necessary for the normal development of both V2a and V2b neurons. We demonstrate that Nkx6.1 and Dll4 positively regulate the activation of Gata2 and Foxn4 in p2 progenitors. Gata2 also participates in the maintenance of p2 domain by repressing motor neuron differentiation and exerting a feedback control on patterning genes. Finally, Gata2 promotes the selective activation of V2b program at the expense of V2a fate. Thus our results provide new insights on the hierarchy and complex interactions between regulators of V2 genetic program.

  18. Computationally driven, quantitative experiments discover genes required for mitochondrial biogenesis.

    Directory of Open Access Journals (Sweden)

    David C Hess

    2009-03-01

    Full Text Available Mitochondria are central to many cellular processes including respiration, ion homeostasis, and apoptosis. Using computational predictions combined with traditional quantitative experiments, we have identified 100 proteins whose deficiency alters mitochondrial biogenesis and inheritance in Saccharomyces cerevisiae. In addition, we used computational predictions to perform targeted double-mutant analysis detecting another nine genes with synthetic defects in mitochondrial biogenesis. This represents an increase of about 25% over previously known participants. Nearly half of these newly characterized proteins are conserved in mammals, including several orthologs known to be involved in human disease. Mutations in many of these genes demonstrate statistically significant mitochondrial transmission phenotypes more subtle than could be detected by traditional genetic screens or high-throughput techniques, and 47 have not been previously localized to mitochondria. We further characterized a subset of these genes using growth profiling and dual immunofluorescence, which identified genes specifically required for aerobic respiration and an uncharacterized cytoplasmic protein required for normal mitochondrial motility. Our results demonstrate that by leveraging computational analysis to direct quantitative experimental assays, we have characterized mutants with subtle mitochondrial defects whose phenotypes were undetected by high-throughput methods.

  19. Direct transcriptional regulation of Gata4 during early endoderm specification is controlled by FoxA2 binding to an intronic enhancer.

    Science.gov (United States)

    Rojas, Anabel; Schachterle, William; Xu, Shan-Mei; Martín, Franz; Black, Brian L

    2010-10-15

    The embryonic endoderm is a multipotent progenitor cell population that gives rise to the epithelia of the digestive and respiratory tracts, the liver and the pancreas. Among the transcription factors that have been shown to be important for endoderm development and gut morphogenesis is GATA4. Despite the important role of GATA4 in endoderm development, its transcriptional regulation is not well understood. In this study, we identified an intronic enhancer from the mouse Gata4 gene that directs expression to the definitive endoderm in the early embryo. The activity of this enhancer is initially broad in all endodermal progenitors, as demonstrated by fate mapping analysis using the Cre/loxP system, but becomes restricted to the dorsal foregut and midgut, and associated organs such as dorsal pancreas and stomach. The function of the intronic Gata4 enhancer is dependent upon a conserved Forkhead transcription factor-binding site, which is bound by recombinant FoxA2 in vitro. These studies identify Gata4 as a direct transcriptional target of FoxA2 in the hierarchy of the transcriptional regulatory network that controls the development of the definitive endoderm. Copyright © 2010 Elsevier Inc. All rights reserved.

  20. Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis.

    Directory of Open Access Journals (Sweden)

    Yao Rong

    Full Text Available Glycosylphosphatidylinositol (GPI is synthesized and transferred to proteins in the endoplasmic reticulum (ER. GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identified to be required for the GPI biosynthetic pathway. Here, we aimed to establish a comprehensive screening method to identify genes involved in GPI biosynthesis using mammalian haploid screens. Human haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to GPI-anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of CD59, a GPI-anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known GPI biosynthetic genes, which when mutated are expected to decrease the surface expression of GPI-anchored proteins. Our results indicate that the forward genetic approach using haploid cells is a useful and powerful technique to identify factors involved in phenotypes of interest.

  1. GATA transcription factors associate with a novel class of nuclear bodies in erythroblasts and megakaryocytes.

    NARCIS (Netherlands)

    A.G. Elefanty (Andrew); M. Antoniou (Michael); N. Custodio; M. Carmo-Fonseca; F.G. Grosveld (Frank)

    1996-01-01

    textabstractThe nuclear distribution of GATA transcription factors in murine haemopoietic cells was examined by indirect immunofluorescence. Specific bright foci of GATA-1 fluorescence were observed in erythroleukaemia cells and primary murine erythroblasts and megakaryocytes, in addition to diffuse

  2. A gata2-dependent transcription network regulates uterine progesterone responsiveness and endometrial function

    Science.gov (United States)

    Altered progesterone responsiveness leads to female infertility and cancer, but underlying mechanisms remain unclear. Mice with uterine-specific ablation of GATA binding protein 2 (Gata2) are infertile, showing failures in embryo implantation, endometrial decidualization, and uninhibited estrogen si...

  3. The evolution of cellular deficiency in GATA2 mutation

    Science.gov (United States)

    Dickinson, Rachel E.; Milne, Paul; Jardine, Laura; Zandi, Sasan; Swierczek, Sabina I.; McGovern, Naomi; Cookson, Sharon; Ferozepurwalla, Zaveyna; Langridge, Alexander; Pagan, Sarah; Gennery, Andrew; Heiskanen-Kosma, Tarja; Hämäläinen, Sari; Seppänen, Mikko; Helbert, Matthew; Tholouli, Eleni; Gambineri, Eleonora; Reykdal, Sigrún; Gottfreðsson, Magnús; Thaventhiran, James E.; Morris, Emma; Hirschfield, Gideon; Richter, Alex G.; Jolles, Stephen; Bacon, Chris M.; Hambleton, Sophie; Haniffa, Muzlifah; Bryceson, Yenan; Allen, Carl; Prchal, Josef T.; Dick, John E.; Bigley, Venetia

    2014-01-01

    Constitutive heterozygous GATA2 mutation is associated with deafness, lymphedema, mononuclear cytopenias, infection, myelodysplasia (MDS), and acute myeloid leukemia. In this study, we describe a cross-sectional analysis of 24 patients and 6 relatives with 14 different frameshift or substitution mutations of GATA2. A pattern of dendritic cell, monocyte, B, and natural killer (NK) lymphoid deficiency (DCML deficiency) with elevated Fms-like tyrosine kinase 3 ligand (Flt3L) was observed in all 20 patients phenotyped, including patients with Emberger syndrome, monocytopenia with Mycobacterium avium complex (MonoMAC), and MDS. Four unaffected relatives had a normal phenotype indicating that cellular deficiency may evolve over time or is incompletely penetrant, while 2 developed subclinical cytopenias or elevated Flt3L. Patients with GATA2 mutation maintained higher hemoglobin, neutrophils, and platelets and were younger than controls with acquired MDS and wild-type GATA2. Frameshift mutations were associated with earlier age of clinical presentation than substitution mutations. Elevated Flt3L, loss of bone marrow progenitors, and clonal myelopoiesis were early signs of disease evolution. Clinical progression was associated with increasingly elevated Flt3L, depletion of transitional B cells, CD56bright NK cells, naïve T cells, and accumulation of terminally differentiated NK and CD8+ memory T cells. These studies provide a framework for clinical and laboratory monitoring of patients with GATA2 mutation and may inform therapeutic decision-making. PMID:24345756

  4. GATA-3 function in innate and adaptive immunity.

    Science.gov (United States)

    Tindemans, Irma; Serafini, Nicolas; Di Santo, James P; Hendriks, Rudi W

    2014-08-21

    The zinc-finger transcription factor GATA-3 has received much attention as a master regulator of T helper 2 (Th2) cell differentiation, during which it controls interleukin-4 (IL-4), IL-5, and IL-13 expression. More recently, GATA-3 was shown to contribute to type 2 immunity through regulation of group 2 innate lymphoid cell (ILC2) development and function. Furthermore, during thymopoiesis, GATA-3 represses B cell potential in early T cell precursors, activates TCR signaling in pre-T cells, and promotes the CD4(+) T cell lineage after positive selection. GATA-3 also functions outside the thymus in hematopoietic stem cells, regulatory T cells, CD8(+) T cells, thymic natural killer cells, and ILC precursors. Here we discuss the varied functions of GATA-3 in innate and adaptive immune cells, with emphasis on its activity in T cells and ILCs, and examine the mechanistic basis for the dose-dependent, developmental-stage- and cell-lineage-specific activity of this transcription factor. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Activation of Hypodermal Differentiation in the Caenorhabditis elegans Embryo by GATA Transcription Factors ELT-1 and ELT-3

    OpenAIRE

    Gilleard, J.S.; McGhee, J D

    2001-01-01

    The Caenorhabditis elegans GATA transcription factor genes elt-1 and elt-3 are expressed in the embryonic hypodermis (also called the epidermis). elt-1 is expressed in precursor cells and is essential for the production of most hypodermal cells (22). elt-3 is expressed in all of the major hypodermal cells except the lateral seam cells, and expression is initiated immediately after the terminal division of precursor lineages (13). Although this expression pattern suggests a role for ELT-3 in h...

  6. No evidence that GATA3 rs570613 SNP modifies breast cancer risk.

    NARCIS (Netherlands)

    Johnatty, S.E.; Couch, F.J.; Fredericksen, Z.; Tarrell, R.; Spurdle, A.B.; Beesley, J.; Chen, X.; Gschwantler-Kaulich, D.; Singer, C.F.; Fuerhauser, C.; Fink-Retter, A.; Domchek, S.M.; Nathanson, K.L.; Pankratz, V.S.; Lindor, N.; Godwin, A.K.; Caligo, M.A.; Hopper, J.; Southey, M.C.; Giles, G.G.; Justenhoven, C.; Brauch, H.; Hamann, U.; Ko, Y.D.; Heikkinen, T.; Aaltonen, K.; Aittomaki, K.; Blomqvist, C.; Nevanlinna, H.; Hall, P.; Czene, K.; Liu, J.; Peock, S.; Cook, M.; Platte, R.; Evans, D.G.; Lalloo, F.; Eeles, R.; Pichert, G.; Eccles, D.; Davidson, R.; Cole, T.; Cook, J.; Douglas, F.; Chu, C.; Hodgson, S.; Paterson, J.; Hogervorst, F.B.L.; Rookus, M.A.; Seynaeve, C.; Wijnen, J.; Vreeswijk, M.; Ligtenberg, M.J.L.; Luijt, R.B. van der; Os, T.A. van; Gille, H.J.; Blok, M.J.; Issacs, C.; Humphreys, M.K.; McGuffog, L.; Healey, S.; Sinilnikova, O.M.; Antoniou, A.C.; Easton, D.F.; Chenevix-Trench, G.

    2009-01-01

    GATA-binding protein 3 (GATA3) is a transcription factor that is crucial to mammary gland morphogenesis and differentiation of progenitor cells, and has been suggested to have a tumor suppressor function. The rs570613 single nucleotide polymorphism (SNP) in intron 4 of GATA3 was previously found to

  7. 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 h...

  8. Plasmid genes required for microcin B17 production.

    Science.gov (United States)

    San Millán, J L; Kolter, R; Moreno, F

    1985-09-01

    The production of the antibiotic substance microcin B17 (Mcc) is determined by a 3.5-kilobase DNA fragment from plasmid pMccB17. Several Mcc- mutations on plasmid pMccB17 were obtained by both transposon insertion and nitrosoguanidine mutagenesis. Plasmids carrying these mutations were tested for their ability to complement Mcc- insertion or deletion mutations on pMM102 (pMM102 is a pBR322 derivative carrying the region encoding microcin B17). Results from these experiments indicate that at least four plasmid genes are required for microcin production.

  9. B-GATA transcription factors - insights into their structure, regulation and role in plant development

    Directory of Open Access Journals (Sweden)

    Claus eSchwechheimer

    2015-02-01

    Full Text Available GATA transcription factors are evolutionarily conserved transcriptional regulators that recognize promoter elements with a G-A-T-A core sequence. In comparison to animal genomes, the GATA transcription factor family in plants is comparatively large with approximately 30 members. In spite of a long-standing interest of plant molecular biologists in GATA factors, only research conducted in the last years has led to reliable insights into their functions during plant development. Here, we review the current knowledge on B-GATAs, one of four GATA factor subfamilies from Arabidopsis thaliana. We show that B-GATAs can be subdivided based on structural features and their biological function into family members with a C-terminal LLM- (leucine-leucine-methionine domain or an N-terminal HAN- (HANABA TARANU domain. The paralogous GNC (GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM INVOLVED and CGA1/GNL (CYTOKININ-INDUCED GATA1/GNC-LIKE are introduced as LLM-domain containing B-GATAs from Arabidopsis that control germination, greening, senescence and flowering time downstream from several growth regulatory signals including light and the hormones gibberellin, auxin, and cytokinin. Arabidopsis HAN and its monocot-specific paralogs from rice (NECK LEAF1, maize (TASSEL SHEATH1, and barley (THIRD OUTER GLUME are HAN-domain-containing B-GATAs with a predominant role in embryo development and floral development. We also review GATA23, a regulator of lateral root initiation from Arabidopsis, that is closely related to GNC and GNL but has a degenerate LLM-domain that is seemingly specific for the Brassicaceae family. The Brassicaceae-specific GATA23 together with the above-mentioned monocot-specific HAN-domain GATAs provide evidence that neofunctionalization of the B-GATAs was used during plant evolution to expand the functional repertoire of these transcription factors.

  10. Identification of a novel pathway involving a GATA transcription factor in yeast and possibly in plant Zn uptake and homeostasis

    Institute of Scientific and Technical Information of China (English)

    Matthew J. Milner; Nicole S. Pence; Jiping Liu; Leon V. Kochian

    2014-01-01

    To gain a better understanding of the regulation of Zn homeostasis in plants and the degree of conservation of Zn homeostasis between plants and yeast, a cDNA library from the Zn/Cd hyperaccumulating plant species, Noccaea caerules-cens, was screened for its ability to restore growth under Zn limiting conditions in the yeast mutant zap1D. ZAP1 is a transcription factor that activates the Zn dependent transcrip-tion of yeast genes involved in Zn uptake, including ZRT1, the yeast high affinity Zn transporter. From this screen two members of the E2F family of transcription factors were found to activate ZRT1 expression in a Zn independent manner. The activation of ZRT1 by the plant E2F proteins involves E2F-mediated activation of a yeast GATA transcription factor which in turn activates ZRT1 expression. A ZRT1 promoter region necessary for activation by E2F and GATA proteins is upstream of two zinc responsive elements previously shown to bind ZAP1 in ZRT1. This activation may not involve direct binding of E2F to the ZRT1 promoter. The expression of E2F genes in yeast does not replace function of ZAP1; instead it appears to activate a novel GATA regulatory pathway involved in Zn uptake and homeostasis that is not Zn responsive.

  11. Common increase of GATA-3 level in PC-12 cells by three teratogens causing autism spectrum disorders.

    Science.gov (United States)

    Rout, Ujjwal K; Clausen, Pete

    2009-06-01

    Autism spectrum disorder (ASD) is a disease of neuro-developmental origin of uncertain etiology. The current understanding is that both genetic and environmental factors contribute to the development of ASD. Exposure to valproate, thalidomide and alcohol during gestation are amongst the environmental triggers that are associated with the development of ASD. These teratogens may disturb the ontogeny of the brain by altering the expression pattern of genes that regulate the normal development of the brain. In this study, a neuron-like PC-12 cell model was used to examine the effects of these compounds on the binding potential of 50 different transcription factors to understand the molecular mechanism/s that may be involved in the teratogenesis caused by these agents. Cells in culture were treated with low or high concentrations of teratogens within a range that are reported in the blood of individuals. A pronounced increase in GATA transcription factor binding was observed for all three teratogens. Furthermore, Western blot analysis showed that GATA-3 level in the nuclear fractions was enhanced by each of the three teratogens. Results suggest that altered gene expression pattern due to heightened GATA-3 activities in the fetral brains following exposure to these teratogens may contribute to the development of ASD.

  12. Two dynamin-2 genes are required for normal zebrafish development.

    Directory of Open Access Journals (Sweden)

    Elizabeth M Gibbs

    Full Text Available Dynamin-2 (DNM2 is a large GTPase involved in clathrin-mediated endocytosis and related trafficking pathways. Mutations in human DNM2 cause two distinct neuromuscular disorders: centronuclear myopathy and Charcot-Marie-Tooth disease. Zebrafish have been shown to be an excellent animal model for many neurologic disorders, and this system has the potential to inform our understanding of DNM2-related disease. Currently, little is known about the endogenous zebrafish orthologs to human DNM2. In this study, we characterize two zebrafish dynamin-2 genes, dnm2 and dnm2-like. Both orthologs are structurally similar to human DNM2 at the gene and protein levels. They are expressed throughout early development and in all adult tissues examined. Knockdown of dnm2 and dnm2-like gene products resulted in extensive morphological abnormalities during development, and expression of human DNM2 RNA rescued these phenotypes. Our findings suggest that dnm2 and dnm2-like are orthologs to human DNM2, and that they are required for normal zebrafish development.

  13. A gene regulatory network armature for T-lymphocyte specification

    Energy Technology Data Exchange (ETDEWEB)

    Fung, Elizabeth-sharon [Los Alamos National Laboratory

    2008-01-01

    Choice of a T-lymphoid fate by hematopoietic progenitor cells depends on sustained Notch-Delta signaling combined with tightly-regulated activities of multiple transcription factors. To dissect the regulatory network connections that mediate this process, we have used high-resolution analysis of regulatory gene expression trajectories from the beginning to the end of specification; tests of the short-term Notchdependence of these gene expression changes; and perturbation analyses of the effects of overexpression of two essential transcription factors, namely PU.l and GATA-3. Quantitative expression measurements of >50 transcription factor and marker genes have been used to derive the principal components of regulatory change through which T-cell precursors progress from primitive multipotency to T-lineage commitment. Distinct parts of the path reveal separate contributions of Notch signaling, GATA-3 activity, and downregulation of PU.l. Using BioTapestry, the results have been assembled into a draft gene regulatory network for the specification of T-cell precursors and the choice of T as opposed to myeloid dendritic or mast-cell fates. This network also accommodates effects of E proteins and mutual repression circuits of Gfil against Egr-2 and of TCF-l against PU.l as proposed elsewhere, but requires additional functions that remain unidentified. Distinctive features of this network structure include the intense dose-dependence of GATA-3 effects; the gene-specific modulation of PU.l activity based on Notch activity; the lack of direct opposition between PU.l and GATA-3; and the need for a distinct, late-acting repressive function or functions to extinguish stem and progenitor-derived regulatory gene expression.

  14. The leukemia associated nuclear corepressor ETO homologue genes MTG16 and MTGR1 are regulated differently in hematopoietic cells

    Directory of Open Access Journals (Sweden)

    Ajore Ram

    2012-03-01

    Full Text Available Abstract Background MTG16, MTGR1 and ETO are nuclear transcriptional corepressors of the human ETO protein family. MTG16 is implicated in hematopoietic development and in controlling erythropoiesis/megakaryopoiesis. Furthermore, ETO homologue genes are 3'participants in leukemia fusions generated by chromosomal translocations responsible of hematopoietic dysregulation. We tried to identify structural and functional promoter elements of MTG16 and MTGR1 genes in order to find associations between their regulation and hematopoiesis. Results 5' deletion examinations and luciferase reporter gene studies indicated that a 492 bp sequence upstream of the transcription start site is essential for transcriptional activity by the MTG16 promoter. The TATA- and CCAAT-less promoter with a GC box close to the start site showed strong reporter activity when examined in erythroid/megakaryocytic cells. Mutation of an evolutionary conserved GATA -301 consensus binding site repressed promoter function. Furthermore, results from in vitro antibody-enhanced electrophoretic mobility shift assay and in vivo chromatin immunoprecipitation indicated binding of GATA-1 to the GATA -301 site. A role of GATA-1 was also supported by transfection of small interfering RNA, which diminished MTG16 expression. Furthermore, expression of the transcription factor HERP2, which represses GATA-1, produced strong inhibition of the MTG16 promoter reporter consistent with a role of GATA-1 in transcriptional activation. The TATA-less and CCAAT-less MTGR1 promoter retained most of the transcriptional activity within a -308 to -207 bp region with a GC-box-rich sequence containing multiple SP1 binding sites reminiscent of a housekeeping gene with constitutive expression. However, mutations of individual SP1 binding sites did not repress promoter function; multiple active SP1 binding sites may be required to safeguard constitutive MTGR1 transcriptional activity. The observed repression of MTG16

  15. Higher Levels of GATA3 Predict Better Survival in Women with Breast Cancer

    Science.gov (United States)

    Yoon, Nam K.; Maresh, Erin L.; Shen, Dejun; Elshimali, Yahya; Apple, Sophia; Horvath, Steve; Mah, Vei; Bose, Shikha; Chia, David; Chang, Helena R.; Goodglick, Lee

    2010-01-01

    The GATA family members are zinc finger transcription factors involved in cell differentiation and proliferation. GATA3 in particular is necessary for mammary gland maturation, and its loss has been implicated in breast cancer development. Our goal was to validate the ability of GATA3 expression to predict survival in breast cancer patients. Protein expression of GATA3 was analyzed on a high density tissue microarray consisting of 242 cases of breast cancer. We associated GATA3 expression with patient outcomes and clinicopathological variables. Expression of GATA3 was significantly increased in breast cancer, in situ lesions, and hyperplastic tissue compared to normal breast tissue. GATA3 expression decreased with increasing tumor grade. Low GATA3 expression was a significant predictor of disease-related death in all patients, as well as in subgroups of estrogen receptor positive or low grade patients. Additionally, low GATA3 expression correlated with increased tumor size and estrogen and progesterone receptor negativity. GATA3 is an important predictor of disease outcome in breast cancer patients. This finding has been validated in a diverse set of populations. Thus, GATA3 expression has utility as a prognostic indicator in breast cancer. PMID:21078439

  16. CHD5 is required for neurogenesis and has a dual role in facilitating gene expression and polycomb gene repression

    DEFF Research Database (Denmark)

    Egan, Chris M; Nyman, Ulrika; Skotte, Julie

    2013-01-01

    differentiation and leads to an accumulation of undifferentiated progenitors. CHD5 binds a large cohort of genes and is required for facilitating the activation of neuronal genes. It also binds a cohort of Polycomb targets and is required for the maintenance of H3K27me3 on these genes. Interestingly...

  17. Systematic Cellular Disease Models Reveal Synergistic Interaction of Trisomy 21 and GATA1 Mutations in Hematopoietic Abnormalities.

    Science.gov (United States)

    Banno, Kimihiko; Omori, Sayaka; Hirata, Katsuya; Nawa, Nobutoshi; Nakagawa, Natsuki; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Sakuma, Tetsushi; Yamamoto, Takashi; Toki, Tsutomu; Ito, Etsuro; Yamamoto, Toshiyuki; Kokubu, Chikara; Takeda, Junji; Taniguchi, Hidetoshi; Arahori, Hitomi; Wada, Kazuko; Kitabatake, Yasuji; Ozono, Keiichi

    2016-05-10

    Chromosomal aneuploidy and specific gene mutations are recognized early hallmarks of many oncogenic processes. However, the net effect of these abnormalities has generally not been explored. We focused on transient myeloproliferative disorder (TMD) in Down syndrome, which is characteristically associated with somatic mutations in GATA1. To better understand functional interplay between trisomy 21 and GATA1 mutations in hematopoiesis, we constructed cellular disease models using human induced pluripotent stem cells (iPSCs) and genome-editing technologies. Comparative analysis of these engineered iPSCs demonstrated that trisomy 21 perturbed hematopoietic development through the enhanced production of early hematopoietic progenitors and the upregulation of mutated GATA1, resulting in the accelerated production of aberrantly differentiated cells. These effects were mediated by dosage alterations of RUNX1, ETS2, and ERG, which are located in a critical 4-Mb region of chromosome 21. Our study provides insight into the genetic synergy that contributes to multi-step leukemogenesis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  18. GATA3 expression in breast carcinoma: utility in triple-negative, sarcomatoid, and metastatic carcinomas.

    Science.gov (United States)

    Cimino-Mathews, Ashley; Subhawong, Andrea P; Illei, Peter B; Sharma, Rajni; Halushka, Marc K; Vang, Russell; Fetting, John H; Park, Ben Ho; Argani, Pedram

    2013-07-01

    GATA3 plays an integral role in breast luminal cell differentiation and is implicated in breast cancer progression. GATA3 immunohistochemistry is a useful marker of breast cancer; however, its use in specific subtypes is unclear. Here, we evaluate GATA3 expression in 86 invasive ductal carcinomas including triple-negative, Her-2, and luminal subtypes, in addition to 13 metaplastic carcinomas and in 34 fibroepithelial neoplasms. In addition, we report GATA3 expression in matched primary and metastatic breast carcinomas in 30 patients with known estrogen receptor (ER), progesterone receptor (PR), and Her-2 status, including 5 with ER and/or PR loss from primary to metastasis. Tissue microarrays containing 5 to 10 cores per tumor were stained for GATA3, scored as follows: 0 (0-5%), 1+ (6%-25%), 2+ (26%-50%), 3+ (51%-75%), and 4+ (>75%). GATA3 labeling was seen in 67% (66/99) of primary ductal carcinomas including 43% of triple-negative and 54% of metaplastic carcinomas. In contrast, stromal GATA3 labeling was seen in only 1 fibroepithelial neoplasm. GATA3 labeling was seen in 90% (27/30) of primary breast carcinomas in the paired cohort, including 67% of triple-negative carcinomas. GATA3 labeling was overwhelmingly maintained in paired metastases. Notably, GATA3 was maintained in all "luminal loss" metastases, which showed ER and/or PR loss. In conclusion, GATA3 expression is maintained between matched primary and metastatic carcinomas including ER-negative cases. GATA3 can be particularly useful as a marker for metastatic breast carcinoma, especially triple-negative and metaplastic carcinomas, which lack specific markers of mammary origin. Finally, GATA3 labeling may help distinguish metaplastic carcinoma from malignant phyllodes tumors.

  19. Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 enhance the cardio-inducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming.

    Directory of Open Access Journals (Sweden)

    Nicolas Christoforou

    Full Text Available Transient overexpression of defined combinations of master regulator genes can effectively induce cellular reprogramming: the acquisition of an alternative predicted phenotype from a differentiated cell lineage. This can be of particular importance in cardiac regenerative medicine wherein the heart lacks the capacity to heal itself, but simultaneously contains a large pool of fibroblasts. In this study we determined the cardio-inducing capacity of ten transcription factors to actuate cellular reprogramming of mouse embryonic fibroblasts into cardiomyocyte-like cells. Overexpression of transcription factors MYOCD and SRF alone or in conjunction with Mesp1 and SMARCD3 enhanced the basal but necessary cardio-inducing effect of the previously reported GATA4, TBX5, and MEF2C. In particular, combinations of five or seven transcription factors enhanced the activation of cardiac reporter vectors, and induced an upregulation of cardiac-specific genes. Global gene expression analysis also demonstrated a significantly greater cardio-inducing effect when the transcription factors MYOCD and SRF were used. Detection of cross-striated cells was highly dependent on the cell culture conditions and was enhanced by the addition of valproic acid and JAK inhibitor. Although we detected Ca(2+ transient oscillations in the reprogrammed cells, we did not detect significant changes in resting membrane potential or spontaneously contracting cells. This study further elucidates the cardio-inducing effect of the transcriptional networks involved in cardiac cellular reprogramming, contributing to the ongoing rational design of a robust protocol required for cardiac regenerative therapies.

  20. The regulation of troponins I, C and ANP by GATA4 and Nkx2-5 in heart of hibernating thirteen-lined ground squirrels, Ictidomys tridecemlineatus.

    Directory of Open Access Journals (Sweden)

    Bryan E Luu

    Full Text Available Hibernation is an adaptive strategy used by various mammals to survive the winter under situations of low ambient temperatures and limited or no food availability. The heart of hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus has the remarkable ability to descend to low, near 0°C temperatures without falling into cardiac arrest. We hypothesized that the transcription factors GATA4 and Nkx2-5 may play a role in cardioprotection by facilitating the expression of key downstream targets such as troponin I, troponin C, and ANP (atrial natriuretic peptide. This study measured relative changes in transcript levels, protein levels, protein post-translational modifications, and transcription factor binding over six stages: euthermic control (EC, entrance into torpor (EN, early torpor (ET, late torpor (LT, early arousal (EA, and interbout arousal (IA. We found differential regulation of GATA4 whereby transcript/protein expression, post-translational modification (phosphorylation of serine 261, and DNA binding were enhanced during the transitory phases (entrance and arousal of hibernation. Activation of GATA4 was paired with increases in cardiac troponin I, troponin C and ANP protein levels during entrance, while increases in p-GATA4 DNA binding during early arousal was paired with decreases in troponin I and no changes in troponin C and ANP protein levels. Unlike its binding partner, the relative mRNA/protein expression and DNA binding of Nkx2-5 did not change during hibernation. This suggests that either Nkx2-5 does not play a substantial role or other regulatory mechanisms not presently studied (e.g. posttranslational modifications are important during hibernation. The data suggest a significant role for GATA4-mediated gene transcription in the differential regulation of genes which aid cardiac-specific challenges associated with torpor-arousal.

  1. Similarity of GATA-3 Expression between Rat and Human Mammary Glands

    Science.gov (United States)

    Kinoshita, Yuichi; Yoshizawa, Katsuhiko; Emoto, Yuko; Yuki, Michiko; Yuri, Takashi; Shikata, Nobuaki; Tsubura, Airo

    2014-01-01

    The GATA family members are zinc finger transcription factors involved in cell differentiation and proliferation. In particular, GATA-3 is necessary for mammary gland maturation and is a useful marker in the characterization of mammary carcinoma in humans. The expression of GATA-3 protein in normal mammary glands, fibroadenomas and carcinomas was immunohistochemically compared in female rats and humans. In normal mammary glands of rats and humans, scattered luminal cells in the acini and whole ductal epithelial cells were positive for GATA-3 in the nuclei. No positive cells were detected in rat or human fibroadenomas. In rat and human mammary carcinomas, the nuclei of proliferating luminal-derived cancer cells expressed GATA-3. Therefore, GATA-3 protein is a candidate marker for mammary carcinoma in rats as well as humans. PMID:25352719

  2. GATA3 Inhibits Breast Cancer Metastasis through the Reversal of Epithelial-Mesenchymal Transition*

    OpenAIRE

    Yan, Wei; Cao, Qing Jackie; Arenas, Richard B.; Bentley, Brooke; Shao, Rong

    2010-01-01

    GATA3, a transcription factor that regulates T lymphocyte differentiation and maturation, is exclusively expressed in early stage well differentiated breast cancers but not in advanced invasive cancers. However, little is understood regarding its activity and the mechanisms underlying this differential expression in cancers. Here, we employed GATA3-positive, non-invasive (MCF-7) and GATA3-negative, invasive (MDA-MB-231) breast cancer cells to define its role in the transformation between thes...

  3. Significance of GATA-3 expression in outcomes of patients with breast cancer who received systemic chemotherapy and/or hormonal therapy and clinicopathologic features of GATA-3-positive tumors.

    Science.gov (United States)

    Gulbahce, H Evin; Sweeney, Carol; Surowiecka, Maria; Knapp, Dennis; Varghese, Linda; Blair, Cindy K

    2013-11-01

    GATA-3 and estrogen receptor (ER) are involved in a positive cross-regulatory loop and are frequently coexpressed in breast cancers. GATA-3 expression was shown to be an independent predictor of overall and disease-free survival in some studies, whereas others showed no difference. However, the studies used different cutoff values for determining GATA-3 positivity and analyzed outcomes in patients who received systemic therapy together with those who did not. We investigated GATA-3 expression and correlated clinicopathologic findings and outcomes in 516 women who received systemic chemotherapy and/or hormonal therapy. Nuclear staining of 1% or greater was considered positive for GATA-3, ER and progesterone receptor (PR). Of 516 cases, 436 (84.5%) were GATA-3+. GATA-3+ tumors were more likely to be grade 1 or 2, ER+, PR+, non-triple-negative phenotypes (all P chemotherapy and/or hormonal therapy among GATA-3-positive and GATA-3-negative groups. GATA-3+ tumors are correlated with lower grade, ER+, PR+, and non-triple-negative phenotypes. Although there was no difference in OS and BCS between GATA-3-positive and GATA-3-negative groups, there was an adverse effect of GATA-3 expression in the ER-negative subgroup of patients who received systemic therapy. © 2013 Elsevier Inc. All rights reserved.

  4. Expression analysis and localization of wt1, ad4bp/sf-1 and gata4 in the testis of catfish, Clarias batrachus: Impact of wt1-esiRNA silencing.

    Science.gov (United States)

    Murugananthkumar, Raju; Senthilkumaran, Balasubramanian

    2016-08-15

    In teleosts, a comprehensive role or interaction of wt1, ad4bp/sf-1 and gata4 genes in relation to gonadal development and/or recrudescence was never attempted. Present study aimed to identify the involvement of these genes during testicular development of catfish, Clarias batrachus. Dominant expression of wt1 and gata4 was observed in developing and adult testis, while ad4bp/sf-1 showed steady expression. Localization of these genes in adult testis revealed their presence in spermatogonia, spermatocytes and interstitial/Leydig cells. Significant high expression during pre-spawning and spawning phases, and upregulated levels of these genes after hCG induction authenticated gonadotropic regulation. Transient silencing of wt1-esiRNA displayed decrease in wt1 expression, which further downregulated the expression of ad4bp/sf-1 and gata4, and certain steroidogenic enzyme genes related to androgen production. These results suggest that wt1 might target ad4bp/sf-1 and gata4 expression, and also have regulatory influence either indirectly or directly on the steroidogenic enzyme genes of catfish.

  5. Borrelidin Isolated from Streptomyces sp. Inhibited Adipocyte Differentiation in 3T3-L1 Cells via Several Factors Including GATA-Binding Protein 3.

    Science.gov (United States)

    Matsuo, Hirotaka; Kondo, Yoshiyuki; Kawasaki, Takashi; Tokuyama, Shinji; Imamura, Nobutaka

    2015-01-01

    An inhibitor of 3T3-L1 adipocyte differentiation was isolated from Streptomyces sp. TK08330 and identified by spectroscopy as the 18-membered macrolide borrelidin. Treatment with 1.0 μM borrelidin suppressed intracellular lipid accumulation by 80% and inhibited the expression of adipocyte-specific genes. Borrelidin suppressed the mRNA expression of two master regulators of adipocyte differentiation, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein (C/EBPα). Studies on well-known upstream regulators of PPARγ revealed that borrelidin down-regulated C/EBPδ mRNA expression but did not affect expression of C/EBPβ. Borrelidin increased mRNA expression of negative regulators of differentiation such as GATA-binding protein (GATA) 3, Krüppel-like factor (KLF) 3 and KLF7, as well as positive regulators, KLF4, KLF6 and KLF15, at early stages of differentiation. To elucidate a primary mediator of borrelidin differentiation inhibitory activity, small interfering RNA (siRNA) transfection experiments were performed. The mRNA expression of PPARγ, which was down-regulated by borrelidin, was not changed by KLF3 and KLF7 siRNA treatment. In contrast, expression of PPARγ in GATA-3 siRNA-treated cells was not significantly different from that of control siRNA-treated cells. Borrelidin significantly inhibited lipid accumulation in control siRNA-treated cells, and treatment with GATA-3 siRNA slightly reduced the inhibitory effect of borrelidin. These results indicate that borrelidin inhibited adipocyte differentiation partially via GATA-3.

  6. The Caenorhabditis elegans GATA factor elt-1 is essential for differentiation and maintenance of hypodermal seam cells and for normal locomotion.

    Science.gov (United States)

    Smith, Judith A; McGarr, Pamela; Gilleard, John S

    2005-12-15

    The Caenorhabditis elegans GATA transcription factor elt-1 has previously been shown to have a central role in the specification of hypodermal (epidermal) cell fates and acts several cell divisions before the birth of hypodermal cells. Here we report that elt-1 also has essential functions during subsequent development. Reporter gene studies show that elt-1 expression is maintained in lateral seam cells throughout development and elt-1 RNA interference experiments support an essential role for elt-1 in the differentiation of lateral seam cells in the embryo. The maintenance of seam-cell fates in all larval stages including L2d and dauer also requires elt-1. The elt-1 RNAi phenotype shows that seam cells are essential for the structural integrity of adult hermaphrodites in the vulval region and for diametric shrinkage during dauer larval formation. By contrast, severe seam-cell loss in the larval stages has little effect on moulting, indicating that the presence of these cells is not essential for this process. The elt-1 reporter gene is also expressed in neurones of the locomotory circuit. Loss of elt-1 function during postembryonic development results in a hypermotility phenotype whereas overexpression of elt-1 leads to a reciprocal phenotype of reduced motility and paralysis. These results suggest that elt-1 is a key regulator of neuronal function in larvae and adult worms.

  7. Deactivation of the GATA Transcription Factor ELT-2 Is a Major Driver of Normal Aging in C. elegans.

    Directory of Open Access Journals (Sweden)

    Frederick G Mann

    2016-04-01

    Full Text Available To understand the molecular processes underlying aging, we screened modENCODE ChIP-seq data to identify transcription factors that bind to age-regulated genes in C. elegans. The most significant hit was the GATA transcription factor encoded by elt-2, which is responsible for inducing expression of intestinal genes during embryogenesis. Expression of ELT-2 decreases during aging, beginning in middle age. We identified genes regulated by ELT-2 in the intestine during embryogenesis, and then showed that these developmental genes markedly decrease in expression as worms grow old. Overexpression of elt-2 extends lifespan and slows the rate of gene expression changes that occur during normal aging. Thus, our results identify the developmental regulator ELT-2 as a major driver of normal aging in C. elegans.

  8. Deactivation of the GATA Transcription Factor ELT-2 Is a Major Driver of Normal Aging in C. elegans.

    Science.gov (United States)

    Mann, Frederick G; Van Nostrand, Eric L; Friedland, Ari E; Liu, Xiao; Kim, Stuart K

    2016-04-01

    To understand the molecular processes underlying aging, we screened modENCODE ChIP-seq data to identify transcription factors that bind to age-regulated genes in C. elegans. The most significant hit was the GATA transcription factor encoded by elt-2, which is responsible for inducing expression of intestinal genes during embryogenesis. Expression of ELT-2 decreases during aging, beginning in middle age. We identified genes regulated by ELT-2 in the intestine during embryogenesis, and then showed that these developmental genes markedly decrease in expression as worms grow old. Overexpression of elt-2 extends lifespan and slows the rate of gene expression changes that occur during normal aging. Thus, our results identify the developmental regulator ELT-2 as a major driver of normal aging in C. elegans.

  9. Zpl,a new gene in drosophila required for neurogenesis

    Institute of Scientific and Technical Information of China (English)

    ZHAODEBIAO; SERGECOTE; 等

    1994-01-01

    zpl(zip-like) gene mutant embryos showed the cuticular defect with alternative denticle rows and a hole from head to abdomen.zpl mutants also caused the overgrowth of neural cells and axons both in CNS and PNS as well as the wrong pathway of neural fasciculation and the disappearance of hypophysis,as shown by whole mount embryos stained with antibody against HRP and MAb-22C10.Genetic analysis has provided evidence that zpl,located in the right arm of the second chromosome(between 75 and 102 genetic map units),is a new gene closely related to the zip gene.

  10. GATA-4 protects against hypoxia-induced cardiomyocyte injury: effects on mitochondrial membrane potential.

    Science.gov (United States)

    Li, Hong-Xia; Zhou, Ya-Feng; Zhao, Xin; Jiang, Bin; Yang, Xiang-Jun

    2014-08-01

    Our previous studies have suggested that GATA-4 increases the differentiation of bone-marrow-derived mesenchymal stem cells (MSCs) into cardiac phenotypes. This study further investigated whether GATA-4 enhances MSC-mediated cardioprotection following hypoxia. MSCs were harvested from rat bone marrow and transduced with GATA-4 (MSC(GATA-4)). To mimic ischemic injury, cultured cardiomyocytes (CMs) isolated from neonatal rat ventricles were exposed to hypoxia or were pretreated with concentrated conditioned medium (CdM) from MSC(GATA-4) or transduced control MSC (MSC(Null)) for 16 h before exposure to hypoxic culture conditions (low glucose and low oxygen). Myocyte damage was estimated by annexin-V-PE and TUNEL technique and by lactate dehydrogenase (LDH) release. Cell survival was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium (MTT) uptake. Mitochondrial membrane potential was determined using confocal microscopy. ELISA studies indicated that insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) were significantly increased in MSC(GATA-4) compared with MSC(Null). Hypoxia-induced apoptosis/cell death was significantly reduced when CMs were co-cultured with MSC(GATA-4) in a dual-chamber system. Cell protection mediated by MSC(GATA-4) was mimicked by treating CMs with CdM from MSC(GATA-4) and abrogated with IGF-1- and VEGF-neutralizing antibodies. MSC(GATA-4) protects CMs under hypoxic conditions. The release of IGF-1 and VEGF from MSC(GATA-4) is likely to be responsible for protection of CMs.

  11. cAMP-dependent proteolysis of GATA-6 is linked to JNK-signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ushijima, Hironori [Department of Molecular Biology, School of Pharmacy, Iwate Medical University, 2-1-1, Nishitokuta, Yahaba, Shiwagun, Iwate 028-3694 (Japan); Maeda, Masatomo, E-mail: mmaeda@iwate-med.ac.jp [Department of Molecular Biology, School of Pharmacy, Iwate Medical University, 2-1-1, Nishitokuta, Yahaba, Shiwagun, Iwate 028-3694 (Japan)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6. Black-Right-Pointing-Pointer Effect of a JNK activator anisomycin on the proteolysis was examined. Black-Right-Pointing-Pointer Anisomycin stimulated the export of nuclear GATA-6 into the cytoplasm. Black-Right-Pointing-Pointer JNK activated the CRM1 mediated nuclear export of GATA-6. Black-Right-Pointing-Pointer JNK further stimulated slowly the degradation of GATA-6 by cytoplasmic proteasomes. -- Abstract: A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6 by proteasomes around its IC50. We further examined the effects of SP600125 on the degradation of GATA-6 in detail, since an activator of JNK (anisomycin) is available. Interestingly, anisomycin immediately stimulated the export of nuclear GATA-6 into the cytoplasm, and then the cytoplasmic content of GATA-6 decreased slowly through degradation by proteasomes. Such an effect of anisomycin was inhibited by SP600125, indicating that the observed phenomenon might be linked to the JNK signaling pathway. The inhibitory effect of SP600125 could not be ascribed to the inhibition of PKA, since phosphorylation of CREB occurred in the presence of dbcAMP and SP600125. The nuclear export of GATA-6 was inhibited by leptomycin B, suggesting that CRM1-mediated export could be activated by anisomycin. Furthermore, it seems likely that the JNK activated by anisomycin may stimulate not only the nuclear export of GATA-6 through CRM1 but also the degradation of GATA-6 by cytoplasmic proteasomes. In contrast, A-kinase might activate only the latter process through JNK.

  12. YUCCA auxin biosynthetic genes are required for Arabidopsis shade avoidance

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    Patricia Müller-Moulé

    2016-10-01

    Full Text Available Plants respond to neighbor shade by increasing stem and petiole elongation. Shade, sensed by phytochrome photoreceptors, causes stabilization of PHYTOCHROME INTERACTING FACTOR proteins and subsequent induction of YUCCA auxin biosynthetic genes. To investigate the role of YUCCA genes in phytochrome-mediated elongation, we examined auxin signaling kinetics after an end-of-day far-red (EOD-FR light treatment, and found that an auxin responsive reporter is rapidly induced within 2 hours of far-red exposure. YUCCA2, 5, 8, and 9 are all induced with similar kinetics suggesting that they could act redundantly to control shade-mediated elongation. To test this hypothesis we constructed a yucca2, 5, 8, 9 quadruple mutant and found that the hypocotyl and petiole EOD-FR and shade avoidance responses are completely disrupted. This work shows that YUCCA auxin biosynthetic genes are essential for detectable shade avoidance and that YUCCA genes are important for petiole shade avoidance.

  13. YUCCA auxin biosynthetic genes are required for Arabidopsis shade avoidance

    Science.gov (United States)

    Müller-Moulé, Patricia; Nozue, Kazunari; Pytlak, Melissa L.; Palmer, Christine M.; Covington, Michael F.; Wallace, Andreah D.; Harmer, Stacey L.

    2016-01-01

    Plants respond to neighbor shade by increasing stem and petiole elongation. Shade, sensed by phytochrome photoreceptors, causes stabilization of PHYTOCHROME INTERACTING FACTOR proteins and subsequent induction of YUCCA auxin biosynthetic genes. To investigate the role of YUCCA genes in phytochrome-mediated elongation, we examined auxin signaling kinetics after an end-of-day far-red (EOD-FR) light treatment, and found that an auxin responsive reporter is rapidly induced within 2 hours of far-red exposure. YUCCA2, 5, 8, and 9 are all induced with similar kinetics suggesting that they could act redundantly to control shade-mediated elongation. To test this hypothesis we constructed a yucca2, 5, 8, 9 quadruple mutant and found that the hypocotyl and petiole EOD-FR and shade avoidance responses are completely disrupted. This work shows that YUCCA auxin biosynthetic genes are essential for detectable shade avoidance and that YUCCA genes are important for petiole shade avoidance. PMID:27761349

  14. Hypoxia-induced miR-181b enhances angiogenesis of retinoblastoma cells by targeting PDCD10 and GATA6.

    Science.gov (United States)

    Xu, Xiaofang; Ge, Shengfang; Jia, Renbing; Zhou, Yixiong; Song, Xin; Zhang, He; Fan, Xianqun

    2015-06-01

    Previous findings showed that miR-181b is upregulated under hypoxic conditions in retinoblastoma cells. Since hypoxia is a common feature of retinoblastoma that affects tumor progression as well as tumor therapy, in the present study, we investigated the regulatory mechanism of miR-181b under hypoxic conditions, and examined the role of miR-181b in retinoblastoma responses to hypoxia (chemoresistance and angiogenesis) and possible downstream genes. The level of hypoxia-inducible factor-1α (HIF-1α) and miR-181b was detected to examine the link between them. Tube formation and cell cytotoxicity assays were used to clarify the effects of miR-181b on hypoxic responses of retinoblastoma cells. Bioinformatics analysis was performed to predict potential targets of miR-181b and western blotting was used to verify these targets. The results showed a significantly increased expression of HIF-1α in hypoxia-treated retinoblastoma cells. Downregulation of HIF-1α using a small-interfering RNA (siRNA) knockdown technology did not decrease the expression of miR-181b. Through gain- and loss-of-function studies, miR-181b was demonstrated to significantly stimulate the ability of capillary tube formation of endothelial cells. Programmed cell death-10 (PDCD10) and GATA binding protein 6 (GATA6) were identified as the target genes of miR‑181b. To the best of our knowledge, results of the present study provide the first evidence that miR-181b was upregulated by hypoxia in retinoblastoma in an HIF-1α-independent manner. miR-181b increased tumor angiogenesis of retinoblastoma cells. Additionally, miR-181b exerts its angiogenic function, at least in part, by inhibiting PDCD10 and GATA6. Thus, it is a new potentially useful therapeutic target for retinoblastoma.

  15. Haem-regulated eIF2α kinase is necessary for adaptive gene expression in erythroid precursors under the stress of iron deficiency

    Science.gov (United States)

    Liu, Sijin; Bhattacharya, Sanchita; Han, Anping; Suragani, Rajasekhar N. V. S.; Zhao, Wanting; Fry, Rebecca C.; Chen, Jane-Jane

    2016-01-01

    Summary Haem-regulated eIF2α kinase (HRI) is essential for the regulation of globin gene translation and the survival of erythroid precursors in iron/haem deficiency. This study found that that in iron deficiency, fetal definitive erythropoiesis is inhibited at the basophilic erythroblast stage with increased proliferation and elevated apoptosis. This hallmark of ineffective erythropoiesis is more severe in HRI deficiency. Microarray gene profiling analysis showed that HRI was required for adaptive gene expression in erythroid precursors during chronic iron deficiency. The number of genes with expression affected more than twofold increased, from 213 in iron deficiency and 73 in HRI deficiency, to 3135 in combined iron and HRI deficiencies. Many of these genes are regulated by Gata1 and Fog1. We demonstrate for the first time that Gata1 expression in developing erythroid precursors is decreased in iron deficiency, and is decreased further in combined iron and HRI deficiencies. Additionally, Fog1 expression is decreased in combined deficiencies, but not in iron or HRI deficiency alone. Our results indicate that HRI confers adaptive gene expression in developing erythroblasts during iron deficiency through maintaining Gata1/Fog1 expression. PMID:18665838

  16. GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling.

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    Margarita Marroquin-Guzman

    2015-04-01

    Full Text Available Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development.

  17. GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling.

    Science.gov (United States)

    Marroquin-Guzman, Margarita; Wilson, Richard A

    2015-04-01

    Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR) signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development.

  18. GATA-3 is involved in the development of serotonergic neurons in the caudal raphe nuclei

    NARCIS (Netherlands)

    J. van der Wees (Jacqueline); A. Karis (Alar); E. Goedknegt; M. Rutteman; F.G. Grosveld (Frank); J.H. van Doorninck (Hikke); C.I. de Zeeuw (Chris)

    1999-01-01

    textabstractAbstract The GATA-3 transcription factor shows a specific and restricted expression pattern in the developing and adult mouse brain. In the present study we investigated the role of GATA-3 in the caudal raphe system, which is known to operate as a modulator of motor activity. We demonst

  19. Temporal and Spatial Control of Murine GATA-3 Transcription by Promoter Proximal Regulatory Elements.

    NARCIS (Netherlands)

    K.H. Lieuw (Ken); G-L. Li (Guo); F.G. Grosveld (Frank); J.D. Engel (Douglas); Y. Zhou (Yanhua)

    1997-01-01

    textabstractGATA-3 is expressed in a temporally dynamic manner and fulfills vital functions during vertebrate fetal development. Homozygous mGATA-3 mutant embryos die at midgestation, thus complicating the analysis of its contribution to the development of specific cell fates in the many tissues whe

  20. GATA-3 and FOXA1 expression is useful to differentiate breast carcinoma from other carcinomas.

    Science.gov (United States)

    Davis, Drew G; Siddiqui, Momin T; Oprea-Ilies, Gabriela; Stevens, Keith; Osunkoya, Adeboye O; Cohen, Cynthia; Li, Xiaoxian Bill

    2016-01-01

    GATA-3, a member of the GATA family of zinc-finger DNA binding proteins, and FOXA1, a member of the forkhead transcription factor family, are both associated with estrogen receptor expression. Both GATA-3 and FOXA1 are useful markers for breast carcinoma, but their expression in the different breast cancer subtypes and other neoplasms has not been thoroughly evaluated. We examined the expression of GATA-3 and FOXA1 in estrogen receptor-positive, Her2/neu-positive, and triple-negative breast carcinomas as well as in 10 other common carcinomas, including hepatocellular, colonic, pancreatic, gastric, endometrial (endometrioid), lung, prostatic, renal cell, urothelial, and ovarian serous carcinomas. Primary and metastatic melanomas and mesotheliomas were also evaluated. GATA-3 and FOXA1 staining of estrogen receptor-positive breast carcinomas was seen in 96.6% and 96.2%, respectively. In triple-negative breast carcinomas, GATA-3 and FOXA1 staining was seen in 21.6% and 15.9%, respectively. Among the other tumors, GATA-3 staining was only seen in urothelial carcinoma (70.9%) and FOXA1 staining was only seen in prostatic (87.5%), urothelial (5.1%) carcinomas, and mesotheliomas (40.0%). In conclusion, GATA-3 and FOXA1 are excellent breast carcinoma markers; however, their utility is limited in the triple-negative subtype. The utility of FOXA1 in diagnosing prostatic carcinoma and mesothelioma warrants further investigation.

  1. Epigenetic regulation of GATA4 expression by histone modification in AFP-producing gastric adenocarcinoma.

    Science.gov (United States)

    Yamamura, Nobuhisa; Kishimoto, Takashi

    2012-08-01

    AFP-producing adenocarcinoma is a variant of adenocarcinoma with high malignancy. Production of AFP suggests enteroblastic or hepatoid differentiation of cancer cells. GATA4 is a key molecule involved in the prenatal development of the stomach and liver. GATA4 is epigenetically silenced by hypermethylation of primer region in many types of cancers including gastric cancer. The aim of this study is to investigate the expression and epigenetic regulation of GATA4 in AFP-producing adenocarcinoma. Immunohistochemical analysis revealed that GATA4 was positive in 3/8 cases of AFP-producing gastric adenocarcinomas and in 28/30 cases of common type adenocarcinomas. Epigenetic modification of GATA4 promoter region was investigated with 3 AFP-producing and 4 common-type gastric cancer cell lines. GATA4 mRNA was detected in 1/3 of AFP-producing and 2/4 of common-type gastric cancer cell lines by RT-PCR. Methylation-specific PCR revealed no GATA4 methylation in any of the AFP-producing gastric cancers, whereas methylation was consistent with GATA4 expression in the common-type gastric cancers. Chromatin immunoprecipitation assay for AFP-producing gastric cancers revealed that histones H3 and H4 were hypoacetylated in the GATA4-negative cells, while they were hyperacetylated in the GATA4-positive cells. Treatment with trichostain A, an inhibitor for histone deacetylase, induced acetylation of histones H3 and H4, and tri-methylation of lysine 4 of histone H3, which was associated with the active transcription of GATA4 in GATA4-negative AFP-producing cells. These results indicated that histone deacetylation is a silencing mechanism for GATA4 expression in AFP-producing gastric cancer cells. Differences between AFP-producing gastric cancer and common-type gastric cancer in terms of the mechanism of GATA4 regulation may be reflected in the phenotypic deviation of AFP-producing gastric cancer from common-type gastric cancer.

  2. Kzp Regulates the Transcription of gata2 and pu.1 during Primitive Hematopoiesis in Zebrafish Embryos

    Institute of Scientific and Technical Information of China (English)

    Fang Liu; Shaohua Yao; Ting Zhang; Chun Xiao; Yanna Shang; Jin Liu; Xianming Mo

    2012-01-01

    Kaiso zinc finger-containing protein (Kzp),a maternally-derived transcription factor,controls dorsoventral patterning during zebrafish gastrulation.Here,we uncovered a new function for Kzp in zebrafish embryonic primitive hematopoiesis.The depletion of kzp led to defects in primitive hematopoiesis including the development of the erythroid and myeloid lineages.On the other hand,overexpression of kzp caused the ectopic expression of gatal,gata2,and pu.1.Chromosome immunoprecipitation assays revealed that Kzp protein directly binds to gatal,gata2,and pu.1 promoters.Interestingly,the ectopic expression of gata2 was able to rescue the erythroid,but not the myeloid lineage in kzp-depleted zebrafish embryos.gatal expression controlled by Kzp was dependent on gata2 during primitive erythropoiesis.Our results indicate that Kzp is a critical transcriptional factor for the expression of gata2 and pu.1 to modulate primitive hematopoiesis.

  3. Exogenous Nkx2.5- or GATA-4-transfected rabbit bone marrow mesenchymal stem cells and myocardial cell co-culture on the treatment of myocardial infarction in rabbits.

    Science.gov (United States)

    Li, Pu; Zhang, Lei

    2015-08-01

    The present study aimed to investigate the effects of Nkx2.5 or GATA-4 transfection with myocardial extracellular environment co-culture on the transformation of bone marrow mesenchymal stem cells (BMSCs) into differentiated cardiomyocytes. Nkx2.5 or GATA-4 were transfected into myocardial extracellular environment co-cultured BMSCs, and then injected into the periphery of infarcted myocardium of a myocardial infarction rabbit model. The effects of these gene transfections and culture on the infarcted myocardium were observed and the results may provide an experimental basis for the efficient myocardial cell differentiation of BMSCs. The present study also suggested that these cells may provide a source and clinical basis for myocardial injury repair via stem cell transplantation. The present study examined whether Nkx2.5 or GATA-4 exogenous gene transfection with myocardial cell extracellular environment co-culture were able to induce the differentiation of BMSCs into cardiac cells. In addition, the effect of these transfected BMSCs on the repair of the myocardium following myocardial infarction was determined using New Zealand rabbit models. The results demonstrated that myocardial cell differentiation was significantly less effective following exogenous gene transfection of Nkx2.5 or GATA-4 alone compared with that of transfection in combination with extracellular environment co-culture. In addition, the results of the present study showed that exogenous gene transfection of Nkx2.5 or GATA-4 into myocardial cell extracellular environment co-cultured BMSCs was able to significantly enhance the ability to repair, mitigating the death of myocardial cells and activation of the myocardium in rabbits with myocardial infarction compared with those of the rabbits transplanted with untreated BMSCs. In conclusion, the exogenous Nkx2.5 and GATA-4 gene transfection into myocardial extracellular environment co-cultured BMSCs induced increased differentiation into myocardial

  4. Expression of zebrafish GATA 3 (gta3) during gastrulation and neurulation suggests a role in the specification of cell fate.

    Science.gov (United States)

    Neave, B; Rodaway, A; Wilson, S W; Patient, R; Holder, N

    1995-06-01

    In order to understand the role of the transcription factor GATA 3 in vertebrate development, we have examined its expression and some aspects of its regulation during gastrulation and neurulation in the zebrafish. The complete coding sequence of the cDNA encoding the zebrafish GATA 3 homologue, termed gta3, is described. Analysis of expression patterns by in situ hybridisation shows the gene to be expressed during gastrulation in the ventral region of the embryo which includes tissue fated to form the non-neural ectoderm. By the end of gastrulation, there is a clear border to the gta3 expression domain that is close to the edge of the neural plate. Subsequently, gta3 expresses in the pronephric duct and in defined regions of the central nervous system which include specific cells in each segment of the spinal cord and nuclei in the brain. Double labelling embryos with a probe for gta3 and antibodies which identify differentiated neurons suggest that gta3 is dynamically expressed during the early differentiation phase of a subset of neurons but not in the terminal phase. Analysis of gta3 expression in dorsalised embryos and in cyc and spt mutant embryos indicates that the neural expression of the gene is subject to control by signals from the mesoderm, including both the notochord and the somites, which influence the segmental organisation of expression in the spinal cord.

  5. VE-statin/egfl7 expression in endothelial cells is regulated by a distal enhancer and a proximal promoter under the direct control of Erg and GATA-2.

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    Alexandra Le Bras

    Full Text Available Angiogenesis is the process by which new blood vessels arise from existing ones by the budding out of endothelial cell capillaries from the luminal side of blood vessels. Blood vessel formation is essential for organ development during embryogenesis and is associated with several physiological and pathological processes, such as wound healing and tumor development. The VE-statin/egfl7 gene is specifically expressed in endothelial cells during embryonic development and in the adult. We studied here the regulatory mechanisms that control this tissue-specific expression. RT-qPCR analyses showed that the specificity of expression of VE-statin/egfl7 in endothelial cells is not shared with its closest neighbor genes notch1 and agpat2 on the mouse chromosome 2. Chromatin-immunoprecipitation analysis of histone modifications at the VE-statin/egfl7 locus showed that the chromatin is specifically opened in endothelial cells, but not in fibroblasts at the transcription start sites. A 13 kb genomic fragment of promoter was cloned and analyzed by gene reporter assays which showed that two conserved regions are important for the specific expression of VE-statin/egfl7 in endothelial cells; a -8409/-7563 enhancer and the -252/+38 region encompassing the exon-1b transcription start site. The latter contains essential GATA and ETS-binding sites, as assessed by linker-scanning analysis and site-directed mutagenesis. An analysis of expression of the ETS and GATA transcription factors showed that Erg, Fli-1 and GATA-2 are the most highly expressed factors in endothelial cells. Erg and GATA-2 directly control the expression of the endogenous VE-statin/egfl7 while Fli-1 probably exerts an indirect control, as assessed by RNA interference and chromatin immunoprecipitation. This first detailed analysis of the mechanisms that govern the expression of the VE-statin/egfl7 gene in endothelial cells pinpoints the specific importance of ETS and GATA factors in the specific

  6. GATA3 expression is decreased in psoriasis and during epidermal regeneration; induction by narrow-band UVB and IL-4.

    Science.gov (United States)

    Rácz, Emoke; Kurek, Dorota; Kant, Marius; Baerveldt, Ewout M; Florencia, Edwin; Mourits, Sabine; de Ridder, Dick; Laman, Jon D; van der Fits, Leslie; Prens, Errol P

    2011-01-01

    Psoriasis is characterized by hyperproliferation of keratinocytes and by infiltration of activated Th1 and Th17 cells in the (epi)dermis. By expression microarray, we previously found the GATA3 transcription factor significantly downregulated in lesional psoriatic skin. Since GATA3 serves as a key switch in both epidermal and T helper cell differentiation, we investigated its function in psoriasis. Because psoriatic skin inflammation shares many characteristics of epidermal regeneration during wound healing, we also studied GATA3 expression under such conditions.Psoriatic lesional skin showed decreased GATA3 mRNA and protein expression compared to non-lesional skin. GATA3 expression was also markedly decreased in inflamed skin of mice with a psoriasiform dermatitis induced with imiquimod. Tape-stripping of non-lesional skin of patients with psoriasis, a standardized psoriasis-triggering and skin regeneration-inducing technique, reduced the expression of GATA3. In wounded skin of mice, low GATA3 mRNA and protein expression was detected. Taken together, GATA3 expression is downregulated under regenerative and inflammatory hyperproliferative skin conditions. GATA3 expression could be re-induced by successful narrow-band UVB treatment of both human psoriasis and imiquimod-induced psoriasiform dermatitis in mice. The prototypic Th2 cytokine IL-4 was the only cytokine capable of inducing GATA3 in skin explants from healthy donors. Based on these findings we argue that GATA3 serves as a key regulator in psoriatic inflammation, keratinocyte hyperproliferation and skin barrier dysfunction.

  7. GATA-3 expression in the development of anterior chamber associated immune deviation

    Institute of Scientific and Technical Information of China (English)

    FU Tao; YANG Pei-zeng; HUANG Xiang-kun; HUANG Qiang; ZHOU Hong-yan; LI Bing; ZHONG Hua-hong; CHEN Xuan

    2005-01-01

    Background Anterior chamber associated immune deviation (ACAID) is characterized by a Th2 cell response. GATA-3 has been shown to be necessary for the activation of Th2 cells. This study was designed to examine the expression of GATA-3 in the development of ACAID. Methods ACAID was induced by injection of 50 μg interphotoreceptor retinoid binding protein (IRBP) into the anterior chamber (AC) of Wistar rats. Delayed-type hypersensitivity (DTH) was evaluated on day 3, 7, 14, 21, 28 after IRBP inoculation. GATA-3 expression was detected using immunohistochemical staining. The expression of GATA-3 mRNA at different time points after AC injection of IRBP was assayed by reverse transcriptase polymerase chain reaction (RT-PCR). Results A significant DTH reaction was observed in Wistar rats on day 3 and 5 after IRBP inoculation. The DTH reaction was decreased 7 days after IRBP inoculation. GATA-3 expression was weak at both mRNA and protein levels in the normal spleen, but was significantly increased on day 5, 7, 14, and 21 after AC injection of IRBP. Conclusion The expression of GATA-3 is increased during ACAID, suggesting that GATA-3 may be involved in the development of ACAID.

  8. SUMOylation regulates the transcriptional repression activity of FOG-2 and its association with GATA-4.

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    José Perdomo

    Full Text Available Friend of GATA 2 (FOG-2, a co-factor of several GATA transcription factors (GATA-4, -5 and 6, is a critical regulator of coronary vessel formation and heart morphogenesis. Here we demonstrate that FOG-2 is SUMOylated and that this modification modulates its transcriptional activity. FOG-2 SUMOylation occurs at four lysine residues (K324, 471, 915, 955 [corrected]. Three of these residues are part of the characteristic SUMO consensus site (ψKXE, while K955 is found in the less frequent TKXE motif. Absence of SUMOylation did not affect FOG-2's nuclear localization. However, mutation of the FOG-2 SUMOylation sites, or de-SUMOylation, with SENP-1 or SENP-8 resulted in stronger transcriptional repression activity in both heterologous cells and cardiomyocytes. Conversely, increased FOG-2 SUMOylation by overexpression of SUMO-1 or expression of a SUMO-1-FOG-2 fusion protein rendered FOG-2 incapable of repressing GATA-4-mediated activation of the B-type natriuretic peptide (BNP promoter. Moreover, we demonstrate both increased interaction between a FOG-2 SUMO mutant and GATA-4 and enhanced SUMOylation of wild-type FOG-2 by co-expression of GATA-4. These data suggest a new dynamics in which GATA-4 may alter the activity of FOG-2 by influencing its SUMOylation status.

  9. Identification of functional mutations in GATA4 in patients with congenital heart disease.

    Directory of Open Access Journals (Sweden)

    Erli Wang

    Full Text Available Congenital heart disease (CHD is one of the most prevalent developmental anomalies and the leading cause of noninfectious morbidity and mortality in newborns. Despite its prevalence and clinical significance, the etiology of CHD remains largely unknown. GATA4 is a highly conserved transcription factor that regulates a variety of physiological processes and has been extensively studied, particularly on its role in heart development. With the combination of TBX5 and MEF2C, GATA4 can reprogram postnatal fibroblasts into functional cardiomyocytes directly. In the past decade, a variety of GATA4 mutations were identified and these findings originally came from familial CHD pedigree studies. Given that familial and sporadic CHD cases allegedly share a basic genetic basis, we explore the GATA4 mutations in different types of CHD. In this study, via direct sequencing of the GATA4 coding region and exon-intron boundaries in 384 sporadic Chinese CHD patients, we identified 12 heterozygous non-synonymous mutations, among which 8 mutations were only found in CHD patients when compared with 957 controls. Six of these non-synonymous mutations have not been previously reported. Subsequent functional analyses revealed that the transcriptional activity, subcellular localization and DNA binding affinity of some mutant GATA4 proteins were significantly altered. Our results expand the spectrum of GATA4 mutations linked to cardiac defects. Together with the newly reported mutations, approximately 110 non-synonymous mutations have currently been identified in GATA4. Our future analysis will explore why the evolutionarily conserved GATA4 appears to be hypermutable.

  10. Promoter activity 5′ of Dbeta2 is coordinated by E47, Runx1, and GATA-3

    Science.gov (United States)

    McMillan, Ruth E.; Sikes, Michael L.

    2009-01-01

    V(D)J recombination involves the stepwise assembly of B and T cell receptor genes as lymphocytes progress through the early stages of development. While the mechanisms that restrict each step in recombination to its appropriate developmental stage are largely unknown, they share many of the components that regulate transcription. For example, enhancer-dependent modifications in histone acetylation and methylation are essential for both germline transcription and rearrangement of antigen receptor genes. Promoters positioned proximal to individual D and J gene segments in Tcra, Tcrb, Tcrd, IgH, and Igk also contribute to antigen receptor gene assembly, though their effects appear more localized than those of enhancers. Tcrb assembly initiates with D-to-J joining at each of two D-J-C gene segment clusters in DN1/2 thymocytes. DJ joints are fused with Vβ elements to complete Tcrb recombination in DN3 cells. We have previously shown that Dβ2 is flanked by upstream and downstream promoters, with the 5′ promoter being held inactive until D-to-J recombination deletes the NFκB-dependent 3′ promoter. We now report that activity of the 5′ promoter reflects a complex interplay between Runx1, GATA-3, and E47 transcription factors. In particular, while multiple E47 and Runx1 binding sites clustered near the Dβ2 5′RS and overlapping inr elements define the core 5′PDβ2, they act in concert with an array of upstream GATA-3 sites to overcome the inhibitory effects of a 110 bp distal polypurine·polypyrimidine (R·Y) tract. The dependence of 5′PDβ2 on E47 is consistent with the reported role of E proteins in post-DN1 thymocyte development and V-to-DJβ recombination. PMID:19592096

  11. Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

    Science.gov (United States)

    Oh, So-Young; Lunderberg, J. Mark; Chateau, Alice; Schneewind, Olaf

    2016-01-01

    ABSTRACT The secondary cell wall polysaccharide (SCWP) is thought to be essential for vegetative growth and surface (S)-layer assembly in Bacillus anthracis; however, the genetic determinants for the assembly of its trisaccharide repeat structure are not known. Here, we report that WpaA (BAS0847) and WpaB (BAS5274) share features with membrane proteins involved in the assembly of O-antigen lipopolysaccharide in Gram-negative bacteria and propose that WpaA and WpaB contribute to the assembly of the SCWP in B. anthracis. Vegetative forms of the B. anthracis wpaA mutant displayed increased lengths of cell chains, a cell separation defect that was attributed to mislocalization of the S-layer-associated murein hydrolases BslO, BslS, and BslT. The wpaB mutant was defective in vegetative replication during early logarithmic growth and formed smaller colonies. Deletion of both genes, wpaA and wpaB, did not yield viable bacilli, and when depleted of both wpaA and wpaB, B. anthracis could not maintain cell shape, support vegetative growth, or assemble SCWP. We propose that WpaA and WpaB fulfill overlapping glycosyltransferase functions of either polymerizing repeat units or transferring SCWP polymers to linkage units prior to LCP-mediated anchoring of the polysaccharide to peptidoglycan. IMPORTANCE The secondary cell wall polysaccharide (SCWP) is essential for Bacillus anthracis growth, cell shape, and division. SCWP is comprised of trisaccharide repeats (→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→) with α-Gal and β-Gal substitutions; however, the genetic determinants and enzymes for SCWP synthesis are not known. Here, we identify WpaA and WpaB and report that depletion of these factors affects vegetative growth, cell shape, and S-layer assembly. We hypothesize that WpaA and WpaB are involved in the assembly of SCWP prior to transfer of this polymer onto peptidoglycan. PMID:27795328

  12. Eomesodermin controls interleukin-5 production in memory T helper 2 cells through inhibition of activity of the transcription factor GATA3.

    Science.gov (United States)

    Endo, Yusuke; Iwamura, Chiaki; Kuwahara, Makoto; Suzuki, Akane; Sugaya, Kaoru; Tumes, Damon J; Tokoyoda, Koji; Hosokawa, Hiroyuki; Yamashita, Masakatsu; Nakayama, Toshinori

    2011-11-23

    The regulation of memory CD4(+) helper T (Th) cell function, such as polarized cytokine production, remains unclear. Here we show that memory T helper 2 (Th2) cells are divided into four subpopulations by CD62L and CXCR3 expression. All four subpopulations produced interleukin-4 (IL-4) and IL-13, whereas only the CD62L(lo)CXCR3(lo) population produced IL-5 accompanied by increased H3-K4 methylation at the Il5 gene locus. The transcription factor Eomesodermin (encoded by Eomes) was highly expressed in memory Th2 cells, whereas its expression was selectively downregulated in the IL-5-producing cells. Il5 expression was enhanced in Eomes-deficient cells, and Eomesodermin was shown to interact with the transcription factor GATA3, preventing GATA3 binding to the Il5 promoter. Memory Th2 cell-dependent airway inflammation was attenuated in the absence of the CD62L(lo)CXCR3(lo) population but was enhanced by Eomes-deficient memory Th2 cells. Thus, IL-5 production in memory Th2 cells is regulated by Eomesodermin via the inhibition of GATA3 activity.

  13. Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Harashima, S; Hinnebusch, A G

    1986-11-01

    GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

  14. Vaginal Smear TNF-alpha, IL18, TLR4, and GATA3 mRNA Levels Correlate with Local Inflammation

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    Olga Bourmenskaya

    2014-12-01

    Full Text Available Background: A molecular approach to estimation of local inflammatory response associated with dysbiotic conditions of the vagina is reported. This approach is based on immune response gene transcription levels measured by qPCR assay. Methods and Results: qPCR analysis of 24 immune response gene transcripts in vaginal smears was performed for 215 women with vaginitis and 95 healthy controls. The data were sorted by comparison to local inflammatory response profiles assessed by conventional methods. The local inflammatory response in the vagina is found to correlate with TNF-alpha, IL18, TLR4, and GATA3 transcript levels. Sensitivity and specificity of the approach, as validated in accordance with conventional clinical examination results, are 94.9% and 93.7%, respectively. Conclusions: The noninvasive diagnostic approach to vaginal pathology based solely on its molecular characterization may prove to be clinically relevant.

  15. Gene expression differences among three Neurospora species reveal genes required for sexual reproduction in Neurospora crassa.

    Science.gov (United States)

    Lehr, Nina A; Wang, Zheng; Li, Ning; Hewitt, David A; López-Giráldez, Francesc; Trail, Frances; Townsend, Jeffrey P

    2014-01-01

    Many fungi form complex three-dimensional fruiting bodies, within which the meiotic machinery for sexual spore production has been considered to be largely conserved over evolutionary time. Indeed, much of what we know about meiosis in plant and animal taxa has been deeply informed by studies of meiosis in Saccharomyces and Neurospora. Nevertheless, the genetic basis of fruiting body development and its regulation in relation to meiosis in fungi is barely known, even within the best studied multicellular fungal model Neurospora crassa. We characterized morphological development and genome-wide transcriptomics in the closely related species Neurospora crassa, Neurospora tetrasperma, and Neurospora discreta, across eight stages of sexual development. Despite diverse life histories within the genus, all three species produce vase-shaped perithecia. Transcriptome sequencing provided gene expression levels of orthologous genes among all three species. Expression of key meiosis genes and sporulation genes corresponded to known phenotypic and developmental differences among these Neurospora species during sexual development. We assembled a list of genes putatively relevant to the recent evolution of fruiting body development by sorting genes whose relative expression across developmental stages increased more in N. crassa relative to the other species. Then, in N. crassa, we characterized the phenotypes of fruiting bodies arising from crosses of homozygous knockout strains of the top genes. Eight N. crassa genes were found to be critical for the successful formation of perithecia. The absence of these genes in these crosses resulted in either no perithecium formation or in arrested development at an early stage. Our results provide insight into the genetic basis of Neurospora sexual reproduction, which is also of great importance with regard to other multicellular ascomycetes, including perithecium-forming pathogens, such as Claviceps purpurea, Ophiostoma ulmi, and

  16. Aberrant JAK/STAT Signaling Suppresses TFF1 and TFF2 through Epigenetic Silencing of GATA6 in Gastric Cancer

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    Cheng-Shyong Wu

    2016-09-01

    Full Text Available Aberrant Janus kinase (JAK/signal transducer and activator of transcription (STAT signaling is crucial to the development of gastric cancer. In this study, we examined the role of STAT3 in the expression and methylation of its targets in gastric cancer patients. Results from RNA sequencing identified an inverse correlation between the expression of STAT3 and GATA6 in 23 pairs of gastric cancer patient samples. We discovered that the expression of GATA6 is epigenetically silenced through promoter methylation in gastric cancer cell lines. Interestingly, the inhibition of STAT3 using a novel STAT3 inhibitor restored the expression of GATA6 and its targets, trefoil factors 1 and 2 (TFF1/2. Moreover, disruption of STAT3 binding to GATA6 promoter by small hairpin RNA restored GATA6 expression in AGS cells. A clinically significant correlation was also observed between the expression of GATA6 and TFF1/2 among tissue samples from 60 gastric cancer patients. Finally, bisulfite pyrosequencing revealed GATA6 methylation in 65% (39/60 of the patients, and those with higher GATA6 methylation tended to have shorter overall survival. In conclusion, we demonstrated that aberrant JAK/STAT signaling suppresses TFF1/2 partially through the epigenetic silencing of GATA6. Therapeutic intervention of STAT3 in reversing the epigenetic status of GATA6 could benefit the treatment of gastric cancer and is worthy of further investigation.

  17. Histone demethylase LSD1-mediated repression of GATA-2 is critical for erythroid differentiation

    Directory of Open Access Journals (Sweden)

    Guo Y

    2015-06-01

    Full Text Available Yidi Guo,1 Xueqi Fu,1,2 Yue Jin,1 Jing Sun,1 Ye Liu,1 Bo Huo,1 Xiang Li,1 Xin Hu1–31School of Life Sciences, Jilin University, 2Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, 3National Engineering Laboratory of AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, People’s Republic of ChinaBackground: The transcription factor GATA-2 is predominantly expressed in hematopoietic stem and progenitor cells and counteracts the erythroid-specific transcription factor GATA-1, to modulate the proliferation and differentiation of hematopoietic cells. During hematopoietic cell differentiation, GATA-2 exhibits dynamic expression patterns, which are regulated by multiple transcription factors.Methods: Stable LSD1-knockdown cell lines were established by growing murine erythroleukemia (MEL or mouse embryonic stem cells together with virus particles, in the presence of Polybrene® at 4 µg/mL, for 24–48 hours followed by puromycin selection (1 µg/mL for 2 weeks. Real-time polymerase chain reaction (PCR-based quantitative chromatin immunoprecipitation (ChIP analysis was used to test whether the TAL1 transcription factor is bound to 1S promoter in the GATA-2 locus or whether LSD1 colocalizes with TAL1 at the 1S promoter. The sequential ChIP assay was utilized to confirm the role of LSD1 in the regulation of H3K4me2 at the GATA-2 locus during erythroid differentiation. Western blot analysis was employed to detect the protein expression. The alamarBlue® assay was used to examine the proliferation of the cells, and the absorbance was monitored at optical density (OD 570 nm and OD 600 nm.Results: In this study, we showed that LSD1 regulates the expression of GATA-2 during erythroid differentiation. Knockdown of LSD1 results in increased GATA-2 expression and inhibits the differentiation of MEL and embryonic stem cells. Furthermore, we demonstrated that LSD1 binds to the 1S promoter of the

  18. Usefulness of GATA-3 as a marker of seminal epithelium in prostate biopsies.

    Science.gov (United States)

    Ortiz-Rey, J A; Chantada-de la Fuente, D; Peteiro-Cancelo, M Á; Gómez-de María, C; San Miguel-Fraile, M P

    2017-04-28

    The incidental presence of seminal vesicle epithelium in prostate needle biopsies is generally recognisable through routine microscopy. However, the biopsy can sometimes be erroneously interpreted as malignant due to its architectural and cytological characteristics, and immunohistochemistry can be useful for correctly identifying the biopsy. Our objective was to analyse the potential usefulness of GATA-3 as a marker of seminal epithelium. Through immunohistochemistry with a monoclonal anti-GATA-3 antibody (clone L50-823), we studied seminal vesicle sections from 20 prostatectomy specimens, 12 prostate needle biopsies that contained seminal vesicle tissue and 68 prostate biopsies without seminal vesicle epithelium, 36 of which showed adenocarcinoma. Staining for GATA-3 was intense in the 20 seminal vesicles of the prostatectomy specimens and in the 12 prostate needle biopsies that contained seminal epithelium. In the 60 biopsies without a seminal vesicle, GATA-3 was positive in the prostate basal cells and even in the secretory cells (57 cases), although with less intensity in 55 of the cases. One of the 36 prostatic adenocarcinomas tested positive for GATA-3. The intense immunohistochemical expression of GATA-3 in the seminal vesicle epithelium can help identify the epithelium in prostate biopsies. This marker is also positive in the basal cells of healthy prostates and, with less intensity, in the secretory cells. Positivity, weak or moderate, is observed on rare occasions in prostatic adenocarcinomas. Copyright © 2017 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.

  19. Anisomycin-induced GATA-6 degradation accompanying a decrease of proliferation of colorectal cancer cell

    Energy Technology Data Exchange (ETDEWEB)

    Ushijima, Hironori; Horyozaki, Akiko; Maeda, Masatomo, E-mail: mmaeda@nupals.ac.jp

    2016-09-09

    Transcription factor GATA-6 plays a key role in normal cell differentiation of the mesoderm and endoderm. On the other hand, GATA-6 is abnormally overexpressed in many clinical gastrointestinal cancer tissue samples, and accelerates cell proliferation or an anti-apoptotic response in cancerous tissues. We previously showed that activation of the JNK signaling cascade causes proteolysis of GATA-6. In this study, we demonstrated that anisomycin, a JNK activator, stimulates nuclear export of GATA-6 in a colorectal cancer cell line, DLD-1. Concomitantly, anisomycin remarkably inhibits the proliferation of DLD-1 cells via G2/M arrest in a plate culture. However, it did not induce apoptosis under growth arrest conditions. Furthermore, the growth of DLD-1 cells in a spheroid culture was suppressed by anisomycin. Although 5-FU showed only a slight inhibitory effect on 3D spheroid cultures, the same concentration of 5-FU together with a low concentration of anisomycin exhibited strong growth inhibition. These results suggest that the induction of GATA-6 dysfunction may be more effective for chemotherapy for colorectal cancer, although the mechanism underlying the synergistic effect of 5-FU and anisomycin remains unknown. - Highlights: • Anisomycin induces proteolysis of GATA-6 in DLD-1 cells. • Anisomycin remarkably inhibits the proliferation of DLD-1 cells via G2/M arrest. • Anisomycin suppresses the growth of spheroids of DLD-1, and enhances the effect of 5-FU.

  20. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Si, Lina; Shi, Jin; Gao, Wenqun [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Zheng, Min [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Liu, Lingjuan; Zhu, Jing [Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Tian, Jie, E-mail: jietian@cqmu.edu.cn [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China)

    2014-07-18

    Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of

  1. Regulation of GATA-binding protein 2 levels via ubiquitin-dependent degradation by Fbw7: involvement of cyclin B-cyclin-dependent kinase 1-mediated phosphorylation of THR176 in GATA-binding protein 2.

    Science.gov (United States)

    Nakajima, Tomomi; Kitagawa, Kyoko; Ohhata, Tatsuya; Sakai, Satoshi; Uchida, Chiharu; Shibata, Kiyoshi; Minegishi, Naoko; Yumimoto, Kanae; Nakayama, Keiichi I; Masumoto, Kazuma; Katou, Fuminori; Niida, Hiroyuki; Kitagawa, Masatoshi

    2015-04-17

    A GATA family transcription factor, GATA-binding protein 2 (GATA2), participates in cell growth and differentiation of various cells, such as hematopoietic stem cells. Although its expression level is controlled by transcriptional induction and proteolytic degradation, the responsible E3 ligase has not been identified. Here, we demonstrate that F-box/WD repeat-containing protein 7 (Fbw7/Fbxw7), a component of Skp1, Cullin 1, F-box-containing complex (SCF)-type E3 ligase, is an E3 ligase for GATA2. GATA2 contains a cell division control protein 4 (Cdc4) phosphodegron (CPD), a consensus motif for ubiquitylation by Fbw7, which includes Thr(176). Ectopic expression of Fbw7 destabilized GATA2 and promoted its proteasomal degradation. Substitution of threonine 176 to alanine in GATA2 inhibited binding with Fbw7, and the ubiquitylation and degradation of GATA2 by Fbw7 was suppressed. The CPD kinase, which mediates the phosphorylation of Thr(176), was cyclin B-cyclin-dependent kinase 1 (CDK1). Moreover, depletion of endogenous Fbw7 stabilized endogenous GATA2 in K562 cells. Conditional Fbw7 depletion in mice increased GATA2 levels in hematopoietic stem cells and myeloid progenitors at the early stage. Increased GATA2 levels in Fbw7-conditional knock-out mice were correlated with a decrease in a c-Kit high expressing population of myeloid progenitor cells. Our results suggest that Fbw7 is a bona fide E3 ubiquitin ligase for GATA2 in vivo.

  2. Requirements in screening cDNA libraries for new genes and solutions offered by SBH technology

    Energy Technology Data Exchange (ETDEWEB)

    Drmanac, R.; Drmanac, S.; Labat, I.; Stavropoulos, N.

    1993-12-31

    Under different assumptions about the total number of genes, the number of housekeeping and tissue-specific genes, and the difference in the number of mRNAs per cell for functional and nonfunctional genes, significantly different results can be expected from screening random cDNA clones. We have developed gene expression models as a guide for interpretation of experimental results. For statistical, biological, and technical reasons, the search for 100,000 plus genes and discrimination between nonfunctional, housekeeping, and tissue-specific genes requires the analysis of up to 10 million clones from 20 to 50 tissues. Oligonucleotide hybridization of dense clone blots is an inexpensive and fast way to screen such large clone sets. Our preliminary results on control clones and thousands of cDNA clones from an infant brain library demonstrate the feasibility of the method. We present several models of gene expression and analyze the main factors which can influence the hunt for new genes via the screening of random cDNA libraries. The basic steps in the preparation and use of dense DNA dot arrays are described, and some results that demonstrate the feasibility and efficiency of gene inventorying by oligonucleotide hybridization are presented. Furthermore, partial SBH and single-pass gel sequencing are compared and a gene analysis scheme that combines the two approaches is discussed.

  3. MED GATA factors promote robust development of the C. elegans endoderm

    Science.gov (United States)

    Maduro, Morris F.; Broitman-Maduro, Gina; Choi, Hailey; Carranza, Francisco; Wu, Allison Chia-Yi; Rifkin, Scott A.

    2015-01-01

    The MED-1,2 GATA factors contribute to specification of E, the progenitor of the C. elegans endoderm, through the genes end-1 and end-3, and in parallel with the maternal factors SKN-1, POP-1 and PAL-1. END-1,3 activate elt-2 and elt-7 to initiate a program of intestinal development, which is maintained by positive autoregulation. Here, we advance the understanding of MED-1,2 in E specification. We find that expression of end-1 and end-3 is greatly reduced in med-1,2(−) embryos. We generated strains in which MED sites have been mutated in end-1 and end-3. Without MED input, gut specification relies primarily on POP-1 and PAL-1. 25% of embryos fail to make intestine, while those that do display abnormal numbers of gut cells due to a delayed and stochastic acquisition of intestine fate. Surviving adults exhibit phenotypes consistent with a primary defect in the intestine. Our results establish that MED-1,2 provide robustness to endoderm specification through end-1 and end-3, and reveal that gut differentiation may be more directly linked to specification than previously appreciated. The results argue against an “all-or-none” description of cell specification, and suggest that activation of tissue-specific master regulators, even when expression of these is maintained by positive autoregulation, does not guarantee proper function of differentiated cells. PMID:25959238

  4. Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kawaguchi, Manami [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Kitajima, Kenji [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Kanokoda, Mai [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Suzuki, Hidenori [Division of Morphological and Biomolecular Research, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602 (Japan); Miyashita, Kazuya; Nakajima, Marino [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Nuriya, Hideko [Core Technology and Research Center, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Kasahara, Kohji [Laboratory of Biomembrane, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Hara, Takahiko, E-mail: hara-tk@igakuken.or.jp [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan)

    2016-06-03

    Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit{sup −}Tie2{sup −}CD41{sup +} Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41{sup +}CD42b{sup +}CD61{sup +} platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future. -- Highlights: •Megakaryocytes are efficiently induced by Gata2 from ESC-derived day 5 HECs. •Gata2-induced ESC-derived megakaryocytes are c-Kit{sup −}Tie2{sup −}CD41{sup +}. •Gata2-induced ESC-derived megakaryocytes produce larger discoid-shaped platelets. •Gata2-induced ESC-derived platelets bind fibrinogen upon thrombin stimulation.

  5. Novel frem1-related mouse phenotypes and evidence of genetic interactions with gata4 and slit3.

    Directory of Open Access Journals (Sweden)

    Tyler F Beck

    Full Text Available The FRAS1-related extracellular matrix 1 (FREM1 gene encodes an extracellular matrix protein that plays a critical role in the development of multiple organ systems. In humans, recessive mutations in FREM1 cause eye defects, congenital diaphragmatic hernia, renal anomalies and anorectal malformations including anteriorly placed anus. A similar constellation of findings-microphthalmia, cryptophthalmos, congenital diaphragmatic hernia, renal agenesis and rectal prolapse-have been described in FREM1-deficient mice. In this paper, we identify a homozygous Frem1 missense mutation (c.1687A>T, p.Ile563Phe in an N-ethyl-N-nitrosourea (ENU-derived mouse strain, crf11, with microphthalmia, cryptophthalmos, renal agenesis and rectal prolapse. This mutation affects a highly conserved residue in FREM1's third CSPG domain. The p.Ile563Phe change is predicted to be deleterious and to cause decreased FREM1 protein stability. The crf11 allele also fails to complement the previously described eyes2 allele of Frem1 (p.Lys826* providing further evidence that the crf11 phenotype is due to changes affecting Frem1 function. We then use mice bearing the crf11 and eyes2 alleles to identify lung lobulation defects and decreased anogenital distance in males as novel phenotypes associated with FREM1 deficiency in mice. Due to phenotypic overlaps between FREM1-deficient mice and mice that are deficient for the retinoic acid-responsive transcription factor GATA4 and the extracellular matrix protein SLIT3, we also perform experiments to look for in vivo genetic interactions between the genes that encode these proteins. These experiments reveal that Frem1 interacts genetically with Gata4 in the development of lung lobulation defects and with Slit3 in the development of renal agenesis. These results demonstrate that FREM1-deficient mice faithfully recapitulate many of the phenotypes seen in individuals with FREM1 deficiency and that variations in GATA4 and SLIT3 expression

  6. Hematopoietic transcription factor GATA-2 promotes upregulation of alpha globin and cell death in FL5.12 cells.

    Science.gov (United States)

    Brecht, K; Simonen, M; Kamke, M; Heim, J

    2005-10-01

    Recently we showed that alpha globin is a novel pro-apoptotic factor in programmed cell death in the pro-B cell line, FL5.12. Alpha globin was also upregulated in various other cell lines after different apoptotic stimuli. Under withdrawal of IL-3, overexpression of alpha globin accelerated apoptosis in FL5.12. Here, we have studied how transcription of alpha globin is placed in the broader context of apoptosis. We used Affymetrix chip technology and RT QPCR to compare expression patterns of FL5.12 cells growing with or without IL-3 to search for transcription factors which were concomitantly upregulated with alpha globin. The erythroid-specific transcription factor GATA-2 was the earliest and most prominently upregulated candidate. GATA-1 was expressed at low levels and was weakly induced while GATA-3 was completely absent. To evaluate the influence of GATA-2 on alpha globin expression and cell viability we overexpressed GATA-2 in FL5.12 cells. Interestingly, high expression of GATA-2 resulted in cell death and elevated alpha globin levels in FL5.12 cells. Transduction of antisense GATA-2 prevented both increase of GATA-2 and alpha globin under apoptotic conditions and delayed cell death. We suggest a role of GATA-2 in apoptosis besides its function in maintenance and proliferation of immature hematopoietic progenitors.

  7. Friend of GATA (FOG interacts with the nucleosome remodeling and deacetylase complex (NuRD to support primitive erythropoiesis in Xenopus laevis.

    Directory of Open Access Journals (Sweden)

    Mizuho S Mimoto

    Full Text Available Friend of GATA (FOG plays many diverse roles in adult and embryonic hematopoiesis, however the mechanisms by which it functions and the roles of potential interaction partners are not completely understood. Previous work has shown that overexpression of FOG in Xenopus laevis causes loss of blood suggesting that in contrast to its role in mammals, FOG might normally function to repress erythropoiesis in this species. Using loss-of-function analysis, we demonstrate that FOG is essential to support primitive red blood cell (RBC development in Xenopus. Moreover, we show that it is specifically required to prevent excess apoptosis of circulating primitive RBCs and that in the absence of FOG, the pro-apoptotic gene Bim-1 is strongly upregulated. To identify domains of FOG that are essential for blood development and, conversely, to begin to understand the mechanism by which overexpressed FOG represses primitive erythropoiesis, we asked whether FOG mutants that are unable to interact with known co-factors retain their ability to rescue blood formation in FOG morphants and whether they repress erythropoiesis when overexpressed in wild type embryos. We find that interaction of FOG with the Nucleosome Remodeling and Deacetylase complex (NuRD, but not with C-terminal Binding Protein, is essential for normal primitive RBC development. In contrast, overexpression of all mutant and wild type constructs causes a comparable repression of primitive erythropoiesis. Together, our data suggest that a requirement for FOG and its interaction with NuRD during primitive erythropoiesis are conserved in Xenopus and that loss of blood upon FOG overexpression is due to a dominant-interfering effect.

  8. Identification of genes required for nonhost resistance to Xanthomonas oryzae pv. oryzae reveals novel signaling components.

    Directory of Open Access Journals (Sweden)

    Wen Li

    Full Text Available BACKGROUND: Nonhost resistance is a generalized, durable, broad-spectrum resistance exhibited by plant species to a wide variety of microbial pathogens. Although nonhost resistance is an attractive breeding strategy, the molecular basis of this form of resistance remains unclear for many plant-microbe pathosystems, including interactions with the bacterial pathogen of rice, Xanthomonas oryzae pv. oryzae (Xoo. METHODS AND FINDINGS: Virus-induced gene silencing (VIGS and an assay to detect the hypersensitive response (HR were used to screen for genes required for nonhost resistance to Xoo in N. benthamiana. When infiltrated with Xoo strain YN-1, N. benthamiana plants exhibited a strong necrosis within 24 h and produced a large amount of H(2O(2 in the infiltrated area. Expression of HR- and defense-related genes was induced, whereas bacterial numbers dramatically decreased during necrosis. VIGS of 45 ACE (Avr/Cf-elicited genes revealed identified seven genes required for nonhost resistance to Xoo in N. benthamiana. The seven genes encoded a calreticulin protein (ACE35, an ERF transcriptional factor (ACE43, a novel Solanaceous protein (ACE80, a hydrolase (ACE117, a peroxidase (ACE175 and two proteins with unknown function (ACE95 and ACE112. The results indicate that oxidative burst and calcium-dependent signaling pathways play an important role in nonhost resistance to Xoo. VIGS analysis further revealed that ACE35, ACE80, ACE95 and ACE175, but not the other three ACE genes, interfered with the Cf-4/Avr4-dependent HR. CONCLUSIONS/SIGNIFICANCE: N. benthamiana plants inoculated with Xoo respond by rapidly eliciting an HR and nonhost resistance. The oxidative burst and other signaling pathways are pivotal in Xoo-N. benthamiana nonhost resistance, and genes involved in this response partially overlap with those involved in Cf/Avr4-dependent HR. The seven genes required for N. benthamiana-mediated resistance to Xoo provide a basis for further dissecting

  9. Identification of Drosophila gene products required for phagocytosis of Candida albicans.

    Directory of Open Access Journals (Sweden)

    Shannon L Stroschein-Stevenson

    2006-01-01

    Full Text Available Phagocytosis is a highly conserved aspect of innate immunity. We used Drosophila melanogaster S2 cells as a model system to study the phagocytosis of Candida albicans, the major fungal pathogen of humans, by screening an RNAi library representing 7,216 fly genes conserved among metazoans. After rescreening the initial genes identified and eliminating certain classes of housekeeping genes, we identified 184 genes required for efficient phagocytosis of C. albicans. Diverse biological processes are represented, with actin cytoskeleton regulation, vesicle transport, signaling, and transcriptional regulation being prominent. Secondary screens using Escherichia coli and latex beads revealed several genes specific for C. albicans phagocytosis. Characterization of one of those gene products, Macroglobulin complement related (Mcr, shows that it is secreted, that it binds specifically to the surface of C. albicans, and that it promotes its subsequent phagocytosis. Mcr is closely related to the four Drosophila thioester proteins (Teps, and we show that TepII is required for efficient phagocytosis of E. coli (but not C. albicans or Staphylococcus aureus and that TepIII is required for the efficient phagocytosis of S. aureus (but not C. albicans or E. coli. Thus, this family of fly proteins distinguishes different pathogens for subsequent phagocytosis.

  10. Gene expression induced by Toll-like receptors in macrophages requires the transcription factor NFAT5.

    Science.gov (United States)

    Buxadé, Maria; Lunazzi, Giulia; Minguillón, Jordi; Iborra, Salvador; Berga-Bolaños, Rosa; Del Val, Margarita; Aramburu, José; López-Rodríguez, Cristina

    2012-02-13

    Toll-like receptors (TLRs) engage networks of transcriptional regulators to induce genes essential for antimicrobial immunity. We report that NFAT5, previously characterized as an osmostress responsive factor, regulates the expression of multiple TLR-induced genes in macrophages independently of osmotic stress. NFAT5 was essential for the induction of the key antimicrobial gene Nos2 (inducible nitric oxide synthase [iNOS]) in response to low and high doses of TLR agonists but is required for Tnf and Il6 mainly under mild stimulatory conditions, indicating that NFAT5 could regulate specific gene patterns depending on pathogen burden intensity. NFAT5 exhibited two modes of association with target genes, as it was constitutively bound to Tnf and other genes regardless of TLR stimulation, whereas its recruitment to Nos2 or Il6 required TLR activation. Further analysis revealed that TLR-induced recruitment of NFAT5 to Nos2 was dependent on inhibitor of κB kinase (IKK) β activity and de novo protein synthesis, and was sensitive to histone deacetylases. In vivo, NFAT5 was necessary for effective immunity against Leishmania major, a parasite whose clearance requires TLRs and iNOS expression in macrophages. These findings identify NFAT5 as a novel regulator of mammalian anti-pathogen responses.

  11. Histomorfometria do corno uterino de gatas (Felis catus submetidas à ovariosalpingohisterectomia

    Directory of Open Access Journals (Sweden)

    Cristina Maria Rodrigues Monteiro

    2012-06-01

    Full Text Available Dados histomorfométricos foram obtidos de cornos uterinos de gatas nulíparas (n=6, primíparas (n= 6, multíparas (n=6 e de gatas tratadas com contraceptivo (n=6.O material coletado após a cirurgia foi fixado em paraformaldeído e incluído em resina Paraplast® para se proceder a microtomia. Os cortes adquiridos foram submetidos à coloração rotineira com hematoxilina e eosina e examinados ao microscópio de luz para a realização das seguintes mensurações:espessura total (μm da parede uterina, endométrio total, diâmetro das glândulas endometriais e altura dos respectivos epitélios glandulares, espessura total do miométrio, do miométrio interno,miométrio externo e estrato vascular.Concluiu-se que: uso de anticoncepcionais e inúmeras gestações alteraram a estrutura uterina; uma única gestação parece não afetar significativamente as estruturas da parede uterina como ocorre nas gatas multíparas; não houve variação significativa das estruturas entre as gatas nulíparas e primíparas, com exceção para miométrio interno; o miométrio total e o endométrio apresentaram variações semelhantes, com exceção para o grupo das gatas tratadas; o miométrio externo apresentou alterações mais marcantes nas gatas tratadas; as variações morfológicas menos marcantes foram relativas ao diâmetro e epitélio glandulares; a presença de glândulas endometriais dilatadas foi encontrada somente nas gatas tratadas.

  12. Four gene products are required for the fungal synthesis of the indole-diterpene, paspaline.

    Science.gov (United States)

    Saikia, Sanjay; Parker, Emily J; Koulman, Albert; Scott, Barry

    2006-03-06

    Paspaline belongs to a large, structurally and functionally diverse group of indole-diterpenes synthesized by filamentous fungi. However, the identity of the gene products required for the biosynthesis of paspaline, a key intermediate for the synthesis of paxilline and other indole-diterpenes, is not known. Transfer of constructs containing different pax gene combinations into a paxilline negative deletion derivative of Penicillium paxilli demonstrated that just four proteins, PaxG, a geranylgeranyl diphosphate synthase, PaxM, a FAD-dependent monooxygenase, PaxB, a putative membrane protein, and PaxC, a prenyl transferase, are required for the biosynthesis of paspaline.

  13. The expression levels of T -bet mRNA and GATA3 mRNA in peripheral blood of patients with preeclampsia%子痫前期患者外周血T - bet mRNA和GATA3mRNA的表达水平

    Institute of Scientific and Technical Information of China (English)

    李净; 杨海澜; 何银芳

    2011-01-01

    from peripheral blood samples of normal pregnant women and patients with preeclampsia, total RNA was abstracted, SYBR Green I fluorescence quantitative PCR was used in the study, β - actin was used as an internal control gene to detect the expression levels of T - bet Mrna and GATA3 Mrna. Results: The expression levels of GATA3 Mrna in normal pregnancy group, mild preeclampsia group and severe preeclampsia group decreased with the aggravation of the disease, there was significant difference between severe preeclampsia group and normal pregnancy group (P 0. 05) . The expression levels of T - bet Mrna increased with the aggravation of the disease, there was significant difference between severe preeclampsia group and normal pregnancy group, mild preeclampsia group (P 0. 05); the ratio of T - bet Mrna/GATA3 Mrna increased with the aggravation of the disease, and there was significant difference between severe preeclampsia group and normal pregnancy group (P 0. 05) . Conclusion: The expression of T-bet Mrna which plays an immune killing role in peripheral blood of patients with preeclampsia increases, while the expression of GAT A3 Mrna which plays immune protection or immune nutrition roles in peripheral blood of patients with preeclampsia decreases, which indicate that the high expression of T - bet Mrna in peripheral blood of patients with preeclampsia is related to occurrence of preeclampsia, the effect of GATA3 Mrna on occurrence and development of preeclampsia is not obvious as same as T - bet Mrna; the ratio of T - bet Mrna/GATA3 Mrna increases with the aggravation of the disease, which indicates that Th1l/ Th2 transcription factor imbalance may be one of the causes of preeclampsia.

  14. Regulation of carotenoid and bacteriochlorophyll biosynthesis genes and identification of an evolutionarily conserved gene required for bacteriochlorophyll accumulation.

    Science.gov (United States)

    Armstrong, G A; Cook, D N; Ma, D; Alberti, M; Burke, D H; Hearst, J E

    1993-05-01

    The temporal expression of ten clustered genes required for carotenoid (crt) and bacteriochlorophyll (bch) biosynthesis was examined during the transition from aerobic respiration to anaerobiosis requisite for the development of the photosynthetic membrane in the bacterium Rhodobacter capsulatus. Accumulation of crtA, crtC, crtD, crtE, crtF, crtK, bchC and bchD mRNAs increased transiently and coordinately, up to 12-fold following removal of oxygen from the growth medium, paralleling increases in mRNAs encoding pigment-binding polypeptides of the photosynthetic apparatus. The crtB and crtI genes, in contrast, were expressed similarly in the presence or absence of oxygen. The regulation patterns of promoters for the crtA and crtI genes and the bchCXYZ operon were characterized using lacZ transcriptional fusion and qualitatively reflected the corresponding mRNA accumulation patterns. We also report that the bchI gene product, encoded by a DNA sequence previously considered to be a portion of crtA, shares 49% sequence identity with the nuclear-encoded Arabidopsis thaliana Cs chloroplast protein required for normal pigmentation in plants.

  15. Reduced Leukocyte Infiltration in Absence of Eosinophils Correlates with Decreased Tissue Damage and Disease Susceptibility in ΔdblGATA Mice during Murine Neurocysticercosis

    Science.gov (United States)

    Mishra, Pramod K.; Li, Qun; Munoz, Luis E.; Mares, Chris A.; Morris, Elizabeth G.; Teale, Judy M.; Cardona, Astrid E.

    2016-01-01

    absence of eosinophils correlates with attenuated tissue damage and longer survival of ΔdblGATA mice. Therefore, our study suggests that approaches to clear NCC will require strategies to tightly control the host immune response while eradicating the parasite with minimal damage to brain tissue. PMID:27332553

  16. NHR-23 dependent collagen and hedgehog-related genes required for molting

    Energy Technology Data Exchange (ETDEWEB)

    Kouns, Nathaniel A.; Nakielna, Johana; Behensky, Frantisek [Laboratory of Model Systems, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague (Czech Republic); Krause, Michael W. [Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD (United States); Kostrouch, Zdenek [Laboratory of Model Systems, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague (Czech Republic); Kostrouchova, Marta, E-mail: marta.kostrouchova@lf1.cuni.cz [Laboratory of Model Systems, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague (Czech Republic)

    2011-10-07

    Highlights: {yields} NHR-23 is a critical regulator of nematode development and molting. {yields} The manuscript characterizes the loss-of-function phenotype of an nhr-23 mutant. {yields} Whole genome expression analysis identifies new potential targets of NHR-23. {yields} Hedgehog-related genes are identified as NHR-23 dependent genes. {yields} New link between sterol mediated signaling and regulation by NHR-23 is found. -- Abstract: NHR-23, a conserved member of the nuclear receptor family of transcription factors, is required for normal development in Caenorhabditis elegans where it plays a critical role in growth and molting. In a search for NHR-23 dependent genes, we performed whole genome comparative expression microarrays on both control and nhr-23 inhibited synchronized larvae. Genes that decreased in response to nhr-23 RNAi included several collagen genes. Unexpectedly, several hedgehog-related genes were also down-regulated after nhr-23 RNAi. A homozygous nhr-23 deletion allele was used to confirm the RNAi knockdown phenotypes and the changes in gene expression. Our results indicate that NHR-23 is a critical co-regulator of functionally linked genes involved in growth and molting and reveal evolutionary parallels among the ecdysozoa.

  17. Abnormal P-selectin localization during megakaryocyte development determines thrombosis in the gata1low model of myelofibrosis.

    Science.gov (United States)

    Zetterberg, Eva; Verrucci, Maria; Martelli, Fabrizio; Zingariello, Maria; Sancillo, Laura; D'Amore, Emanuela; Rana, Rosa Alba; Migliaccio, Anna Rita

    2014-01-01

    Patients with primary myelofibrosis have increased risk for bleeding and thrombosis. It is debated whether propensity to thrombosis is due to increased numbers of platelet microparticles and/or to pathological platelet-neutrophil interactions. Platelet neutrophil interactions are mediated by P-selectin and even though the megakaryocytes of myelofibrosis patients express normal levels of P-selectin, it remains abnormally localized to the demarcation membrane system rather than being assembled into the α-granules in platelets. Mice carrying the hypomorphic Gata1(low) mutation express the same megakaryocyte abnormalities presented by primary myelofibrosis patients, including abnormal P-selectin localization to the DMS and develop with age myelofibrosis, a disease that closely resembles human primary myelofibrosis. Whether these mice would also develop thrombosis has not been investigated as yet. The aim of this study was to determine whether Gata1(low) mice would develop thrombosis with age and, in this case, the role played by P-selectin in the development of the trait. To this aim, Gata1(low) mice were crossed with P-sel(null) mice according to standard genetic protocols and Gata1(low)P-sel(wt), Gata1(low)P-sel(null) and Gata1(WT)P-sel(null) or Gata1(wt)P-sel(wt) (as controls) littermates obtained. It was shown that platelet counts, but not hematocrit, are reduced in Gata1(low) mice. Moreover, platelet microparticles are reduced in Gata1(low) mice and P-selectin positive platelet microparticles were not found. To determine the phenotypic implications of the different mutations, bleeding time was estimated by a tail cut procedure. Mutant mice were sacrificed and presence of thrombosis was determined by immunohistological staining of organs. Gata1(low) mice with or without the P-selectin null trait had a prolonged bleeding time compared to wild type mice. However, in Gata1(low) mice significantly higher frequency of thrombotic events was seen in adult and old Gata1

  18. GATA3 Is a Sensitive and Specific Marker of Benign and Malignant Mesonephric Lesions in the Lower Female Genital Tract.

    Science.gov (United States)

    Howitt, Brooke E; Emori, Megan M; Drapkin, Ronny; Gaspar, Cynthia; Barletta, Justine A; Nucci, Marisa R; McCluggage, W Glenn; Oliva, Esther; Hirsch, Michelle S

    2015-10-01

    GATA3 is a transcription factor critical for embryogenesis, development, and cell differentiation. Recent studies have suggested that GATA3 is a sensitive and relatively specific biomarker for urothelial and breast carcinomas, with most Müllerian carcinomas being negative. We investigated GATA3 expression in mesonephric/Wolffian remnants and tumors in the female genital tract. A western blot was performed to assess specificity for the GATA3 antibody. GATA3 immunohistochemistry was performed on 59 formalin-fixed paraffin-embedded mesonephric samples, including 17 mesonephric remnants (MR; 11 cervical and 6 fallopian tube), 15 mesonephric hyperplasias, 21 mesonephric carcinomas, and 6 female adnexal tumors of probable Wolffian origin. Thirty conventional endocervical adenocarcinomas (ENDO-CA), 9 gastric-type cervical adenocarcinomas, and 165 endometrial adenocarcinomas (EM-CA) were also evaluated. GATA3 nuclear intensity and extent of staining was evaluated. The western blot revealed GATA3 expression in seminal vesicle and cell lines derived from breast and urothelial carcinomas, but not in other cell lines including ovarian, cervical, and endometrial cancers. All cervical MRs and mesonephric hyperplasias, 5/6 (83%) fallopian tube MRs, and 20/21 (95%) mesonephric carcinomas were GATA3 positive, although with great variability in both intensity (weak to strong) and extent (1+ to 3+) of staining. Only 1/6 (17%) female adnexal tumors of probable Wolffian origin showed weak multifocal staining. One of 30 (3%) usual-type ENDO-CAs and 3/165 EM-CAs exhibited weak-moderate GATA3 immunoreactivity; all gastric-type cervical adenocarcinomas were negative. GATA3 is a highly sensitive and specific marker for mesonephric lesions in the lower genital tract; however, its utility in the upper genital tract may be more limited. In addition, GATA3 can aid in distinguishing lower genital mesonephric lesions from usual-type and gastric-type ENDO-CAs and uterine EM-CAs.

  19. Systematic yeast synthetic lethal and synthetic dosage lethal screens identify genes required for chromosome segregation

    OpenAIRE

    Measday, Vivien; Baetz, Kristin; Guzzo, Julie; Yuen, Karen; Kwok, Teresa; Sheikh, Bilal; Ding, Huiming; Ueta, Ryo; Hoac, Trinh; Cheng, Benjamin; Pot, Isabelle; Tong, Amy; Yamaguchi-Iwai, Yuko; Boone, Charles; Hieter, Phil

    2005-01-01

    Accurate chromosome segregation requires the execution and coordination of many processes during mitosis, including DNA replication, sister chromatid cohesion, and attachment of chromosomes to spindle microtubules via the kinetochore complex. Additional pathways are likely involved because faithful chromosome segregation also requires proteins that are not physically associated with the chromosome. Using kinetochore mutants as a starting point, we have identified genes with roles in chromosom...

  20. Interferon regulatory factor 1 is required for mouse Gbp gene activation by gamma interferon.

    OpenAIRE

    1995-01-01

    Full-scale transcriptional activation of the mouse Gbp genes by gamma interferon (IFN-gamma) requires protein synthesis in embryonic fibroblasts. Although the Gbp-1 and Gbp-2 promoters contain binding sites for transcription factors Stat1 and IFN regulatory factor 1 (IRF-1), deletion analysis revealed that the Stat1 binding site is dispensable for IFN-gamma inducibility of Gbp promoter constructs in transfected fibroblasts. However, activation of the mouse Gbp promoter by IFN-gamma requires t...

  1. Analysis of transcription regulatory regions of embryonic chicken pepsinogen (ECPg) gene.

    Science.gov (United States)

    Watanuki, Kumiko; Yasugi, Sadao

    2003-09-01

    Genes encoding pepsinogens, zymogens of digestive enzyme pepsins, are expressed specifically in the gland epithelial cells of the vertebrate stomach, and their expression is also developmentally regulated, therefore providing a good model for the analysis of transcriptional regulation of genes. In the development of chicken embryonic stomach, the epithelium invaginates into the mesenchyme and forms glands and gland epithelial cells then begin to express embryonic chicken pepsinogen (ECPg) gene. It has been shown that cGATA5 binds directly GATA binding sites located within 1.1-kbp upstream of ECPg gene and activates its transcription. To find more precisely the sequences necessary for ECPg gene transcription, we carried out deletion and mutation analysis with 1.1-kbp upstream region. The results suggest that binding of GATA factor to three GATA binding sites within the upstream region -656 to -419 synergistically regulates ECPg expression in the gland epithelial cells.

  2. GATA transcription factors as tissue-specific master regulators for induced responses.

    Science.gov (United States)

    Block, Dena Hs; Shapira, Michael

    2015-01-01

    GATA transcription factors play important roles in directing developmental genetic programs and cell differentiation, and are conserved in animals, plants and fungi. C. elegans has 11 GATA-type transcription factors that orchestrate development of the gut, epidermis and vulva. However, the expression of certain GATA proteins persists into adulthood, where their function is less understood. Accumulating evidence demonstrates contributions of 2 terminal differentiation GATA transcription factors, ELT-2 and ELT-3, to epithelial immune responses in the adult intestine and epidermis (hypodermis), respectively. Involvement in other stress responses has also been documented. We recently showed that ELT-2 acted as a tissue-specific master regulator, cooperating with 2 transcription factors activated by the p38 pathway, ATF-7 and SKN-1, to control immune responses in the adult C. elegans intestine. Here, we discuss the broader implications of these findings for understanding the involvement of GATA transcription factors in adult stress responses, and draw parallels between ELT-2 and ELT-3 to speculate that the latter may fulfill similar tissue-specific functions in the epidermis.

  3. Cloning and characterization of peter pan, a novel Drosophila gene required for larval growth.

    Science.gov (United States)

    Migeon, J C; Garfinkel, M S; Edgar, B A

    1999-06-01

    We identified a new Drosophila gene, peter pan (ppan), in a screen for larval growth-defective mutants. ppan mutant larvae do not grow and show minimal DNA replication but can survive until well after their heterozygotic siblings have pupariated. We cloned the ppan gene by P-element plasmid rescue. ppan belongs to a highly conserved gene family that includes Saccharomyces cerevisiae SSF1 and SSF2, as well as Schizosaccharomyces pombe, Arabidopsis, Caenorhabditis elegans, mouse, and human homologues. Deletion of both SSF1 and SSF2 in yeast is lethal, and depletion of the gene products causes cell division arrest. Mosaic analysis of ppan mutant clones in Drosophila imaginal disks and ovaries demonstrates that ppan is cell autonomous and required for normal mitotic growth but is not absolutely required for general biosynthesis or DNA replication. Overexpression of the wild-type gene causes cell death and disrupts the normal development of adult structures. The ppan gene family appears to have an essential and evolutionarily conserved role in cell growth.

  4. Genes required for the common miracle of fertilization in Caenorhabditis elegans.

    Science.gov (United States)

    Singson, Andrew; Hang, Julie S; Parry, Jean M

    2008-01-01

    Fertilization involves multiple layers of sperm-egg interactions that lead to gamete fusion and egg activation. There must be specific molecules required for these interactions. The challenge is to determine the identity of the genes encoding these molecules and how their protein products function. The nematode worm Caenorhabditis elegans has emerged as an efficient model system for gene discovery and understanding the molecular mechanisms of fertilization. The primary advantage of the C. elegans system is the ability to isolate and maintain mutants that affect sperm or eggs and no other cells. In this review we describe progress and challenges in the analysis of genes required for gamete interactions and egg activation in the worm.

  5. The study on the role of transcription factor GATA binding protein 3 in familial Alzheimer's disease pathogenesis%转录因子GATA结合蛋白3在家族性阿尔茨海默病发病机制中的作用

    Institute of Scientific and Technical Information of China (English)

    秦伟; 周爱红; 左秀美; 王芬; 程哲; 贾建平

    2011-01-01

    Objective To investigate the mechanisms of decreasing insulin degrading enzyme (IDE) level by mutation V97L in the gene presenilin 1 (PS-1).Methods Transcription factor GATA binding protein 3 (GATA-3) activity was assessed by protein/DNA array and verified by Western blot in SH-SY5Y cells transfected by PS-1 mutation V97L.Results Protein/DNA array and Western blot revealed that there was increased transcript factor activity (5.5 times high) and protein level of GATA-3 in V97L-PS-1 transfected SH-SY5Y cells.Transcription factor GATA-3 can bind to the IDE promoter and negatively control the IDE transcription level.Conclusion PS-1 mutation V97L may regulate the transcription of IDE via GATA-3, and subsequently involve in deposition of Aβ42 and development of Alzheimer's disease.%目的 探讨家族性阿尔茨海默病(Alzheimer's disease,AD)相关的早老素1(presenilin-1,PS-1)V97L突变引起胰岛素降解酶(insulin degrading enzyme,IDE)降低的机制.方法 应用转录因子活性芯片技术检测转录因子活性的差异,再用Western b1ot法验证芯片结果.结果 芯片结合MatInspector软件分析发现,上调了5.5倍的转录因子GATA结合蛋白3(GATA binding protein 3,GATA-3)在IDE启动子上有结合位点,并对IDE起负性调控作用.Westem b1ot也证实稳定转染PS-1 V97L细胞系中GATA-3蛋白表达明显增高.结论 PS-1 V97L突变可能通过上调转录因子GATA-3抑制IDE的表达,影响β-淀粉样蛋白42含量进而参与AD的发生.

  6. TRAUCO, a Trithorax-group gene homologue, is required for early embryogenesis in Arabidopsis thaliana.

    Science.gov (United States)

    Aquea, Felipe; Johnston, Amal J; Cañon, Paola; Grossniklaus, Ueli; Arce-Johnson, Patricio

    2010-02-01

    Embryogenesis is a critical stage during the plant life cycle in which a unicellular zygote develops into a multicellular organism. Co-ordinated gene expression is thus necessary for proper embryo development. Polycomb and Trithorax group genes are members of evolutionarily conserved machinery that maintains the correct expression patterns of key developmental regulators by repressing and activating gene transcription. TRAUCO (TRO), a gene homologous to the Trithorax group of genes that can functionally complement a BRE2P yeast mutant, has been identified in Arabidopsis thaliana. It is demonstrated that TRO is a nuclear gene product expressed during embryogenesis, and loss of TRO function leads to impaired early embryo development. Embryos that arrested at the globular stage in the tro-1 mutant allele were fully rescued by a TRO expression clone, a demonstration that the tro-1 mutation is a true loss-of-function in TRO. Our data have established that TRO is the first trithorax-group gene homologue in plants that is required for early embryogenesis.

  7. Evaluation of the effect of genetic variations in GATA-4 on the phenprocoumon and acenocoumarol maintenance dose

    NARCIS (Netherlands)

    Schie, R.M. van; Wessels, J.A.M.; Verhoef, T.I.; Schalekamp, T.; Cessie, S. le; Meer, F.J. van der; Rosendaal, F.R.; Visser, L.E.; Teichert, M.; Hofman, A.; Buhre, P.N.; Boer, A. de; Maitland-van der Zee, A.H.

    2012-01-01

    Aim: To investigate whether the phenprocoumon and acenocoumarol maintenance doses are influenced by genetic variations in GATA-4, a transcription factor of CYP2C9. Patients & methods: The influence of seven GATA-4 SNPs on the coumarin maintenance dose was investigated by performing an analysis of va

  8. GATA3 Expression Is Decreased in Psoriasis and during Epidermal Regeneration: Induction by Narrow-Band UVB and IL-4

    NARCIS (Netherlands)

    Rácz, E.; Kurek, D.; Kant, M.; Baerveldt, E.M.; Florencia, E.; Mourits, S.; De Ridder, D.; Laman, J.D.; Van der Fits, L.; Prens, E.P.

    2011-01-01

    Psoriasis is characterized by hyperproliferation of keratinocytes and by infiltration of activated Th1 and Th17 cells in the (epi)dermis. By expression microarray, we previously found the GATA3 transcription factor significantly downregulated in lesional psoriatic skin. Since GATA3 serves as a key s

  9. GATA3 expression is decreased in psoriasis and during epidermal regeneration; induction by narrow-band UVB and IL-4

    NARCIS (Netherlands)

    E. Rácz (Emőke); D. Kurek (Dorota); M. Kant (Marius); E.M. Baerveldt (Ewout M.); E. Florencia (Edwin); S. Mourits (Sabine); D. de Ridder (Dick); J.D. Laman (Jon); L. van der Fits (Leslie); E.P. Prens (Errol)

    2011-01-01

    textabstractPsoriasis is characterized by hyperproliferation of keratinocytes and by infiltration of activated Th1 and Th17 cells in the (epi)dermis. By expression microarray, we previously found the GATA3 transcription factor significantly downregulated in lesional psoriatic skin. Since GATA3 serve

  10. The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development

    NARCIS (Netherlands)

    R. Keijzer (Richard); W.G. van Tuyl (Minke); C. Meijers (Carel); M.R. Post (Martin); D. Tibboel (Dick); M. Koutsourakis (Manousos); F.G. Grosveld (Frank)

    2001-01-01

    textabstractRecent loss-of-function studies in mice show that the transcription factor GATA6 is important for visceral endoderm differentiation. It is also expressed in early bronchial epithelium and the observation that this tissue does not receive any contribution from Gata6 double mutant embryoni

  11. Loss of c-Kit and bone marrow failure upon conditional removal of the GATA-2 C-terminal zinc finger domain in adult mice.

    Science.gov (United States)

    Li, Haiyan S; Jin, Jin; Liang, Xiaoxuan; Matatall, Katie A; Ma, Ying; Zhang, Huiyuan; Ullrich, Stephen E; King, Katherine Y; Sun, Shao-Cong; Watowich, Stephanie S

    2016-09-01

    Heterozygous mutations in the transcriptional regulator GATA-2 associate with multilineage immunodeficiency, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML). The majority of these mutations localize in the zinc finger (ZnF) domains, which mediate GATA-2 DNA binding. Deregulated hematopoiesis with GATA-2 mutation frequently develops in adulthood, yet GATA-2 function in the bone marrow remains unresolved. To investigate this, we conditionally deleted the GATA-2 C-terminal ZnF (C-ZnF) coding sequences in adult mice. Upon Gata2 C-ZnF deletion, we observed rapid peripheral cytopenia, bone marrow failure, and decreased c-Kit expression on hematopoietic progenitors. Transplant studies indicated GATA-2 has a cell-autonomous role in bone marrow hematopoiesis. Moreover, myeloid lineage populations were particularly sensitive to Gata2 hemizygosity, while molecular assays indicated GATA-2 regulates c-Kit expression in multilineage progenitor cells. Enforced c-Kit expression in Gata2 C-ZnF-deficient hematopoietic progenitors enhanced myeloid colony activity, suggesting GATA-2 sustains myelopoiesis via a cell intrinsic role involving maintenance of c-Kit expression. Our results provide insight into mechanisms regulating hematopoiesis in bone marrow and may contribute to a better understanding of immunodeficiency and bone marrow failure associated with GATA-2 mutation.

  12. A Targetable GATA2-IGF2 Axis Confers Aggressiveness in Lethal Prostate Cancer

    Science.gov (United States)

    Vidal, Samuel J.; Rodriguez-Bravo, Veronica; Quinn, S. Aidan; Rodriguez-Barrueco, Ruth; Lujambio, Amaia; Williams, Estrelania; Sun, Xiaochen; de la Iglesia-Vicente, Janis; Lee, Albert; Readhead, Ben; Chen, Xintong; Galsky, Matthew; Esteve, Berta; Petrylak, Daniel P.; Dudley, Joel T.; Rabadan, Raul; Silva, Jose M.; Hoshida, Yujin; Lowe, Scott W.; Cordon-Cardo, Carlos; Domingo-Domenech, Josep

    2015-01-01

    SUMMARY Elucidating the determinants of aggressiveness in lethal prostate cancer may stimulate therapeutic strategies that improve clinical outcomes. We used experimental models and clinical databases to identify GATA2 as a regulator of chemotherapy resistance and tumorigenicity in this context. Mechanistically, direct upregulation of the growth hormone IGF2 emerged as a mediator of the aggressive properties regulated by GATA2. IGF2 in turn activated IGF1R and INSR as well as a downstream polykinase program. The characterization of this axis prompted a combination strategy whereby dual IGF1R/INSR inhibition restored the efficacy of chemotherapy and improved survival in preclinical models. These studies reveal a GATA2-IGF2 aggressiveness axis in lethal prostate cancer and identify a therapeutic opportunity in this challenging disease. PMID:25670080

  13. Dynamic regulatory interactions of Polycomb group genes: MEDEA autoregulation is required for imprinted gene expression in Arabidopsis.

    Science.gov (United States)

    Baroux, Célia; Gagliardini, Valeria; Page, Damian R; Grossniklaus, Ueli

    2006-05-01

    The imprinted Arabidopsis Polycomb group (PcG) gene MEDEA (MEA), which is homologous to Enhancer of Zeste [E(Z)], is maternally required for normal seed development. Here we show that, unlike known mammalian imprinted genes, MEA regulates its own imprinted expression: It down-regulates the maternal allele around fertilization and maintains the paternal allele silent later during seed development. Autorepression of the maternal MEA allele is direct and independent of the MEA-FIE (FERTILIZATION-INDEPENDENT ENDOSPERM) PcG complex, which is similar to the E(Z)-ESC (Extra sex combs) complex of animals, suggesting a novel mechanism. A complex network of cross-regulatory interactions among the other known members of the MEA-FIE PcG complex implies distinct functions that are dynamically regulated during reproduction.

  14. Identification of genes specifically required for the anaerobic metabolism of benzene in Geobacter metallireducens

    Directory of Open Access Journals (Sweden)

    Tian eZhang

    2014-05-01

    Full Text Available Although the biochemical pathways for the anaerobic degradation of many of the hydrocarbon constituents in petroleum reservoirs have been elucidated, the mechanisms for anaerobic activation of benzene, a very stable molecule, are not known. Previous studies have demonstrated that Geobacter metallireducens can anaerobically oxidize benzene to carbon dioxide with Fe(III as the sole electron acceptor and that phenol is an intermediate in benzene oxidation. In an attempt to identify enzymes that might be involved in the conversion of benzene to phenol, whole-genome gene transcript abundance was compared in cells metabolizing benzene and cells metabolizing phenol. Eleven genes had significantly higher transcript abundance in benzene-metabolizing cells. Five of these genes had annotations suggesting that they did not encode proteins that could be involved in benzene metabolism and were not further studied. Strains were constructed in which one of the remaining six genes was deleted. The strain in which the monocistronic gene Gmet 0232 was deleted metabolized phenol, but not benzene. Transcript abundance of the adjacent monocistronic gene, Gmet 0231, predicted to encode a zinc-containing oxidoreductase, was elevated in cells metabolizing benzene, although not at a statistically significant level. However, deleting Gmet 0231 also yielded a strain that could metabolize phenol, but not benzene. Although homologs of Gmet 0231 and Gmet 0232 are found in microorganisms not known to anaerobically metabolize benzene, the adjacent localization of these genes is unique to G. metallireducens. The discovery of genes that are specifically required for the metabolism of benzene, but not phenol in G. metallireducens is an important step in potentially identifying the mechanisms for anaerobic benzene activation.

  15. CRISPR/Cas9-Directed Reassignment of the GATA1 Initiation Codon in K562 Cells to Recapitulate AML in Down Syndrome

    Directory of Open Access Journals (Sweden)

    Kevin M. Bloh

    2017-06-01

    Full Text Available Using a CRISPR/Cas9 system, we have reengineered a translational start site in the GATA1 gene in K562 cells. This mutation accounts largely for the onset of myeloid leukemia in Down syndrome (ML-DS. For this reengineering, we utilized CRISPR/Cas9 to generate mammalian cell lines that express truncated versions of the Gata1s protein similar to that seen in ML-DS, as determined by analyzing specific genetic alterations resulting from CRISPR/Cas9 cleavage. During this work, 73 cell lines were clonally expanded, with allelic variance analyzed. Using Tracking of Indels by DEcomposition (TIDE and Sanger sequencing, we defined the DNA sequence and variations within each allele. We found significant heterogeneity between alleles in the same clonally expanded cell, as well as among alleles from other clonal expansions. Our data demonstrate and highlight the importance of the randomness of resection promoted by non-homologous end joining after CRISPR/Cas9 cleavage in cells undergoing genetic reengineering. Such heterogeneity must be fully characterized to predict altered functionality inside target tissues and to accurately interpret the associated phenotype. Our data suggest that in cases where the objective is to rearrange specific nucleotides to redirect gene expression in human cells, it is imperative to analyze genetic composition at the individual allelic level.

  16. A zinc transporter gene required for development of the nervous system.

    Science.gov (United States)

    Chowanadisai, Winyoo; Graham, David M; Keen, Carl L; Rucker, Robert B; Messerli, Mark A

    2013-11-01

    The essentiality of zinc for normal brain development is well established. It has been suggested that primary and secondary zinc deficiencies can contribute to the occurrence of numerous human birth defects, including many involving the central nervous system. In a recent study, we searched for zinc transporter genes that were critical for neurodevelopment. We confirmed that ZIP12 is a zinc transporter encoded by the gene slc39a12 that is highly expressed in the central nervous systems of human, mouse, and frog (Xenopus tropicalis).Using loss-of-function methods, we determined that ZIP12 is required for neuronal differentiation and neurite outgrowth and necessary for neurulation and embryonic viability. These results highlight an essential need for zinc regulation during embryogenesis and nervous system development. We suggest that slc39a12 is a candidate gene for inherited neurodevelopmental defects in humans.

  17. Requirement of histone deacetylase activity for the expression of critical photoreceptor genes

    Directory of Open Access Journals (Sweden)

    Cepko Constance L

    2007-06-01

    Full Text Available Abstract Background Histone deacetylases (HDACs play a major role in the regulation of gene transcription, often leading to transcriptional repression, as well as other effects following deacetylation of non-histone proteins. Results To investigate the role of HDACs in the developing mammalian retina, a general inhibitor of HDACs, trichostatin-A (TSA, was used to treat newborn murine retinae in explant cultures. Inhibition of HDAC activity resulted in a reduction in RNA levels for genes that regulate retinal development, as well as cell cycle regulators. Several of the genes encode transcription factors essential for rod photoreceptor development, Otx2, Nrl, and Crx. Using luciferase reporter assays, the promoter activity of both Nrl and Crx was found to be compromised by HDAC inhibition. Furthermore, downregulation of gene expression by HDAC inhibition didn't require de novo protein synthesis, and was associated with hyperacetylation of histones and non-histone proteins. Finally, HDAC inhibition in retinal explant cultures resulted in increased cell death, reduction in proliferation, a complete loss of rod photoreceptors and Müller glial cells, and an increase in bipolar cells. Conclusion HDAC activity is required for the expression of critical pro-rod transcription factors and the development of rod photoreceptor cells.

  18. Extracellular Matrix-Regulated Gene Expression RequiresCooperation of SWI/SNF and Transcription Factors

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ren; Spencer, Virginia A.; Bissell, Mina J.

    2006-05-25

    Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation (ChIP) assays and mammary-specific genes as models, we show here that extracellular matrix (ECM) molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the {beta}- and ?-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both {beta}- and ?-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. ChIP analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Coimmunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, C/EBP{beta}, and glucocorticoid receptor (GR). Thus, ECM- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors.

  19. CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

    Science.gov (United States)

    Převorovský, Martin; Oravcová, Martina; Zach, Róbert; Jordáková, Anna; Bähler, Jürg; Půta, František; Folk, Petr

    2016-11-16

    For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.

  20. U2 snRNP is required for expression of the 3' end of genes.

    Directory of Open Access Journals (Sweden)

    Mitsunori Koga

    Full Text Available Pre-mRNA in eukaryotes is subjected to mRNA processing, which includes capping, polyadenylation, and splicing. Transcription and mRNA processing are coupled, and this coupling stimulates mRNA processing; however, the effects of mRNA processing on transcription are not fully understood. In this study, we found that inhibition of U2 snRNP by a splicing inhibitor, spliceostatin A (SSA, or by an antisense oligonucleotide to U2 snRNA, caused gene-specific 3'-end down-regulation. Removal of SSA from the culture media restored expression of the 3' ends of genes, suggesting that U2 snRNP is required for expression of the 3' end of genes. Finally, we found that SSA treatment caused accumulation of Pol II near the 5' end of 3'-end down regulated genes, such as CDK6, SMEK2 and EGFR, indicating that SSA treatment led to transcription elongation arrest on these genes. These findings suggest that U2 snRNP is important for production of full length mRNA probably through regulation of transcription elongation, and that a novel checkpoint mechanism prevents pre-mRNA from accumulating as a result of splicing deficiencies, and thereby prevents production of aberrant proteins that might be translated from pre-mRNAs through the arrest of transcription elongation.

  1. U2 snRNP Is Required for Expression of the 3′ End of Genes

    Science.gov (United States)

    Koga, Mitsunori; Satoh, Takayuki; Takasaki, Ichiro; Kawamura, Yumi; Yoshida, Minoru; Kaida, Daisuke

    2014-01-01

    Pre-mRNA in eukaryotes is subjected to mRNA processing, which includes capping, polyadenylation, and splicing. Transcription and mRNA processing are coupled, and this coupling stimulates mRNA processing; however, the effects of mRNA processing on transcription are not fully understood. In this study, we found that inhibition of U2 snRNP by a splicing inhibitor, spliceostatin A (SSA), or by an antisense oligonucleotide to U2 snRNA, caused gene-specific 3′-end down-regulation. Removal of SSA from the culture media restored expression of the 3′ ends of genes, suggesting that U2 snRNP is required for expression of the 3′ end of genes. Finally, we found that SSA treatment caused accumulation of Pol II near the 5′ end of 3′-end down regulated genes, such as CDK6, SMEK2 and EGFR, indicating that SSA treatment led to transcription elongation arrest on these genes. These findings suggest that U2 snRNP is important for production of full length mRNA probably through regulation of transcription elongation, and that a novel checkpoint mechanism prevents pre-mRNA from accumulating as a result of splicing deficiencies, and thereby prevents production of aberrant proteins that might be translated from pre-mRNAs through the arrest of transcription elongation. PMID:24845214

  2. U2 snRNP is required for expression of the 3' end of genes.

    Science.gov (United States)

    Koga, Mitsunori; Satoh, Takayuki; Takasaki, Ichiro; Kawamura, Yumi; Yoshida, Minoru; Kaida, Daisuke

    2014-01-01

    Pre-mRNA in eukaryotes is subjected to mRNA processing, which includes capping, polyadenylation, and splicing. Transcription and mRNA processing are coupled, and this coupling stimulates mRNA processing; however, the effects of mRNA processing on transcription are not fully understood. In this study, we found that inhibition of U2 snRNP by a splicing inhibitor, spliceostatin A (SSA), or by an antisense oligonucleotide to U2 snRNA, caused gene-specific 3'-end down-regulation. Removal of SSA from the culture media restored expression of the 3' ends of genes, suggesting that U2 snRNP is required for expression of the 3' end of genes. Finally, we found that SSA treatment caused accumulation of Pol II near the 5' end of 3'-end down regulated genes, such as CDK6, SMEK2 and EGFR, indicating that SSA treatment led to transcription elongation arrest on these genes. These findings suggest that U2 snRNP is important for production of full length mRNA probably through regulation of transcription elongation, and that a novel checkpoint mechanism prevents pre-mRNA from accumulating as a result of splicing deficiencies, and thereby prevents production of aberrant proteins that might be translated from pre-mRNAs through the arrest of transcription elongation.

  3. A GATA transcription factor recruits Hda1 in response to reduced Tor1 signaling to establish a hyphal chromatin state in Candida albicans.

    Directory of Open Access Journals (Sweden)

    Yang Lu

    Full Text Available Candida albicans is an important opportunistic fungal pathogen of immunocompromised individuals. One critical virulence attribute is its morphogenetic plasticity. Hyphal development requires two temporally linked changes in promoter chromatin, which is sequentially regulated by temporarily clearing the transcription inhibitor Nrg1 upon activation of the cAMP/PKA pathway and promoter recruitment of the histone deacetylase Hda1 under reduced Tor1 signaling. Molecular mechanisms for the temporal connection and the link to Tor1 signaling are not clear. Here, through a forward genetic screen, we report the identification of the GATA family transcription factor Brg1 as the factor that recruits Hda1 to promoters of hypha-specific genes during hyphal elongation. BRG1 expression requires both the removal of Nrg1 and a sub-growth inhibitory level of rapamycin; therefore, it is a sensitive readout of Tor1 signaling. Interestingly, promoters of hypha-specific genes are not accessible to Brg1 in yeast cells. Furthermore, ectopic expression of Brg1 cannot induce hyphae, but can sustain hyphal development. Nucleosome mapping of a hypha-specific promoter shows that Nrg1 binding sites are in nucleosome free regions in yeast cells, whereas Brg1 binding sites are occupied by nucleosomes. Nucleosome disassembly during hyphal initiation exposes the binding sites for both regulators. During hyphal elongation, Brg1-mediated Hda1 recruitment causes nucleosome repositioning and occlusion of Nrg1 binding sites. We suggest that nucleosome repositioning is the underlying mechanism for the yeast-hyphal transition. The hypha-specific regulator Ume6 is a key downstream target of Brg1 and functions after Brg1 as a built-in positive feedback regulator of the hyphal transcriptional program to sustain hyphal development. With the levels of Nrg1 and Brg1 dynamically and sensitively controlled by the two major cellular growth pathways, temporal changes in nucleosome positioning

  4. iPSCs Offer a New Look at GATA1-Trisomy 21 Cooperation.

    Science.gov (United States)

    McNulty, Maureen; Crispino, John D

    2016-05-05

    GATA1 mutations and trisomy 21 are inextricably linked in the neonatal leukemia of children with Down syndrome (DS). A recent report by Banno et al. (2016) sheds new light on the mechanism of the synergy between these genetic alterations by modeling hematopoietic abnormalities with patient-derived induced pluripotent stem cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Gata3 drives development of RORγt+ group 3 innate lymphoid cells

    NARCIS (Netherlands)

    N. Serafini (Nicolas); R.G.J. Klein Wolterink (Roel); N. Satoh-Takayama (Naoko); W. Xu (Weiwei); C.A. Vosshenrich (Christian); R.W. Hendriks (Rudi); J.P. di Santo (James)

    2014-01-01

    textabstractGroup 3 innate lymphoid cells (ILC3) include IL-22-producing NKp46+ cells and IL-17A/IL-22-producing CD4+ lymphoid tissue inducerlike cells that express RORγt and are implicated in protective immunity at mucosal surfaces. Whereas the transcription factor Gata3 is essential for T cell and

  6. SATB1 packages densely-looped, transciptionally-active chromatinfor coordinated expression of cytokine genes

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Shutao; Lee, Charles C.; Kohwi-Shigematsu, Terumi

    2006-05-23

    SATB1 is an important regulator of nuclear architecture that anchors specialized DNA sequences onto its cage-like network and recruits chromatin remodeling/modifying factors to control gene transcription. We studied the role of SATB1 in regulating the coordinated expression of Il5, Il4, and Il13 from the 200kb cytokine gene cluster region of mouse chromosome 11 during T-helper 2 (Th2)-cell activation. We show that upon cell activation, SATB1 is rapidly induced to form a unique transcriptionally-active chromatin structure that includes the cytokine gene region. Chromatin is folded into numerous small loops all anchored by SATB1, is histone H3 acetylated at lysine 9/14, and associated with Th2-specific factors, GATA3, STAT6, c-Maf, the chromatin-remodeling enzyme Brg-1, and RNA polymerase II across the 200kb region. Before activation, the chromatin displays some of these features, such as association with GATA3 and STAT6, but these were insufficient for cytokine gene expression. Using RNA interference (RNAi), we show that upon cell activation, SATB1 is not only required for chromatin folding into dense loops, but also for c-Maf induction and subsequently for Il4, Il5, and Il13 transcription. Our results show that SATB1 is an important determinant for chromatin architecture that constitutes a novel higher-order, transcriptionally-active chromatin structure upon Th2-cell activation.

  7. Glutathione S-transferase Ya subunit gene: identification of regulatory elements required for basal level and inducible expression.

    OpenAIRE

    Telakowski-Hopkins, C A; King, R. G.; Pickett, C B

    1988-01-01

    The function of the 5'-flanking region of a rat glutathione S-transferase Ya subunit structural gene has been examined in homologous and heterologous cells. By using the 5'-flanking region of the Ya subunit gene fused to the structural gene encoding chloramphenicol acetyltransferase, we have identified two cis-acting regulatory elements in the upstream region of the Ya gene. One element is required for maximum basal level expression in homologous cells, whereas maximum basal level expression ...

  8. Paralysis and killing of Caenorhabditis elegans by enteropathogenic Escherichia coli requires the bacterial tryptophanase gene.

    Science.gov (United States)

    Anyanful, Akwasi; Dolan-Livengood, Jennifer M; Lewis, Taiesha; Sheth, Seema; Dezalia, Mark N; Sherman, Melanie A; Kalman, Lisa V; Benian, Guy M; Kalman, Daniel

    2005-08-01

    Pathogenic Escherichia coli, including enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) are major causes of food and water-borne disease. We have developed a genetically tractable model of pathogenic E. coli virulence based on our observation that these bacteria paralyse and kill the nematode Caenorhabditis elegans. Paralysis and killing of C. elegans by EPEC did not require direct contact, suggesting that a secreted toxin mediates the effect. Virulence against C. elegans required tryptophan and bacterial tryptophanase, the enzyme catalysing the production of indole and other molecules from tryptophan. Thus, lack of tryptophan in growth media or deletion of tryptophanase gene failed to paralyse or kill C. elegans. While known tryptophan metabolites failed to complement an EPEC tryptophanase mutant when presented extracellularly, complementation was achieved with the enzyme itself expressed either within the pathogen or within a cocultured K12 strains. Thus, an unknown metabolite of tryptophanase, derived from EPEC or from commensal non-pathogenic strains, appears to directly or indirectly regulate toxin production within EPEC. EPEC strains containing mutations in the locus of enterocyte effacement (LEE), a pathogenicity island required for virulence in humans, also displayed attenuated capacity to paralyse and kill nematodes. Furthermore, tryptophanase activity was required for full activation of the LEE1 promoter, and for efficient formation of actin-filled membranous protrusions (attaching and effacing lesions) that form on the surface of mammalian epithelial cells following attachment and which depends on LEE genes. Finally, several C. elegans genes, including hif-1 and egl-9, rendered C. elegans less susceptible to EPEC when mutated, suggesting their involvement in mediating toxin effects. Other genes including sek-1, mek-1, mev-1, pgp-1,3 and vhl-1, rendered C. elegans more

  9. Intraflagellar Transport and Functional Analysis of Genes Required for Flagellum Formation in Trypanosomes

    Science.gov (United States)

    Absalon, Sabrina; Blisnick, Thierry; Kohl, Linda; Toutirais, Géraldine; Doré, Gwénola; Julkowska, Daria; Tavenet, Arounie

    2008-01-01

    Intraflagellar transport (IFT) is the bidirectional movement of protein complexes required for cilia and flagella formation. We investigated IFT by analyzing nine conventional IFT genes and five novel putative IFT genes (PIFT) in Trypanosoma brucei that maintain its existing flagellum while assembling a new flagellum. Immunostaining against IFT172 or expression of tagged IFT20 or green fluorescent protein GFP::IFT52 revealed the presence of IFT proteins along the axoneme and at the basal body and probasal body regions of both old and new flagella. IFT particles were detected by electron microscopy and exhibited a strict localization to axonemal microtubules 3–4 and 7–8, suggesting the existence of specific IFT tracks. Rapid (>3 μm/s) bidirectional intraflagellar movement of GFP::IFT52 was observed in old and new flagella. RNA interference silencing demonstrated that all individual IFT and PIFT genes are essential for new flagellum construction but the old flagellum remained present. Inhibition of IFTB proteins completely blocked axoneme construction. Absence of IFTA proteins (IFT122 and IFT140) led to formation of short flagella filled with IFT172, indicative of defects in retrograde transport. Two PIFT proteins turned out to be required for retrograde transport and three for anterograde transport. Finally, flagellum membrane elongation continues despite the absence of axonemal microtubules in all IFT/PIFT mutant. PMID:18094047

  10. MOB1, an essential yeast gene required for completion of mitosis and maintenance of ploidy.

    Science.gov (United States)

    Luca, F C; Winey, M

    1998-01-01

    Mob1p is an essential Saccharomyces cerevisiae protein, identified from a two-hybrid screen, that binds Mps1p, a protein kinase essential for spindle pole body duplication and mitotic checkpoint regulation. Mob1p contains no known structural motifs; however MOB1 is a member of a conserved gene family and shares sequence similarity with a nonessential yeast gene, MOB2. Mob1p is a phosphoprotein in vivo and a substrate for the Mps1p kinase in vitro. Conditional alleles of MOB1 cause a late nuclear division arrest at restrictive temperature. MOB1 exhibits genetic interaction with three other yeast genes required for the completion of mitosis, LTE1, CDC5, and CDC15 (the latter two encode essential protein kinases). Most haploid mutant mob1 strains also display a complete increase in ploidy at permissive temperature. The mechanism for the increase in ploidy may occur through MPS1 function. One mob1 strain, which maintains stable haploidy at both permissive and restrictive temperature, diploidizes at permissive temperature when combined with the mps1-1 mutation. Strains containing mob2Delta also display a complete increase in ploidy when combined with the mps1-1 mutation. Perhaps in addition to, or as part of, its essential function in late mitosis, MOB1 is required for a cell cycle reset function necessary for the initiation of the spindle pole body duplication.

  11. Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor

    Science.gov (United States)

    Boulin, Thomas; Gielen, Marc; Richmond, Janet E.; Williams, Daniel C.; Paoletti, Pierre; Bessereau, Jean-Louis

    2008-01-01

    Levamisole-sensitive acetylcholine receptors (L-AChRs) are ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junctions of nematodes. They constitute a major drug target for anthelminthic treatments because they can be activated by nematode-specific cholinergic agonists such as levamisole. Genetic screens conducted in Caenorhabditis elegans for resistance to levamisole toxicity identified genes that are indispensable for the biosynthesis of L-AChRs. These include 5 genes encoding distinct AChR subunits and 3 genes coding for ancillary proteins involved in assembly and trafficking of the receptors. Despite extensive analysis of L-AChRs in vivo, pharmacological and biophysical characterization of these receptors has been greatly hampered by the absence of a heterologous expression system. Using Xenopus laevis oocytes, we were able to reconstitute functional L-AChRs by coexpressing the 5 distinct receptor subunits and the 3 ancillary proteins. Strikingly, this system recapitulates the genetic requirements for receptor expression in vivo because omission of any of these 8 genes dramatically impairs L-AChR expression. We demonstrate that 3 α- and 2 non-α-subunits assemble into the same receptor. Pharmacological analysis reveals that the prototypical cholinergic agonist nicotine is unable to activate L-AChRs but rather acts as a potent allosteric inhibitor. These results emphasize the role of ancillary proteins for efficient expression of recombinant neurotransmitter receptors and open the way for in vitro screening of novel anthelminthic agents. PMID:19020092

  12. Congenital erythropoietic porphyria due to a mutation in GATA1: the first trans-acting mutation causative for a human porphyria.

    Science.gov (United States)

    Phillips, John D; Steensma, David P; Pulsipher, Michael A; Spangrude, Gerald J; Kushner, James P

    2007-03-15

    Congenital erythropoietic porphyria (CEP), an autosomal recessive disorder, is due to mutations of uroporphyrinogen III synthase (UROS). Deficiency of UROS results in excess uroporphyrin I, which causes photosensitization. We evaluated a 3-year-old boy with CEP. A hypochromic, microcytic anemia was present from birth, and platelet counts averaged 70 x 10(9)/L (70,000/microL). Erythrocyte UROS activity was 21% of controls. Red cell morphology and globin chain labeling studies were compatible with beta-thalassemia. Hb electrophoresis revealed 36.3% A, 2.4% A(2), 59.5% F, and 1.8% of an unidentified peak. No UROS or alpha- and beta-globin mutations were found in the child or the parents. The molecular basis of the phenotype proved to be a mutation of GATA1, an X-linked transcription factor common to globin genes and heme biosynthetic enzymes in erythrocytes. A mutation at codon 216 in the child and on one allele of his mother changed arginine to tryptophan (R216W). This is the first report of a human porphyria due to a mutation in a trans-acting factor and the first association of CEP with thalassemia and thrombocytopenia. The Hb F level of 59.5% suggests a role for GATA-1 in globin switching. A bone marrow allograft corrected both the porphyria and the thalassemia.

  13. GATA-4/-6 and HNF-1/-4 families of transcription factors control the transcriptional regulation of the murine Muc5ac mucin during stomach development and in epithelial cancer cells.

    Science.gov (United States)

    Jonckheere, Nicolas; Vincent, Audrey; Franquet-Ansart, Hélène; Witte-Bouma, Janneke; Korteland-van Male, Anita; Leteurtre, Emmanuelle; Renes, Ingrid B; Van Seuningen, Isabelle

    2012-08-01

    During human embryonic and fetal development of the gastrointestinal tract, the gene encoding the MUC5AC mucin has a spatio-temporal pattern of expression restricted to the stomach. In order to better understand the molecular mechanisms responsible for this restricted pattern of expression, we have studied Muc5ac expression in the developing stomach of the mouse and correlated it to that of transcription factors known to be involved in cell differentiation programs during development. Our results indicate that GATA-6 and HNF-4α expression increased concomitantly with the induction of Muc5ac expression in embryonic stomach. We then studied Muc5ac transcriptional regulation by these transcription factors and showed that they all transactivate Muc5ac promoter. We also identified several active GATA-4/-5/-6 and HNF-1/-4 cis-elements using gel shift assays, chromatin immunoprecipitation and site-directed mutagenesis. Among all Muc5ac regulators, only GATA-6 and HNF-4a expression was concomitant to that of Muc5ac in the developing stomach. This is thus in favor of an important role for these two transcription factors as regulators of expression of the Muc5ac mucin during stomach development and in epithelial cancer cells.

  14. Association between KCNJ6 (GIRK2) gene polymorphisms and postoperative analgesic requirements after major abdominal surgery.

    Science.gov (United States)

    Nishizawa, Daisuke; Nagashima, Makoto; Katoh, Ryoji; Satoh, Yasuo; Tagami, Megumi; Kasai, Shinya; Ogai, Yasukazu; Han, Wenhua; Hasegawa, Junko; Shimoyama, Naohito; Sora, Ichiro; Hayashida, Masakazu; Ikeda, Kazutaka

    2009-09-16

    Opioids are commonly used as effective analgesics for the treatment of acute and chronic pain. However, considerable individual differences have been widely observed in sensitivity to opioid analgesics. We focused on a G-protein-activated inwardly rectifying potassium (GIRK) channel subunit, GIRK2, that is an important molecule in opioid transmission. In our initial polymorphism search, a total of nine single-nucleotide polymorphisms (SNPs) were identified in the whole exon, 5'-flanking, and exon-intron boundary regions of the KCNJ6 gene encoding GIRK2. Among them, G-1250A and A1032G were selected as representative SNPs for further association studies. In an association study of 129 subjects who underwent major open abdominal surgery, the A/A genotype in the A1032G SNP and -1250G/1032A haplotype were significantly associated with increased postoperative analgesic requirements compared with other genotypes and haplotypes. The total dose (mean+/-SEM) of rescue analgesics converted to equivalent oral morphine doses was 20.45+/-9.27 mg, 10.84+/-2.24 mg, and 13.07+/-2.39 mg for the A/A, A/G, and G/G genotypes in the A1032G SNP, respectively. Additionally, KCNJ6 gene expression levels in the 1032A/A subjects were significantly decreased compared with the 1032A/G and 1032G/G subjects in a real-time quantitative PCR analysis using human brain tissues, suggesting that the 1032A/A subjects required more analgesics because of lower KCNJ6 gene expression levels and consequently insufficient analgesic effects. The results indicate that the A1032G SNP and G-1250A/A1032G haplotype could serve as markers that predict increased analgesic requirements. Our findings will provide valuable information for achieving satisfactory pain control and open new avenues for personalized pain treatment.

  15. Association between KCNJ6 (GIRK2 gene polymorphisms and postoperative analgesic requirements after major abdominal surgery.

    Directory of Open Access Journals (Sweden)

    Daisuke Nishizawa

    Full Text Available Opioids are commonly used as effective analgesics for the treatment of acute and chronic pain. However, considerable individual differences have been widely observed in sensitivity to opioid analgesics. We focused on a G-protein-activated inwardly rectifying potassium (GIRK channel subunit, GIRK2, that is an important molecule in opioid transmission. In our initial polymorphism search, a total of nine single-nucleotide polymorphisms (SNPs were identified in the whole exon, 5'-flanking, and exon-intron boundary regions of the KCNJ6 gene encoding GIRK2. Among them, G-1250A and A1032G were selected as representative SNPs for further association studies. In an association study of 129 subjects who underwent major open abdominal surgery, the A/A genotype in the A1032G SNP and -1250G/1032A haplotype were significantly associated with increased postoperative analgesic requirements compared with other genotypes and haplotypes. The total dose (mean+/-SEM of rescue analgesics converted to equivalent oral morphine doses was 20.45+/-9.27 mg, 10.84+/-2.24 mg, and 13.07+/-2.39 mg for the A/A, A/G, and G/G genotypes in the A1032G SNP, respectively. Additionally, KCNJ6 gene expression levels in the 1032A/A subjects were significantly decreased compared with the 1032A/G and 1032G/G subjects in a real-time quantitative PCR analysis using human brain tissues, suggesting that the 1032A/A subjects required more analgesics because of lower KCNJ6 gene expression levels and consequently insufficient analgesic effects. The results indicate that the A1032G SNP and G-1250A/A1032G haplotype could serve as markers that predict increased analgesic requirements. Our findings will provide valuable information for achieving satisfactory pain control and open new avenues for personalized pain treatment.

  16. Expression and characterization of streptococcal rgp genes required for rhamnan synthesis in Escherichia coli.

    Science.gov (United States)

    Shibata, Yukie; Yamashita, Yoshihisa; Ozaki, Kazuhisa; Nakano, Yoshio; Koga, Toshihiko

    2002-06-01

    Six genes (rgpA through rgpF) that were involved in assembling the rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans were previously identified (Y. Yamashita, Y. Tsukioka, K. Tomihisa, Y. Nakano, and T. Koga, J. Bacteriol. 180:5803-5807, 1998). The group-specific antigens of Lancefield group A, C, and E streptococci and the polysaccharide antigen of Streptococcus sobrinus have the same rhamnan backbone as the RGP of S. mutans. Escherichia coli harboring plasmid pRGP1 containing all six rgp genes did not synthesize complete RGP. However, E. coli carrying a plasmid with all of the rgp genes except for rgpE synthesized the rhamnan backbone of RGP without glucose side chains, suggesting that in addition to rgpE, another gene is required for glucose side-chain formation. Synthesis of the rhamnan backbone in E. coli required the initiation of transfer of N-acetylglucosamine to a lipid carrier and the expression of the rgpC and rgpD genes encoding the putative ABC transporter specific for RGP. The similarities in RGP synthesis between E. coli and S. mutans suggest common pathways for rhamnan synthesis. Therefore, we evaluated the rhamnosyl polymerization process in E. coli by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the lipooligosaccharide (LOS). An E. coli transformant harboring rgpA produced the LOS modified by the addition of a single rhamnose residue. Furthermore, the rgpA, rgpB, and rgpF genes of pRGP1 were independently mutated by an internal deletion, and the LOS chemotypes of their transformants were examined. The transformant with an rgpA deletion showed the same LOS profile as E. coli without a plasmid. The transformant with an rgpB deletion showed the same LOS profile as E. coli harboring rgpA alone. The transformant with an rgpF deletion showed the LOS band with the most retarded migration. On the basis of these results, we speculated that RgpA, RgpB, and RgpF, in that order, function in rhamnan polymerization.

  17. Expression and Characterization of Streptococcal rgp Genes Required for Rhamnan Synthesis in Escherichia coli†

    Science.gov (United States)

    Shibata, Yukie; Yamashita, Yoshihisa; Ozaki, Kazuhisa; Nakano, Yoshio; Koga, Toshihiko

    2002-01-01

    Six genes (rgpA through rgpF) that were involved in assembling the rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans were previously identified (Y. Yamashita, Y. Tsukioka, K. Tomihisa, Y. Nakano, and T. Koga, J. Bacteriol. 180:5803-5807, 1998). The group-specific antigens of Lancefield group A, C, and E streptococci and the polysaccharide antigen of Streptococcus sobrinus have the same rhamnan backbone as the RGP of S. mutans. Escherichia coli harboring plasmid pRGP1 containing all six rgp genes did not synthesize complete RGP. However, E. coli carrying a plasmid with all of the rgp genes except for rgpE synthesized the rhamnan backbone of RGP without glucose side chains, suggesting that in addition to rgpE, another gene is required for glucose side-chain formation. Synthesis of the rhamnan backbone in E. coli required the initiation of transfer of N-acetylglucosamine to a lipid carrier and the expression of the rgpC and rgpD genes encoding the putative ABC transporter specific for RGP. The similarities in RGP synthesis between E. coli and S. mutans suggest common pathways for rhamnan synthesis. Therefore, we evaluated the rhamnosyl polymerization process in E. coli by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the lipooligosaccharide (LOS). An E. coli transformant harboring rgpA produced the LOS modified by the addition of a single rhamnose residue. Furthermore, the rgpA, rgpB, and rgpF genes of pRGP1 were independently mutated by an internal deletion, and the LOS chemotypes of their transformants were examined. The transformant with an rgpA deletion showed the same LOS profile as E. coli without a plasmid. The transformant with an rgpB deletion showed the same LOS profile as E. coli harboring rgpA alone. The transformant with an rgpF deletion showed the LOS band with the most retarded migration. On the basis of these results, we speculated that RgpA, RgpB, and RgpF, in that order, function in rhamnan polymerization

  18. Geminin is required for zygotic gene expression at the Xenopus mid-blastula transition.

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    Sarah L Kerns

    Full Text Available In many organisms early development is under control of the maternal genome and zygotic gene expression is delayed until the mid-blastula transition (MBT. As zygotic transcription initiates, cell cycle checkpoints become activated and the tempo of cell division slows. The mechanisms that activate zygotic transcription at the MBT are incompletely understood, but they are of interest because they may resemble mechanisms that cause stem cells to stop dividing and terminally differentiate. The unstable regulatory protein Geminin is thought to coordinate cell division with cell differentiation. Geminin is a bi-functional protein. It prevents a second round of DNA replication during S and G2 phase by binding and inhibiting the essential replication factor Cdt1. Geminin also binds and inhibits a number of transcription factors and chromatin remodeling proteins and is thought to keep dividing cells in an undifferentiated state. We previously found that the cells of Geminin-deficient Xenopus embryos arrest in G2 phase just after the MBT then disintegrate at the onset of gastrulation. Here we report that they also fail to express most zygotic genes. The gene expression defect is cell-autonomous and is reproduced by over-expressing Cdt1 or by incubating the embryos in hydroxyurea. Geminin deficient and hydroxyurea-treated blastomeres accumulate DNA damage in the form of double stranded breaks. Bypassing the Chk1 pathway overcomes the cell cycle arrest caused by Geminin depletion but does not restore zygotic gene expression. In fact, bypassing the Chk1 pathway by itself induces double stranded breaks and abolishes zygotic transcription. We did not find evidence that Geminin has a replication-independent effect on transcription. We conclude that Geminin is required to maintain genome integrity during the rapid cleavage divisions, and that DNA damage disrupts zygotic gene transcription at the MBT, probably through activation of DNA damage checkpoint pathways.

  19. GATA1-mutant clones are frequent and often unsuspected in babies with Down syndrome: identification of a population at risk of leukemia

    Science.gov (United States)

    Alford, Kate; Hall, Georgina; Juban, Gaetan; Richmond, Helen; Norton, Alice; Vallance, Grant; Perkins, Kelly; Marchi, Emanuele; McGowan, Simon; Roy, Anindita; Cowan, Gillian; Anthony, Mark; Gupta, Amit; Ho, John; Uthaya, Sabita; Curley, Anna; Rasiah, Shree Vishna; Watts, Timothy; Nicholl, Richard; Bedford-Russell, Alison; Blumberg, Raoul; Thomas, Angela; Gibson, Brenda; Halsey, Chris; Lee, Pek-Wan; Godambe, Sunit; Sweeney, Connor; Bhatnagar, Neha; Goriely, Anne; Campbell, Peter

    2013-01-01

    Transient abnormal myelopoiesis (TAM), a preleukemic disorder unique to neonates with Down syndrome (DS), may transform to childhood acute myeloid leukemia (ML-DS). Acquired GATA1 mutations are present in both TAM and ML-DS. Current definitions of TAM specify neither the percentage of blasts nor the role of GATA1 mutation analysis. To define TAM, we prospectively analyzed clinical findings, blood counts and smears, and GATA1 mutation status in 200 DS neonates. All DS neonates had multiple blood count and smear abnormalities. Surprisingly, 195 of 200 (97.5%) had circulating blasts. GATA1 mutations were detected by Sanger sequencing/denaturing high performance liquid chromatography (Ss/DHPLC) in 17 of 200 (8.5%), all with blasts >10%. Furthermore low-abundance GATA1 mutant clones were detected by targeted next-generation resequencing (NGS) in 18 of 88 (20.4%; sensitivity ∼0.3%) DS neonates without Ss/DHPLC-detectable GATA1 mutations. No clinical or hematologic features distinguished these 18 neonates. We suggest the term “silent TAM” for neonates with DS with GATA1 mutations detectable only by NGS. To identify all babies at risk of ML-DS, we suggest GATA1 mutation and blood count and smear analyses should be performed in DS neonates. Ss/DPHLC can be used for initial screening, but where GATA1 mutations are undetectable by Ss/DHPLC, NGS-based methods can identify neonates with small GATA1 mutant clones. PMID:24021668

  20. Young woman with mild bone marrow dysplasia, GATA2 and ASXL1 mutation treated with allogeneic hematopoietic stem cell transplantation

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    Anna Lübking

    2015-01-01

    We describe a case of symptomatic DCML deficiency and rather discrete bone marrow findings due to GATA2 mutation. Exome sequencing revealed a somatic ASXL1 mutation and the patient underwent allogeneic stem cell transplantation successfully.

  1. Expression of T - bet and GATA3 in The Pathogenesis of Psoriasis%银屑病患者皮损T-bet/GATA3表达的免疫组化研究

    Institute of Scientific and Technical Information of China (English)

    陈晋广; 刘玉峰; 任小丽

    2007-01-01

    目的 检测Th1/Th2细胞特异性转录因子T-bet/GATA3在寻常性银屑病患者皮损中表达,探讨T-bet/GATA3在银屑病发病机制中的作用及意义.方法 采用免疫组化ABC法研究银屑病患者皮损中T-bet、GATA3表达,并以15例正常人作为对照组.通过图像分析技术进行定量分析.结果 银屑病皮损中既可表达T-bet,又可表达GATA3,而在正常皮肤不表达.定量分析显示,皮损中T-bet表达水平显著高于GATA3(P<0.05).结论 T-bet/GATA3表达的失衡和T-bet高表达可能参与了银屑病发病过程.

  2. Msx genes define a population of mural cell precursors required for head blood vessel maturation.

    Science.gov (United States)

    Lopes, Miguel; Goupille, Olivier; Saint Cloment, Cécile; Lallemand, Yvan; Cumano, Ana; Robert, Benoît

    2011-07-01

    Vessels are primarily formed from an inner endothelial layer that is secondarily covered by mural cells, namely vascular smooth muscle cells (VSMCs) in arteries and veins and pericytes in capillaries and veinules. We previously showed that, in the mouse embryo, Msx1(lacZ) and Msx2(lacZ) are expressed in mural cells and in a few endothelial cells. To unravel the role of Msx genes in vascular development, we have inactivated the two Msx genes specifically in mural cells by combining the Msx1(lacZ), Msx2(lox) and Sm22α-Cre alleles. Optical projection tomography demonstrated abnormal branching of the cephalic vessels in E11.5 mutant embryos. The carotid and vertebral arteries showed an increase in caliber that was related to reduced vascular smooth muscle coverage. Taking advantage of a newly constructed Msx1(CreERT2) allele, we demonstrated by lineage tracing that the primary defect lies in a population of VSMC precursors. The abnormal phenotype that ensues is a consequence of impaired BMP signaling in the VSMC precursors that leads to downregulation of the metalloprotease 2 (Mmp2) and Mmp9 genes, which are essential for cell migration and integration into the mural layer. Improper coverage by VSMCs secondarily leads to incomplete maturation of the endothelial layer. Our results demonstrate that both Msx1 and Msx2 are required for the recruitment of a population of neural crest-derived VSMCs.

  3. Transcriptional repression of the yeast CHA1 gene requires the chromatin-remodeling complex RSC

    DEFF Research Database (Denmark)

    Moreira, José Manuel Alfonso; Holmberg, S

    1999-01-01

    In eukaryotes, DNA is packaged into chromatin, a compact structure that must be disrupted when genes are transcribed by RNA polymerase II. For transcription to take place, chromatin is remodeled via nucleosome disruption or displacement, a fundamental transcriptional regulatory mechanism in eukar......In eukaryotes, DNA is packaged into chromatin, a compact structure that must be disrupted when genes are transcribed by RNA polymerase II. For transcription to take place, chromatin is remodeled via nucleosome disruption or displacement, a fundamental transcriptional regulatory mechanism...... in eukaryotic organisms. Here we show that the yeast chromatin-remodeling complex, RSC (remodels the structure of chromatin), isolated on the basis of homology to the SWI/SNF complex, is required for proper transcriptional regulation and nucleosome positioning in the highly inducible CHA1 promoter...... of the CHA1 promoter is disrupted, an architectural change normally only observed during transcriptional induction. In addition, deletion of the gene-specific activator Cha4p did not affect derepression of CHA1 in cells depleted for Swh3p. Thus, CHA1 constitutes a target for the RSC complex, and we propose...

  4. Comprehensive identification of Salmonella enterica serovar typhimurium genes required for infection of BALB/c mice.

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    Roy R Chaudhuri

    2009-07-01

    Full Text Available Genes required for infection of mice by Salmonella Typhimurium can be identified by the interrogation of random transposon mutant libraries for mutants that cannot survive in vivo. Inactivation of such genes produces attenuated S. Typhimurium strains that have potential for use as live attenuated vaccines. A quantitative screen, Transposon Mediated Differential Hybridisation (TMDH, has been developed that identifies those members of a large library of transposon mutants that are attenuated. TMDH employs custom transposons with outward-facing T7 and SP6 promoters. Fluorescently-labelled transcripts from the promoters are hybridised to whole-genome tiling microarrays, to allow the position of the transposon insertions to be determined. Comparison of microarray data from the mutant library grown in vitro (input with equivalent data produced after passage of the library through mice (output enables an attenuation score to be determined for each transposon mutant. These scores are significantly correlated with bacterial counts obtained during infection of mice using mutants with individual defined deletions of the same genes. Defined deletion mutants of several novel targets identified in the TMDH screen are effective live vaccines.

  5. Genome-wide functional profiling reveals genes required for tolerance to benzene metabolites in yeast.

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    Matthew North

    Full Text Available Benzene is a ubiquitous environmental contaminant and is widely used in industry. Exposure to benzene causes a number of serious health problems, including blood disorders and leukemia. Benzene undergoes complex metabolism in humans, making mechanistic determination of benzene toxicity difficult. We used a functional genomics approach to identify the genes that modulate the cellular toxicity of three of the phenolic metabolites of benzene, hydroquinone (HQ, catechol (CAT and 1,2,4-benzenetriol (BT, in the model eukaryote Saccharomyces cerevisiae. Benzene metabolites generate oxidative and cytoskeletal stress, and tolerance requires correct regulation of iron homeostasis and the vacuolar ATPase. We have identified a conserved bZIP transcription factor, Yap3p, as important for a HQ-specific response pathway, as well as two genes that encode putative NAD(PH:quinone oxidoreductases, PST2 and YCP4. Many of the yeast genes identified have human orthologs that may modulate human benzene toxicity in a similar manner and could play a role in benzene exposure-related disease.

  6. Normal Ciliogenesis Requires Synergy between the Cystic Kidney Disease Genes MKS-3 and NPHP-4

    Science.gov (United States)

    Williams, Corey L.; Masyukova, Svetlana V.

    2010-01-01

    Cilia dysfunction contributes to renal cyst formation in multiple human syndromes including nephronophthisis (NPHP), Meckel-Gruber syndrome (MKS), Joubert syndrome (JBTS), and Bardet-Beidl syndrome (BBS). Although genetically heterogeneous, these diseases share several loci that affect cilia and/or basal body proteins, but the functions and interactions of these gene products are incompletely understood. Here, we report that the ciliated sensory neurons (CSNs) of C. elegans express the putative transmembrane protein MKS-3, which localized to the distal end of their dendrites and to the cilium base but not to the cilium itself. Localization of MKS-3 and other known MKS and NPHP proteins partially overlapped. By analyzing mks-3 mutants, we found that ciliogenesis did not require MKS-3; instead, cilia elongated and cilia-mediated chemoreception was abnormal. Genetic analysis indicated that mks-3 functions in a pathway with other mks genes. Furthermore, mks-1 and mks-3 genetically interacted with a separate pathway (involving nphp-1 and nphp-4) to influence proper positioning, orientation, and formation of cilia. Combined disruption of nphp and mks pathways had cell nonautonomous effects on C. elegans sensilla. Taken together, these data demonstrate the importance of mutational load on the presentation and severity of ciliopathies and expand the understanding of the interactions between ciliopathy genes. PMID:20150540

  7. The Drosophila gene Medea demonstrates the requirement for different classes of Smads in dpp signaling.

    Science.gov (United States)

    Das, P; Maduzia, L L; Wang, H; Finelli, A L; Cho, S H; Smith, M M; Padgett, R W

    1998-04-01

    Signals from transforming growth factor-beta (TGF-beta) ligands are transmitted within the cell by members of the Smad family, which can be grouped into three classes based on sequence similarities. Our previous identification of both class I and II Smads functioning in a single pathway in C. elegans, raised the issue of whether the requirement for Smads derived from different classes is a general feature of TGF-beta signaling. We report here the identification of a new Drosophila class II Smad, Medea, a close homolog of the human tumor-suppressor gene DPC4. Embryos from germline clones of both Medea and Mad (a class I Smad) are ventralized, as are embryos null for the TGF-beta-like ligand decapentaplegic (dpp). Loss of Medea also blocks dpp signaling during later development, suggesting that Medea, like Mad, is universally required for dpp signaling. Furthermore, we show that the necessity for these two closely related, non-redundant Smads, is due to their different signaling properties - upon activation of the Dpp pathway, Mad is required to actively translocate Medea into the nucleus. These results provide a paradigm for, and distinguish between, the requirement for class I and II Smads in Dpp/BMP signaling.

  8. Suppression of GATA-3 nuclear import and phosphorylation: a novel mechanism of corticosteroid action in allergic disease.

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    Kittipong Maneechotesuwan

    2009-05-01

    Full Text Available BACKGROUND: GATA-3 plays a critical role in regulating the expression of the cytokines interleukin (IL-4, IL-5, and IL-13 from T helper-2 (Th2 cells and therefore is a key mediator of allergic diseases. Corticosteroids are highly effective in suppressing allergic inflammation, but their effects on GATA-3 are unknown. We investigated the effect of the corticosteroid fluticasone propionate on GATA-3 regulation in human T-lymphocytes in vitro and in vivo. METHODS AND FINDINGS: In a T lymphocyte cell line (HuT-78 and peripheral blood mononuclear cells stimulated by anti-CD3 and anti-CD28 in vitro we demonstrated that fluticasone inhibits nuclear translocation of GATA-3 and expression of Th2 cytokines via a mechanism independent of nuclear factor-kappaB and is due, in part, to competition between GATA-3 and the ligand-activated glucocorticoid receptor for nuclear transport through the nuclear importer importin-alpha. In addition, fluticasone induces the expression of mitogen-activated protein kinase (MAPK phosphatase-1 (MKP-1, the endogenous inhibitor of p38 MAPK, which is necessary for GATA-3 nuclear translocation. These inhibitory effects of fluticasone are rapid, potent, and prolonged. We also demonstrated that inhaled fluticasone inhibits GATA-3 nuclear translocation in peripheral blood lymphocytes of patients with asthma in vivo. CONCLUSIONS: Corticosteroids have a potent inhibitory effect on GATA-3 via two interacting mechanisms that potently suppress Th2 cytokine expression. This novel mechanism of action of corticosteroids may account for the striking clinical efficacy of corticosteroids in the treatment of allergic diseases.

  9. Drosophila Dynein intermediate chain gene, Dic61B, is required for spermatogenesis.

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    Roshan Fatima

    Full Text Available This study reports the identification and characterization of a novel gene, Dic61B, required for male fertility in Drosophila. Complementation mapping of a novel male sterile mutation, ms21, isolated in our lab revealed it to be allelic to CG7051 at 61B1 cytogenetic region, since two piggyBac insertion alleles, CG7051(c05439 and CG7051(f07138 failed to complement. CG7051 putatively encodes a Dynein intermediate chain. All three mutants, ms21, CG7051(c05439 and CG7051(f07138, exhibited absolute recessive male sterility with abnormally coiled sperm axonemes causing faulty sperm individualization as revealed by Phalloidin staining in Don Juan-GFP background. Sequencing of PCR amplicons uncovered two point mutations in ms21 allele and confirmed the piggyBac insertions in CG7051(c05439 and CG7051(f07138 alleles to be in 5'UTR and 4(th exon of CG7051 respectively, excision of which reverted the male sterility. In situ hybridization to polytene chromosomes demonstrated CG7051 to be a single copy gene. RT-PCR of testis RNA revealed defective splicing of the CG7051 transcripts in mutants. Interestingly, expression of cytoplasmic dynein intermediate chain, α, β, γ tubulins and α-spectrin was normal in mutants while ultra structural studies revealed defects in the assembly of sperm axonemes. Bioinformatics further highlighted the homology of CG7051 to axonemal dynein intermediate chain of various organisms, including DNAI1 of humans, mutations in which lead to male sterility due to immotile sperms. Based on these observations we conclude that CG7051 encodes a novel axonemal dynein intermediate chain essential for male fertility in Drosophila and rename it as Dic61B. This is the first axonemal Dic gene of Drosophila to be characterized at molecular level and shown to be required for spermatogenesis.

  10. Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown

    Science.gov (United States)

    Fakhoury, Johans J.; Edwardson, Thomas G.; Conway, Justin W.; Trinh, Tuan; Khan, Farhad; Barłóg, Maciej; Bazzi, Hassan S.; Sleiman, Hanadi F.

    2015-12-01

    Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into `gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing.Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable

  11. Sample size requirements for indirect association studies of gene-environment interactions (G x E).

    Science.gov (United States)

    Hein, Rebecca; Beckmann, Lars; Chang-Claude, Jenny

    2008-04-01

    Association studies accounting for gene-environment interactions (G x E) may be useful for detecting genetic effects. Although current technology enables very dense marker spacing in genetic association studies, the true disease variants may not be genotyped. Thus, causal genes are searched for by indirect association using genetic markers in linkage disequilibrium (LD) with the true disease variants. Sample sizes needed to detect G x E effects in indirect case-control association studies depend on the true genetic main effects, disease allele frequencies, whether marker and disease allele frequencies match, LD between loci, main effects and prevalence of environmental exposures, and the magnitude of interactions. We explored variables influencing sample sizes needed to detect G x E, compared these sample sizes with those required to detect genetic marginal effects, and provide an algorithm for power and sample size estimations. Required sample sizes may be heavily inflated if LD between marker and disease loci decreases. More than 10,000 case-control pairs may be required to detect G x E. However, given weak true genetic main effects, moderate prevalence of environmental exposures, as well as strong interactions, G x E effects may be detected with smaller sample sizes than those needed for the detection of genetic marginal effects. Moreover, in this scenario, rare disease variants may only be detectable when G x E is included in the analyses. Thus, the analysis of G x E appears to be an attractive option for the detection of weak genetic main effects of rare variants that may not be detectable in the analysis of genetic marginal effects only.

  12. Blistered: a gene required for vein/intervein formation in wings of Drosophila.

    Science.gov (United States)

    Fristrom, D; Gotwals, P; Eaton, S; Kornberg, T B; Sturtevant, M; Bier, E; Fristrom, J W

    1994-09-01

    We have characterized the blistered (bs) locus phenotypically, genetically and developmentally using a set of new bs alleles. Mutant defects range from wings with ectopic veins and intervein blisters to completely ballooned wings where the distinction between vein and intervein is lost. Mosaic analyses show that severe bs alleles behave largely autonomously; homozygous patches having vein-like properties. Developmental analyses were undertaken using light and electron microscopy of wild-type and bs wings as well as confocal microscopy of phalloidin- and laminin-stained preparations. bs defects were first seen early in the prepupal period with the failure of apposition of dorsal and ventral wing epithelia. Correspondingly, during definitive vein/intervein differentiation in the pupal period (18-36 hours after puparium formation), the extent of dorsal/ventral reapposition is reduced in bs wings. Regions of the wing that fail to become apposed differentiate properties of vein cells; i.e. become constricted apically and acquire a laminin-containing matrix basally. To further understand bs function, we examined genetic interactions between various bs alleles and mutants of two genes whose products have known functions in wing development. (i) rhomboid, a component of the EGF-R signalling pathway, is expressed in vein cells and is required for specification of vein cell fate. rhove mutations (lacking rhomboid in wings) suppress the excess vein formation and associated with bs. Conversely, rho expression in prepupal and pupal bs wings is expanded in the regions of increased vein formation. (ii) The integrin genes, inflated and myospheroid, are expressed in intervein cells and are required for adhesion between the dorsal and ventral wing surfaces. Loss of integrin function results in intervein blisters. Integrin mutants interact with bs mutants to increase the frequency of intervein blisters but do not typically enhance vein defects. Both developmental and genetic analyses

  13. rFTR1 is Required for Pathogenesis, and appears to be an Essential Gene, of Rhizopus oryzae

    Science.gov (United States)

    BACKGROUND: Rhizopus oryzae is a multinucleated fungus responsible for the majority of cases of mucormycosis. The high affinity iron permease gene (rFTR1) is required for R. oryzae iron transport in iron-limited environments. We sought to disrupt the gene to define its role in virulence. METHODS: ...

  14. The cardiac transcription network modulated by Gata4, Mef2a, Nkx2.5, Srf, histone modifications, and microRNAs.

    Directory of Open Access Journals (Sweden)

    Jenny Schlesinger

    2011-02-01

    Full Text Available The transcriptome, as the pool of all transcribed elements in a given cell, is regulated by the interaction between different molecular levels, involving epigenetic, transcriptional, and post-transcriptional mechanisms. However, many previous studies investigated each of these levels individually, and little is known about their interdependency. We present a systems biology study integrating mRNA profiles with DNA-binding events of key cardiac transcription factors (Gata4, Mef2a, Nkx2.5, and Srf, activating histone modifications (H3ac, H4ac, H3K4me2, and H3K4me3, and microRNA profiles obtained in wild-type and RNAi-mediated knockdown. Finally, we confirmed conclusions primarily obtained in cardiomyocyte cell culture in a time-course of cardiac maturation in mouse around birth. We provide insights into the combinatorial regulation by cardiac transcription factors and show that they can partially compensate each other's function. Genes regulated by multiple transcription factors are less likely differentially expressed in RNAi knockdown of one respective factor. In addition to the analysis of the individual transcription factors, we found that histone 3 acetylation correlates with Srf- and Gata4-dependent gene expression and is complementarily reduced in cardiac Srf knockdown. Further, we found that altered microRNA expression in Srf knockdown potentially explains up to 45% of indirect mRNA targets. Considering all three levels of regulation, we present an Srf-centered transcription network providing on a single-gene level insights into the regulatory circuits establishing respective mRNA profiles. In summary, we show the combinatorial contribution of four DNA-binding transcription factors in regulating the cardiac transcriptome and provide evidence that histone modifications and microRNAs modulate their functional consequence. This opens a new perspective to understand heart development and the complexity cardiovascular disorders.

  15. Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl.

    Science.gov (United States)

    Zhu, Wei; Pao, Gerald M; Satoh, Akira; Cummings, Gillian; Monaghan, James R; Harkins, Timothy T; Bryant, Susan V; Randal Voss, S; Gardiner, David M; Hunter, Tony

    2012-10-01

    The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration-wound healing, cellular dedifferentiation and blastemal formation-will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells to a germline-like state. Two genes that are required for self-renewal of germ cells in mice and flies, Piwi-like 1 (PL1) and Piwi-like 2 (PL2), are expressed in limb blastemal cells, the basal layer keratinocytes and the thickened apical epithelial cap in the wound epidermis in the regenerating limb. Depletion of PL1 and PL2 by morpholino oligonucleotides decreased cell proliferation and increased cell death in the blastema leading to a significant retardation of regeneration. Examination of key molecules that are known to be required for limb development or regeneration further revealed that FGF8 is transcriptionally downregulated in the presence of the morpholino oligos, indicating PL1 and PL2 might participate in FGF signaling during limb regeneration. Given the requirement for FGF signaling in limb development and regeneration, the results suggest that PL1 and PL2 function to establish a unique germline-like state that is associated with successful regeneration.

  16. Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl

    Science.gov (United States)

    Zhu, Wei; Pao, Gerald M; Satoh, Akira; Cummings, Gillian; Monaghan, James R; Harkins, Timothy T; Bryant, Susan V; Voss, S Randal; Gardiner, David M; Hunter, Tony

    2013-01-01

    The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration – wound healing, cellular dedifferentiation and blastemal formation – will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells to a germline-like state. Two genes that are required for self-renewal of germ cells in mice and flies, Piwi-like 1 (PL1) and Piwi-like 2 (PL2), are expressed in limb blastemal cells, the basal layer keratinocytes and the thickened apical epithelial cap in the wound epidermis in the regenerating limb. Depletion of PL1 and PL2 by morpholino oligonucleotides decreased cell proliferation and increased cell death in the blastema leading to a significant retardation of regeneration. Examination of key molecules that are known to be required for limb development or regeneration further revealed that FGF8 is transcriptionally downregulated in the presence of the morpholino oligos, indicating PL1 and PL2 might participate in FGF signaling during limb regeneration. Given the requirement for FGF signaling in limb development and regeneration, the results suggest that PL1 and PL2 function to establish a unique germline-like state that is associated with successful regeneration. PMID:22841627

  17. The requirement of multiple defense genes in soybean Rsv1-mediated extreme resistance to soybean mosaic virus.

    Science.gov (United States)

    Zhang, Chunquan; Grosic, Sehiza; Whitham, Steven A; Hill, John H

    2012-10-01

    Soybean mosaic virus (SMV) is a major viral pathogen of soybean. Among the three SMV resistance genes, Rsv1 mediates extreme resistance (ER) against most SMV strains, including the β-glucuronidase-tagged G2 isolate that was previously used in studies of Rsv1. Using virus-induced gene silencing (VIGS), we screened 82 VIGS constructs to identify genes that play a role in Rsv1-mediated ER to SMV infection. The target genes included putative Rsv1 candidate genes, soybean orthologs to known defense-signaling genes, and 62 WRKY transcription factors. We identified eight VIGS constructs that compromised Rsv1-mediated resistance when the target genes were silenced, including GmEDR1, GmEDS1, GmHSP90, GmJAR1, GmPAD4, and two WRKY transcription factors. Together, our results provide new insight into the soybean signaling network required for ER against SMV.

  18. Gut microbiota inhibit Asbt-dependent intestinal bile acid reabsorption via Gata4

    Science.gov (United States)

    Out, Carolien; Patankar, Jay V.; Doktorova, Marcela; Boesjes, Marije; Bos, Trijnie; de Boer, Sanna; Havinga, Rick; Wolters, Henk; Boverhof, Renze; van Dijk, Theo H.; Smoczek, Anna; Bleich, André; Sachdev, Vinay; Kratky, Dagmar; Kuipers, Folkert; Verkade, Henkjan J.; Groen, Albert K.

    2017-01-01

    Background & Aims Regulation of bile acid homeostasis in mammals is a complex process regulated via extensive cross-talk between liver, intestine and intestinal microbiota. Here we studied the effects of gut microbiota on bile acid homeostasis in mice. Methods Bile acid homeostasis was assessed in four mouse models. Germfree mice, conventionally-raised mice, Asbt-KO mice and intestinal-specific Gata4-iKO mice were treated with antibiotics (bacitracin, neomycin and vancomycin; 100 mg/kg) for five days and subsequently compared with untreated mice. Results Attenuation of the bacterial flora by antibiotics strongly reduced fecal excretion and synthesis of bile acids, but increased the expression of the bile acid synthesis enzyme CYP7A1. Similar effects were seen in germfree mice. Intestinal bile acid absorption was increased and accompanied by increases in plasma bile acid levels, biliary bile acid secretion and enterohepatic cycling of bile acids. In the absence of microbiota, the expression of the intestinal bile salt transporter Asbt was strongly increased in the ileum and was also expressed in more proximal parts of the small intestine. Most of the effects of antibiotic treatment on bile acid homeostasis could be prevented by genetic inactivation of either Asbt or the transcription factor Gata4. Conclusions Attenuation of gut microbiota alters Gata4-controlled expression of Asbt, increasing absorption and decreasing synthesis of bile acids. Our data support the concept that under physiological conditions microbiota stimulate Gata4, which suppresses Asbt expression, limiting the expression of this transporter to the terminal ileum. Our studies expand current knowledge on the bacterial control of bile acid homeostasis. PMID:26022694

  19. Chromohalobacter salarius sp. nov., a moderately halophilic bacterium isolated from a solar saltern in Cabo de Gata, Almeria, southern Spain.

    Science.gov (United States)

    Aguilera, Margarita; Cabrera, Antonio; Incerti, Claudia; Fuentes, Susana; Russell, Nick J; Ramos-Cormenzana, Alberto; Monteoliva-Sánchez, Mercedes

    2007-06-01

    A moderately halophilic, Gram-negative bacterium (strain CG4.1(T)), which was isolated from a solar saltern at Cabo de Gata, a wildlife reserve located in the province of Almería, southern Spain, was subjected to a polyphasic taxonomic study. This organism was an aerobic, motile rod that produced colonies with a yellow pigment. Strain CG4.1(T) grew at salinities of 3-25 % (w/v), at 15-45 degrees C and at pH 5-9. The organism reduced nitrate, hydrolysed starch and had phenylalanine deaminase activity. The major fatty acids were C(18 : 1)omega7c, C(16 : 0) and C(19 : 0) cyclo omega8c. The DNA G+C content was 63.6 mol%. On the basis of phenotypic and phylogenetic data, strain CG4.1(T) appears to be a member of the genus Chromohalobacter and clustered closely with Chromohalobacter species, with 95-96 % similarity between their 16S rRNA gene sequences. However, DNA-DNA relatedness between the isolate and the type strains of Chromohalobacter species was low. Therefore, it is proposed that strain CG4.1(T) represents a novel species, Chromohalobacter salarius sp. nov. The type strain is strain CG4.1(T) (=CECT 5903(T)=LMG 23626(T)).

  20. Expression and significance of GATA3 in epithelial ovarian cancer%GATA3在上皮性卵巢癌中的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    马杰; 赵卫东; 邹强; 金夏; 张丽娜; 曹振平; 冯定庆

    2011-01-01

    目的:探讨GATA3在上皮性卵巢癌组织中的表达及其意义.方法:应用RT-PCR和Western Blotting检测30例上皮性卵巢癌标本和18例正常卵巢组织中GATA3 mRNA及蛋白水平表达情况,应用免疫组化检测肿瘤组织和正常卵巢组织中GATA3蛋白定位及表达情况.结果:RT-PCR提示上皮性卵巢癌组织中GATA3 mRNA表达升高,在卵巢癌组织和正常卵巢组织中的阳性表达率分别为90.0%和44.4%,差异有统计学意义(P<0.05);Western Blotting和免疫组化提示卵巢癌组织中GATA3在蛋白水平的表达明显降低,正常卵巢组织中定位在细胞核内,而卵巢癌组织中细胞核内表达不明显.结论:卵巢癌组织中GATA3在基因水平表达较正常卵巢组织上调,其翻译的蛋白表达却降低,可能是GATA3 mRNA合成的蛋白大多由核内转移而出,从而促进肿瘤的发生与发展,为进一步探讨上皮性卵巢癌的发病机理提供了实验依据.%Objective: To explore the expression and significance of GATA3 in epithelial ovarian cancer tissue. Methods; RT -PCR and Western Blotting were used to detect the expression levels of GATA3 mRNA and protein in 30 cases with epithelial ovarian cancer and 18 cases with normal ovarian tissue, immunohistoehemical method was used to detect the locations and expressions of GATA3 protein in epithelial ovarian cancer tissue and normal ovarian tissue. Results: The result of RT - PCR showed that the expression level of GAT A3 mRNA in epithelial ovarian cancer tissue increased, the positive expression rates of GATA3 mRNA in epithelial ovarian cancer tissue and normal ovarian tissue were 90. 0% and 44. 4% , respectively, there was significant difference between the two groups (P < 0. 05 ) ; the results of Western Blotting and immunohistoehemical method indicated that the expression level of GAT A3 protein in epithelial ovarian cancer tissue decreased significantly, GAT A3 protein located in cell nucleus of normal ovarian tissue

  1. The nephronophthisis-related gene ift-139 is required for ciliogenesis in Caenorhabditis elegans

    Science.gov (United States)

    Niwa, Shinsuke

    2016-01-01

    Defects in cilia cause a spectrum of diseases known as ciliopathies. Nephronophthisis, a ciliopathy, is the most common genetic cause of renal disease. Here, I cloned and analysed a nephronophthisis-related gene ift-139 in Caenorhabditis elegans. ift-139 was exclusively expressed in ciliated neurons in C. elegans. Genetic and cellular analyses suggest that ift-139 plays a role in retrograde intraflagellar transport and is required for cilia formation. A homologous point mutation that causes ciliopathy disrupted the function of ift-139 in C. elegans. ift-139 is an orthologue of human TTC21B, mutations in which are known to cause nephronophthisis 12 and short-rib thoracic dysplasia 4. These results suggest that ift-139 is evolutionarily conserved and fundamental to the formation of cilia. PMID:27515926

  2. Inositol polyphosphate multikinase is a transcriptional coactivator required for immediate early gene induction.

    Science.gov (United States)

    Xu, Risheng; Paul, Bindu D; Smith, Dani R; Tyagi, Richa; Rao, Feng; Khan, A Basit; Blech, Daniel J; Vandiver, M Scott; Harraz, Maged M; Guha, Prasun; Ahmed, Ishrat; Sen, Nilkantha; Gallagher, Michela; Snyder, Solomon H

    2013-10-01

    Profound induction of immediate early genes (IEGs) by neural activation is a critical determinant for plasticity in the brain, but intervening molecular signals are not well characterized. We demonstrate that inositol polyphosphate multikinase (IPMK) acts noncatalytically as a transcriptional coactivator to mediate induction of numerous IEGs. IEG induction by electroconvulsive stimulation is virtually abolished in the brains of IPMK-deleted mice, which also display deficits in spatial memory. Neural activity stimulates binding of IPMK to the histone acetyltransferase CBP and enhances its recruitment to IEG promoters. Interestingly, IPMK regulation of CBP recruitment and IEG induction does not require its catalytic activities. Dominant-negative constructs, which prevent IPMK-CBP binding, substantially decrease IEG induction. As IPMK is ubiquitously expressed, its epigenetic regulation of IEGs may influence diverse nonneural and neural biologic processes.

  3. The extramacrochaetae gene is required for blastokinesis in silkworm, Bombyx mori.

    Science.gov (United States)

    Liu, Wenbin; Chai, Dezhi; Wang, Cailian; Li, Qing; Lei, Jinfeng; Yang, Min; Dai, Fangyin; Lu, Cheng

    2015-07-01

    In silkworm, Bombyx mori Linnaeus (Lepidoptera: Bombycidae), blastokinesis results in embryo reversal from ventrally to dorsally convex flexion. In this study, we showed that the extramacrochaetae (emc) gene is required for blastokinesis in silkworm. Depletion of Bmemc expression via RNA interference led to severe phenotypic defects in blastokinesis. The defective embryos failed to invert their body sides during blastokinesis. This caused the posterior half of the abdomen to abnormally fold back toward the dorsal side, forming a U-shaped morphology. Dorsal closure was also disrupted. Our results suggest that Bmemc is involved in blastokinesis of silkworm embryos. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 405-409, 2015. © 2015 Wiley Periodicals, Inc.

  4. The fas operon of Rhodococcus fascians encodes new genes required for efficient fasciation of host plants.

    Science.gov (United States)

    Crespi, M; Vereecke, D; Temmerman, W; Van Montagu, M; Desomer, J

    1994-01-01

    Three virulence loci (fas, att, and hyp) of Rhodococcus fascians D188 have been identified on a 200-kb conjugative linear plasmid (pFiD188). The fas locus was delimited to a 6.5-kb DNA fragment by insertion mutagenesis, single homologous disruptive recombination, and in trans complementation of different avirulent insertion mutants. The locus is arranged as a large operon containing six open reading frames whose expression is specifically induced during the interaction with host plants. One predicted protein is homologous to P-450 cytochromes from actinomycetes. The putative ferredoxin component is of a novel type containing additional domains homologous to transketolases from chemoautotrophic, photosynthetic, and methylotrophic microorganisms. Genetic analysis revealed that fas encodes, in addition to the previously identified ipt, at least two new genes that are involved in fasciation development, one of which is only required on older tobacco plants. PMID:8169198

  5. Transportin-SR is required for proper splicing of resistance genes and plant immunity.

    Directory of Open Access Journals (Sweden)

    Shaohua Xu

    2011-06-01

    Full Text Available Transportin-SR (TRN-SR is a member of the importin-β super-family that functions as the nuclear import receptor for serine-arginine rich (SR proteins, which play diverse roles in RNA metabolism. Here we report the identification and cloning of mos14 (modifier of snc1-1, 14, a mutation that suppresses the immune responses conditioned by the auto-activated Resistance (R protein snc1 (suppressor of npr1-1, constitutive 1. MOS14 encodes a nuclear protein with high similarity to previously characterized TRN-SR proteins in animals. Yeast two-hybrid assays showed that MOS14 interacts with AtRAN1 via its N-terminus and SR proteins via its C-terminus. In mos14-1, localization of several SR proteins to the nucleus was impaired, confirming that MOS14 functions as a TRN-SR. The mos14-1 mutation results in altered splicing patterns of SNC1 and another R gene RPS4 and compromised resistance mediated by snc1 and RPS4, suggesting that nuclear import of SR proteins by MOS14 is required for proper splicing of these two R genes and is important for their functions in plant immunity.

  6. gld-1, a tumor suppressor gene required for oocyte development in Caenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    Francis, R.; Schedl, T. [Washington Univ. School of Medicine, St. Louis, MO (United States); Barton, M.K.; Kimble, J. [Univ. of Wisconsin, Madison, WI (United States)

    1995-02-01

    We have characterized 31 mutations in the gld-1 (defective in germline development) gene of Caenorhabditis elegans. In gld-1 (null) hermaphrodites, oogenesis is abolished and a germline tumor forms where oocyte development would normally occur. By contrast, gld-1 (null) males are unaffected. The hermaphrodite germline tumor appears to derive from germ cells that enter the meiotic pathway normally but then exit pachytene and return to the mitotic cycle. Certain gld-1 partial loss-of-function mutations also abolish oogenesis, but germ cells arrest in pachytene rather than returning to mitosis. Our results indicate that gld-1 is a tumor suppressor gene required for oocyte development. The tumorous phenotype suggests that gld-1(+) may function to negatively regulate proliferation during meiotic prophase and/or act to direct progression through meiotic prophase. We also show that gld-1(+) has an additional nonessential role in germline sex determination: promotion of hermaphrodite spermatogenesis. This function of gld-1 is inferred from a haplo-insufficient phenotype and from the properties of gain-of-function gld-1 mutations that cause alterations in the sexual identity of germ cells. 69 refs., 10 figs., 8 tabs.

  7. Multiple Functions of Let-23, a Caenorhabditis Elegans Receptor Tyrosine Kinase Gene Required for Vulval Induction

    Science.gov (United States)

    Aroian, R. V.; Sternberg, P. W.

    1991-01-01

    The let-23 gene, which encodes a putative tyrosine kinase of the epidermal growth factor (EGF) receptor subfamily, has multiple functions during Caenorhabditis elegans development. We show that let-23 function is required for vulval precursor cells (VPCs) to respond to the signal that induces vulval differentiation: a complete loss of let-23 function results in no induction. However, some let-23 mutations that genetically reduce but do not eliminate let-23 function result in VPCs apparently hypersensitive to inductive signal: as many as five of six VPCs can adopt vulval fates, in contrast to the three that normally do. These results suggest that the let-23 receptor tyrosine kinase controls two opposing pathways, one that stimulates vulval differentiation and another that negatively regulates vulval differentiation. Furthermore, analysis of 16 new let-23 mutations indicates that the let-23 kinase functions in at least five tissues. Since various let-23 mutant phenotypes can be obtained independently, the let-23 gene is likely to have tissue-specific functions. PMID:2071015

  8. PRMT1 mediated methylation of TAF15 is required for its positive gene regulatory function

    Energy Technology Data Exchange (ETDEWEB)

    Jobert, Laure; Argentini, Manuela [Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), CNRS UMR 7104, INSERM U 596, Universite Louis Pasteur de Strasbourg, BP 10142 - 67404 Illkirch Cedex, CU de Strasbourg (France); Tora, Laszlo, E-mail: laszlo@igbmc.u-strasbg.fr [Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), CNRS UMR 7104, INSERM U 596, Universite Louis Pasteur de Strasbourg, BP 10142 - 67404 Illkirch Cedex, CU de Strasbourg (France)

    2009-04-15

    TAF15 (formerly TAF{sub II}68) is a nuclear RNA-binding protein that is associated with a distinct population of TFIID and RNA polymerase II complexes. TAF15 harbours an N-terminal activation domain, an RNA recognition motif (RRM) and many Arg-Gly-Gly (RGG) repeats at its C-terminal end. The N-terminus of TAF15 serves as an essential transforming domain in the fusion oncoprotein created by chromosomal translocation in certain human chondrosarcomas. Post-transcriptional modifications (PTMs) of proteins are known to regulate their activity, however, nothing is known on how PTMs affect TAF15 function. Here we demonstrate that endogenous human TAF15 is methylated in vivo at its numerous RGG repeats. Furthermore, we identify protein arginine N-methyltransferase 1 (PRMT1) as a TAF15 interactor and the major PRMT responsible for its methylation. In addition, the RGG repeat-containing C-terminus of TAF15 is responsible for the shuttling between the nucleus and the cytoplasm and the methylation of RGG repeats affects the subcellular localization of TAF15. The methylation of TAF15 by PRMT1 is required for the ability of TAF15 to positively regulate the expression of the studied endogenous TAF15-target genes. Our findings demonstrate that arginine methylation of TAF15 by PRMT1 is a crucial event determining its proper localization and gene regulatory function.

  9. The Wilms' tumor gene Wt1 is required for normal development of the retina.

    Science.gov (United States)

    Wagner, Kay-Dietrich; Wagner, Nicole; Vidal, Valerie P I; Schley, Gunnar; Wilhelm, Dagmar; Schedl, Andreas; Englert, Christoph; Scholz, Holger

    2002-03-15

    The Wilms' tumor gene Wt1 is known for its important functions during genitourinary and mesothelial formation. Here we show that Wt1 is necessary for neuronal development in the vertebrate retina. Mouse embryos with targeted disruption of Wt1 exhibit remarkably thinner retinas than age-matched wild-type animals. A large fraction of retinal ganglion cells is lost by apoptosis, and the growth of optic nerve fibers is severely disturbed. Strikingly, expression of the class IV POU-domain transcription factor Pou4f2 (formerly Brn-3b), which is critical for the survival of most retinal ganglion cells, is lost in Wt1(-/-) retinas. Forced expression of Wt1 in cultured cells causes an up-regulation of Pou4f2 mRNA. Moreover, the Wt1(-KTS) splice variant can activate a reporter construct carrying 5'-regulatory sequences of the human POU4F2. The lack of Pou4f2 and the ocular defects in Wt1(-/-) embryos are rescued by transgenic expression of a 280 kb yeast artificial chromosome carrying the human WT1 gene. Taken together, our findings demonstrate a continuous requirement for Wt1 in normal retina formation with a critical role in Pou4f2-dependent ganglion cell differentiation.

  10. Predicting the size of the progeny mapping population required to positionally clone a gene.

    Science.gov (United States)

    Dinka, Stephen J; Campbell, Matthew A; Demers, Tyler; Raizada, Manish N

    2007-08-01

    A key frustration during positional gene cloning (map-based cloning) is that the size of the progeny mapping population is difficult to predict, because the meiotic recombination frequency varies along chromosomes. We describe a detailed methodology to improve this prediction using rice (Oryza sativa L.) as a model system. We derived and/or validated, then fine-tuned, equations that estimate the mapping population size by comparing these theoretical estimates to 41 successful positional cloning attempts. We then used each validated equation to test whether neighborhood meiotic recombination frequencies extracted from a reference RFLP map can help researchers predict the mapping population size. We developed a meiotic recombination frequency map (MRFM) for approximately 1400 marker intervals in rice and anchored each published allele onto an interval on this map. We show that neighborhood recombination frequencies (R-map, >280-kb segments) extracted from the MRFM, in conjunction with the validated formulas, better predicted the mapping population size than the genome-wide average recombination frequency (R-avg), with improved results whether the recombination frequency was calculated as genes/cM or kb/cM. Our results offer a detailed road map for better predicting mapping population size in diverse eukaryotes, but useful predictions will require robust recombination frequency maps based on sampling more progeny.

  11. Herbaspirillum seropedicae rfbB and rfbC genes are required for maize colonization.

    Science.gov (United States)

    Balsanelli, Eduardo; Serrato, Rodrigo V; de Baura, Valter A; Sassaki, Guilherme; Yates, Marshall G; Rigo, Liu Un; Pedrosa, Fábio O; de Souza, Emanuel M; Monteiro, Rose A

    2010-08-01

    In this study we disrupted two Herbaspirillum seropedicae genes, rfbB and rfbC, responsible for rhamnose biosynthesis and its incoporation into LPS. GC-MS analysis of the H. seropedicae wild-type strain LPS oligosaccharide chain showed that rhamnose, glucose and N-acetyl glucosamine are the predominant monosaccharides, whereas rhamnose and N-acetyl glucosamine were not found in the rfbB and rfbC strains. The electrophoretic pattern of the mutants LPS was drastically altered when compared with the wild type. Knockout of rfbB or rfbC increased the sensitivity towards SDS, polymyxin B sulfate and salicylic acid. The mutants attachment capacity to maize root surface plantlets was 100-fold lower than the wild type. Interestingly, the wild-type capacity to attach to maize roots was reduced to a level similar to that of the mutants when the assay was performed in the presence of isolated wild-type LPS, glucosamine or N-acetyl glucosamine. The mutant strains were also significantly less efficient in endophytic colonization of maize. Expression analysis indicated that the rfbB gene is upregulated by naringenin, apigenin and CaCl(2). Together, the results suggest that intact LPS is required for H. seropedicae attachment to maize root and internal colonization of plant tissues.

  12. Ascl1 is a required downstream effector of Gsx gene function in the embryonic mouse telencephalon

    Directory of Open Access Journals (Sweden)

    Allen Zegary J

    2009-02-01

    Full Text Available Abstract Background The homeobox gene Gsx2 (formerly Gsh2 is known to regulate patterning in the lateral ganglionic eminence (LGE of the embryonic telencephalon. In its absence, the closely related gene Gsx1 (previously known as Gsh1 can partially compensate in the patterning and differentiation of ventral telencephalic structures, such as the striatum. However, the cellular and molecular mechanisms underlying this compensation remain unclear. Results We show here that in the Gsx2 mutants Gsx1 is expressed in only a subset of the ventral telencephalic progenitors that normally express Gsx2. Based on the similarities in the expression of Gsx1 and Ascl1 (Mash1 within the Gsx2 mutant LGE, we examined whether Ascl1 plays an integral part in the Gsx1-based recovery. Ascl1 mutants show only modest alterations in striatal development; however, in Gsx2;Ascl1 double mutants, striatal development is severely affected, similar to that seen in the Gsx1;Gsx2 double mutants. This is despite the fact that Gsx1 is expressed, and even expands, in the Gsx2;Ascl1 mutant LGE, comparable to that seen in the Gsx2 mutant. Finally, Notch signaling has recently been suggested to be required for normal striatal development. In spite of the fact that Notch signaling is severely disrupted in Ascl1 mutants, it actually appears to be improved in the Gsx2;Ascl1 double mutants. Conclusion These results, therefore, reveal a non-proneural requirement of Ascl1 that together with Gsx1 compensates for the loss of Gsx2 in a subset of LGE progenitors.

  13. Modularity of Plant Metabolic Gene Clusters: A Trio of Linked Genes That Are Collectively Required for Acylation of Triterpenes in Oat[W][OA

    Science.gov (United States)

    Mugford, Sam T.; Louveau, Thomas; Melton, Rachel; Qi, Xiaoquan; Bakht, Saleha; Hill, Lionel; Tsurushima, Tetsu; Honkanen, Suvi; Rosser, Susan J.; Lomonossoff, George P.; Osbourn, Anne

    2013-01-01

    Operon-like gene clusters are an emerging phenomenon in the field of plant natural products. The genes encoding some of the best-characterized plant secondary metabolite biosynthetic pathways are scattered across plant genomes. However, an increasing number of gene clusters encoding the synthesis of diverse natural products have recently been reported in plant genomes. These clusters have arisen through the neo-functionalization and relocation of existing genes within the genome, and not by horizontal gene transfer from microbes. The reasons for clustering are not yet clear, although this form of gene organization is likely to facilitate co-inheritance and co-regulation. Oats (Avena spp) synthesize antimicrobial triterpenoids (avenacins) that provide protection against disease. The synthesis of these compounds is encoded by a gene cluster. Here we show that a module of three adjacent genes within the wider biosynthetic gene cluster is required for avenacin acylation. Through the characterization of these genes and their encoded proteins we present a model of the subcellular organization of triterpenoid biosynthesis. PMID:23532069

  14. miR-200c and GATA binding protein 4 regulate human embryonic stem cell renewal and differentiation

    Directory of Open Access Journals (Sweden)

    Hsiao-Ning Huang

    2014-03-01

    Full Text Available Human embryonic stem cells (hESCs are functionally unique for their self-renewal ability and pluripotency, but the molecular mechanisms giving rise to these properties are not fully understood. hESCs can differentiate into embryoid bodies (EBs containing ectoderm, mesoderm, and endoderm. In the miR-200 family, miR-200c was especially enriched in undifferentiated hESCs and significantly downregulated in EBs. The knockdown of the miR-200c in hESCs downregulated Nanog expression, upregulated GATA binding protein 4 (GATA4 expression, and induced hESC apoptosis. The knockdown of GATA4 rescued hESC apoptosis induced by downregulation of miR-200c. miR-200c directly targeted the 3′-untranslated region of GATA4. Interestingly, the downregulation of GATA4 significantly inhibited EB formation in hESCs. Overexpression of miR-200c inhibited EB formation and repressed the expression of ectoderm, endoderm, and mesoderm markers, which could partially be rescued by ectopic expression of GATA4. Fibroblast growth factor (FGF and activin A/nodal can sustain hESC renewal in the absence of feeder layer. Inhibition of transforming growth factor-β (TGF-β/activin A/nodal signaling by SB431542 treatment downregulated the expression of miR-200c. Overexpression of miR-200c partially rescued the expression of Nanog/phospho-Smad2 that was downregulated by SB431542 treatment. Our observations have uncovered novel functions of miR-200c and GATA4 in regulating hESC renewal and differentiation.

  15. Expression of T -bet/GATA -3 in decidual tissue of patients with preeclampsia%T - bet/GATA -3在子痫前期患者蜕膜组织中的表达

    Institute of Scientific and Technical Information of China (English)

    杨艳红; 杨海澜; 韩方

    2011-01-01

    Objective: To compare the expression levels of T - bet/GATA - 3 in decidual tissues between normal pregnant women and patients with preeclampsia, explore their relationships with the occurrence of preeclampsia. Methods; A case - control study was performed, 13 patients with mild preeclampsia and 13 patients with severe preeclampsia were selected as study group, 14 normal pregnant women were selected as control group. Western blotting was used to compare the expression levels of T - bet/GATA - 3 in the three groups. Results: The expression level of T - bet in study group was higher than that in control group, the expression level of T - bet in severe preeclampsia group was higher than that in mild preeclampsia group, there was significant difference (P <0.05) . The expression level of GATA -3 in study group was lower than that in control group, the expression level of GATA -3 in severe preeclampsia group was lower than that in mild preeclampsia group, there was significant difference. There was significant difference in the ratio of T - bet/GATA - 3 among the three groups, and the ratio of T - bet/GATA - 3 increased with the aggravation of the disease. There was a significant negative correlation between T - bet and GATA - 3 (γ = - 0. 895, P < 0.05 ) . Conclusion: The unbalance and disorder of T - bet expression and GATA - 3 expression is an important link in the immune pathogenesis of preeclampsia, Thl/Th2 unbalance may be a pathogenesis of hypertensive disorder complicating pregnancy.%目的:比较T - bet/CATA -3在正常妊娠和子痫前期患者蜕膜组织中的表达水平,并探讨两者与子痫前期发病的关系.方法:采用病例对照研究方法,选择轻度子痫前期和重度子痫前期患者各13例作为研究组,选择14例正常妊娠孕妇为对照组,用Western bloting方法比较三组孕妇蜕膜中T- bet/GATA -3的表达水平.结果:①研究组T- bet表达水平比对照组高,重度子痫前期组比轻度组高,各组比

  16. Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R gene-mediated signaling pathways in Arabidopsis.

    Science.gov (United States)

    Aarts, N; Metz, M; Holub, E; Staskawicz, B J; Daniels, M J; Parker, J E

    1998-08-18

    The Arabidopsis genes EDS1 and NDR1 were shown previously by mutational analysis to encode essential components of race-specific disease resistance. Here, we examined the relative requirements for EDS1 and NDR1 by a broad spectrum of Resistance (R) genes present in three Arabidopsis accessions (Columbia, Landsberg-erecta, and Wassilewskija). We show that there is a strong requirement for EDS1 by a subset of R loci (RPP2, RPP4, RPP5, RPP21, and RPS4), conferring resistance to the biotrophic oomycete Peronospora parasitica, and to Pseudomonas bacteria expressing the avirulence gene avrRps4. The requirement for NDR1 by these EDS1-dependent R loci is either weak or not measurable. Conversely, three NDR1-dependent R loci, RPS2, RPM1, and RPS5, operate independently of EDS1. Another RPP locus, RPP8, exhibits no strong exclusive requirement for EDS1 or NDR1 in isolate-specific resistance to P. parasitica, although resistance is compromised weakly by eds1. Similarly, resistance conditioned by two EDS1-dependent RPP genes, RPP4 and RPP5, is impaired partially by ndr1, implicating a degree of pathway cross-talk. Our results provide compelling evidence for the preferential utilization of either signaling component by particular R genes and thus define at least two disease resistance pathways. The data also suggest that strong dependence on EDS1 or NDR1 is governed by R protein structural type rather than pathogen class.

  17. A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression

    Directory of Open Access Journals (Sweden)

    Amsterdam Adam

    2006-06-01

    Full Text Available Abstract Background Craniofacial birth defects result from defects in cranial neural crest (NC patterning and morphogenesis. The vertebrate craniofacial skeleton is derived from cranial NC cells and the patterning of these cells occurs within the pharyngeal arches. Substantial efforts have led to the identification of several genes required for craniofacial skeletal development such as the endothelin-1 (edn1 signaling pathway that is required for lower jaw formation. However, many essential genes required for craniofacial development remain to be identified. Results Through screening a collection of insertional zebrafish mutants containing approximately 25% of the genes essential for embryonic development, we present the identification of 15 essential genes that are required for craniofacial development. We identified 3 genes required for hyomandibular development. We also identified zebrafish models for Campomelic Dysplasia and Ehlers-Danlos syndrome. To further demonstrate the utility of this method, we include a characterization of the wdr68 gene. We show that wdr68 acts upstream of the edn1 pathway and is also required for formation of the upper jaw equivalent, the palatoquadrate. We also present evidence that the level of wdr68 activity required for edn1 pathway function differs between the 1st and 2nd arches. Wdr68 interacts with two minibrain-related kinases, Dyrk1a and Dyrk1b, required for embryonic growth and myotube differentiation, respectively. We show that a GFP-Wdr68 fusion protein localizes to the nucleus with Dyrk1a in contrast to an engineered loss of function mutation Wdr68-T284F that no longer accumulated in the cell nucleus and failed to rescue wdr68 mutant animals. Wdr68 homologs appear to exist in all eukaryotic genomes. Notably, we found that the Drosophila wdr68 homolog CG14614 could substitute for the vertebrate wdr68 gene even though insects lack the NC cell lineage. Conclusion This work represents a systematic

  18. Rectal Delivery of a DNAzyme That Specifically Blocks the Transcription Factor GATA3 and Reduces Colitis in Mice.

    Science.gov (United States)

    Popp, Vanessa; Gerlach, Katharina; Mott, Stefanie; Turowska, Agnieszka; Garn, Holger; Atreya, Raja; Lehr, Hans-Anton; Ho, I-Cheng; Renz, Harald; Weigmann, Benno; Neurath, Markus F

    2017-01-01

    GATA3 is a transcription factor that regulates T-cell production of cytokines. We investigated the role of GATA3 in development of colitis in mice. We performed quantitative polymerase chain reaction and immunofluorescence analyses of colon tissues from patients with Crohn's disease (n = 61) or ulcerative colitis (UC, n = 74) or from patients without inflammatory bowel diseases (n = 22), to measure levels of GATA3. Colitis was induced by administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid to control mice, mice with T-cell-specific deletion of GATA3, and mice with deletion of tumor necrosis factor receptor (TNFR) 1 and TNFR2 (TNFR double knockouts); some mice were given a GATA3-specific DNAzyme (hgd40) or a control DNAzyme via intrarectal administration, or systemic injections of an antibody to TNF before or during sensitization and challenge phase of colitis induction. Colon tissues were collected and immunofluorescence and histochemical analyses were performed. Lamina propria mononuclear cells and T cells were isolated and analyzed by flow cytometry or cytokine assays. Colonic distribution of labeled DNAzyme and inflammation were monitored by in vivo imaging (endoscopy) of mice. Levels of GATA3 messenger RNA were higher in colon tissues from patients with UC, but not ileal Crohn's disease, than control tissues; levels of GATA3 correlated with levels of inflammatory cytokines (interleukin [IL] 9, IL17A, IL6, IL5, IL4, IL13, and TNF). We observed increased expression of GATA3 by lamina propria T cells from mice with colitis compared with controls. Mice with T-cell-specific deletion of GATA3 did not develop colitis and their colonic tissues did not produce inflammatory cytokines (IL6, IL9, or IL13). The DNAzyme hgd40 inhibited expression of GATA3 messenger RNA by unstimulated and stimulated T cells, and distributed throughout the inflamed colons of mice with colitis. Colon tissues from mice given hgd40 had reduced expression of GATA3 messenger RNA

  19. Evolution of New cis-Regulatory Motifs Required for Cell-Specific Gene Expression in Caenorhabditis.

    Directory of Open Access Journals (Sweden)

    Michalis Barkoulas

    2016-09-01

    Full Text Available Patterning of C. elegans vulval cell fates relies on inductive signaling. In this induction event, a single cell, the gonadal anchor cell, secretes LIN-3/EGF and induces three out of six competent precursor cells to acquire a vulval fate. We previously showed that this developmental system is robust to a four-fold variation in lin-3/EGF genetic dose. Here using single-molecule FISH, we find that the mean level of expression of lin-3 in the anchor cell is remarkably conserved. No change in lin-3 expression level could be detected among C. elegans wild isolates and only a low level of change-less than 30%-in the Caenorhabditis genus and in Oscheius tipulae. In C. elegans, lin-3 expression in the anchor cell is known to require three transcription factor binding sites, specifically two E-boxes and a nuclear-hormone-receptor (NHR binding site. Mutation of any of these three elements in C. elegans results in a dramatic decrease in lin-3 expression. Yet only a single E-box is found in the Drosophilae supergroup of Caenorhabditis species, including C. angaria, while the NHR-binding site likely only evolved at the base of the Elegans group. We find that a transgene from C. angaria bearing a single E-box is sufficient for normal expression in C. elegans. Even a short 58 bp cis-regulatory fragment from C. angaria with this single E-box is able to replace the three transcription factor binding sites at the endogenous C. elegans lin-3 locus, resulting in the wild-type expression level. Thus, regulatory evolution occurring in cis within a 58 bp lin-3 fragment, results in a strict requirement for the NHR binding site and a second E-box in C. elegans. This single-cell, single-molecule, quantitative and functional evo-devo study demonstrates that conserved expression levels can hide extensive change in cis-regulatory site requirements and highlights the evolution of new cis-regulatory elements required for cell-specific gene expression.

  20. A multistep genomic screen identifies new genes required for repair of DNA double-strand breaks in Saccharomyces cerevisiae.

    Science.gov (United States)

    McKinney, Jennifer Summers; Sethi, Sunaina; Tripp, Jennifer DeMars; Nguyen, Thuy N; Sanderson, Brian A; Westmoreland, James W; Resnick, Michael A; Lewis, L Kevin

    2013-04-15

    Efficient mechanisms for rejoining of DNA double-strand breaks (DSBs) are vital because misrepair of such lesions leads to mutation, aneuploidy and loss of cell viability. DSB repair is mediated by proteins acting in two major pathways, called homologous recombination and nonhomologous end-joining. Repair efficiency is also modulated by other processes such as sister chromatid cohesion, nucleosome remodeling and DNA damage checkpoints. The total number of genes influencing DSB repair efficiency is unknown. To identify new yeast genes affecting DSB repair, genes linked to gamma radiation resistance in previous genome-wide surveys were tested for their impact on repair of site-specific DSBs generated by in vivo expression of EcoRI endonuclease. Eight members of the RAD52 group of DNA repair genes (RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, MRE11 and XRS2) and 73 additional genes were found to be required for efficient repair of EcoRI-induced DSBs in screens utilizing both MATa and MATα deletion strain libraries. Most mutants were also sensitive to the clastogenic chemicals MMS and bleomycin. Several of the non-RAD52 group genes have previously been linked to DNA repair and over half of the genes affect nuclear processes. Many proteins encoded by the protective genes have previously been shown to associate physically with each other and with known DNA repair proteins in high-throughput proteomics studies. A majority of the proteins (64%) share sequence similarity with human proteins, suggesting that they serve similar functions. We have used a genetic screening approach to detect new genes required for efficient repair of DSBs in Saccharomyces cerevisiae. The findings have spotlighted new genes that are critical for maintenance of genome integrity and are therefore of greatest concern for their potential impact when the corresponding gene orthologs and homologs are inactivated or polymorphic in human cells.

  1. Identification and characterization of genes required for compensatory growth in Drosophila.

    Science.gov (United States)

    Gerhold, Abigail R; Richter, Daniel J; Yu, Albert S; Hariharan, Iswar K

    2011-12-01

    To maintain tissue homeostasis, some organs are able to replace dying cells with additional proliferation of surviving cells. Such proliferation can be localized (e.g., a regeneration blastema) or diffuse (compensatory growth). The relationship between such growth and the growth that occurs during development has not been characterized in detail. Drosophila melanogaster larval imaginal discs can recover from extensive damage, producing normally sized adult organs. Here we describe a system using genetic mosaics to screen for recessive mutations that impair compensatory growth. By generating clones of cells that carry a temperature-sensitive cell-lethal mutation, we conditionally ablate patches of tissue in the imaginal disc and assess the ability of the surviving sister clones to replace the lost tissue. We have used this system together with a modified whole-genome resequencing (WGS) strategy to identify several mutations that selectively compromise compensatory growth. We find specific alleles of bunched (bun) and Ribonucleoside diphosphate reductase large subunit (RnrL) reduce compensatory growth in the imaginal disc. Other genes identified in the screen, including two alleles of Topoisomerase 3-alpha (Top3α), while also required for developmental growth, appear to have an enhanced requirement during compensatory growth. Compensatory growth occurs at a higher rate than normal growth and may therefore have features in common with some types of overgrowth. Indeed, the RnrL allele identified compromises both these types of altered growth and mammalian ribonucleotide reductase and topoisomerases are targets of anticancer drugs. Finally, the approach we describe is applicable to the study of compensatory growth in diverse tissues in Drosophila.

  2. The pam1 gene is required for meiotic bouquet formation and efficient homologous synapsis in maize (Zea mays L.).

    OpenAIRE

    Golubovskaya, Inna N.; Harper, Lisa C.; Pawlowski, Wojciech P.; Schichnes, Denise; Cande, W. Zacheus

    2002-01-01

    The clustering of telomeres on the nuclear envelope (NE) during meiotic prophase to form the bouquet arrangement of chromosomes may facilitate homologous chromosome synapsis. The pam1 (plural abnormalities of meiosis 1) gene is the first maize gene that appears to be required for telomere clustering, and homologous synapsis is impaired in pam1. Telomere clustering on the NE is arrested or delayed at an intermediate stage in pam1. Telomeres associate with the NE during the leptotene-zygotene t...

  3. Symbiotic plasmid is required for NolR to fully repress nodulation genes in Rhizobium leguminosarum A34

    Institute of Scientific and Technical Information of China (English)

    Fengqing Li; Bihe Hou; Guofan Hong

    2008-01-01

    NolR is a reguiator of noduiation genes present in Rhizobium and Sinorhizobium. However, the mechanism by which NolR participates in the inducible transcription ofnoduiation genes remains unclear. To investigate whether there are other factors regulating the function of NoIR, an insertion mutant of NolR in Rhizobium leguminosarum strain 8401, which lacks the symbiotic plasmid, was constructed by homologous recombination. We investigated the effects of NolR inactivation on the expression of nodulation genes. Three inducible nodulation genes (nodA, nodF and nodM) were expressed constitutively in NoiR-mutant, MRl14. Our results suggested that the symbiotic plasmid is required for NolR to fully repress nodulation genes in Rhizobium ieguminosarum A34. In addition, MRl14 has provided a useful tool for further study of molecular interactions between NolR and other factors.

  4. The type VI secretion system gene cluster of Salmonella typhimurium: required for full virulence in mice.

    Science.gov (United States)

    Liu, Ji; Guo, Ji-Tao; Li, Yong-Guo; Johnston, Randal N; Liu, Gui-Rong; Liu, Shu-Lin

    2013-07-01

    Type VI secretion system (T6SS) has increasingly been believed to participate in the infection process for many bacterial pathogens, but its role in the virulence of Salmonella typhimurium remains unclear. To look into this, we deleted the T6SS cluster from the genome of S. typhimurium 14028s and analyzed the phenotype of the resulting T6SS knockout mutant (T6SSKO mutant) in vitro and in vivo. We found that the T6SSKO mutant exhibited reduced capability in colonizing the spleen and liver in an in vivo colonization competition model in BALB/c mice infected by the oral route. Additionally, infection via intraperitoneal administration also showed that the T6SSKO mutant was less capable of colonizing the mouse spleen and liver than the wild-type strain. We did not detect significant differences between the T6SSKO and wild-type strains in epithelial cell invasion tests. However, in the macrophage RAW264.7 cell line, the T6SSKO mutant survived and proliferated significantly more poorly than the wild-type strain. These findings indicate that T6SS gene cluster is required for full virulence of S. typhimurium 14028s in BALB/c mice, possibly due to its roles in bacterial survival and proliferation in macrophages.

  5. The Ciliopathy Gene ahi1 Is Required for Zebrafish Cone Photoreceptor Outer Segment Morphogenesis and Survival

    Science.gov (United States)

    Lessieur, Emma M.; Fogerty, Joseph; Gaivin, Robert J.; Song, Ping; Perkins, Brian D.

    2017-01-01

    Purpose Joubert syndrome (JBTS) is an autosomal recessive ciliopathy with considerable phenotypic variability. In addition to central nervous system abnormalities, a subset of JBTS patients exhibit retinal dystrophy and/or kidney disease. Mutations in the AHI1 gene are causative for approximately 10% of all JBTS cases. The purpose of this study was to generate ahi1 mutant alleles in zebrafish and to characterize the retinal phenotypes. Methods Zebrafish ahi1 mutants were generated using transcription activator-like effector nucleases (TALENs). Expression analysis was performed by whole-mount in situ hybridization. Anatomic and molecular characterization of photoreceptors was investigated by histology, electron microscopy, and immunohistochemistry. The optokinetic response (OKR) behavior assay was used to assess visual function. Kidney cilia were evaluated by whole-mount immunostaining. Results The ahi1lri46 mutation in zebrafish resulted in shorter cone outer segments but did not affect visual behavior at 5 days after fertilization (dpf). No defects in rod morphology or rhodopsin localization were observed at 5 dpf. By 5 months of age, cone degeneration and rhodopsin mislocalization in rod photoreceptors was observed. The connecting cilium formed normally and Cc2d2a and Cep290 localized properly. Distal pronephric duct cilia were absent in mutant fish; however, only 9% of ahi1 mutants had kidney cysts by 5 dpf, suggesting that the pronephros remained largely functional. Conclusions The results indicate that Ahi1 is required for photoreceptor disc morphogenesis and outer segment maintenance in zebrafish. PMID:28118669

  6. The yeast SEN1 gene is required for the processing of diverse RNA classes.

    Science.gov (United States)

    Ursic, D; Himmel, K L; Gurley, K A; Webb, F; Culbertson, M R

    1997-12-01

    A single base change in the helicase superfamily 1 domain of the yeast Saccharomyces cerevisiae SEN1 gene results in a heat-sensitive mutation that alters the cellular abundance of many RNA species. We compared the relative amounts of RNAs between cells that are wild-type and mutant after temperature-shift. In the mutant several RNAs were found to either decrease or increase in abundance. The affected RNAs include tRNAs, rRNAs and small nuclear and nucleolar RNAs. Many of the affected RNAs have been positively identified and include end-matured precursor tRNAs and the small nuclear and nucleolar RNAs U5 and snR40 and snR45. Several small nucleolar RNAs co-immunoprecipitate with Sen1 but differentially associate with the wild-type and mutant protein. Its inactivation also impairs precursor rRNA maturation, resulting in increased accumulation of 35S and 6S precursor rRNAs and reduced levels of 20S, 23S and 27S rRNA processing intermediates. Thus, Sen1 is required for the biosynthesis of various functionally distinct classes of nuclear RNAs. We propose that Sen1 is an RNA helicase acting on a wide range of RNA classes. Its effects on the targeted RNAs in turn enable ribonuclease activity.

  7. Purine biosynthetic genes are required for cadmium tolerance in Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Speiser, D.M.; Ortiz, D.F.; Kreppel, L.; Scheel, G.; McDonald, G.; Ow, D.W. (Dept. of Agriculture, Albany, CA (United States) Univ. of California, Berkeley (United States))

    1992-12-01

    Phytochelatins (PCs) are metal-chelating peptides produced in plants and some fungi in response to heavy metal exposure. A Cd-sensitive mutant of the fission yeast Schizosaccharomyces pombe, defective in production of a PC-Cd-sulfide complex essential for metal tolerance, was found to harbor mutations in specific genes of the purine biosynthetic pathway. Genetic analysis of the link between metal complex accumulation and purine biosynthesis enzymes revealed that genetic lesions blocking two segments of the pathway, before and after the IMP branchpoint, are required to produce the Cd-sensitive phenotype. The biochemical functions of these two segments of the pathway are similar, and a model based on the alternate use of a sulfur analog substrate is presented. The novel participation of purine biosynthesis enzymes in the conversion of the PC-Cd complex to the PC-Cd-sulfide complex in the fission yeast raises an intriguing possibility that these same enzymes might have a role in sulfur metabolism in the fission yeast S. pombe, and perhaps in other biological systems. 41 refs., 8 figs., 2 tabs.

  8. Cloning and Expression of the Lactococcus lactis purDEK Genes, Required for Growth in Milk

    DEFF Research Database (Denmark)

    Nilsson, Dan; Kilstrup, Mogens

    1998-01-01

    An operon containing the genes purD and purE and part of the purK gene was cloned from the facultative anaerobic gram positive bacterium Lactococcus lactis by complementation of the purD mutation in Escherichia coli SO609. The genes encode enzymes in the de novo pathway of purine nucleotides....... The expression of the genes was regulated approximately 35-fold at the transcription level by the availability of purines in the growth medium. Deletion analysis of the nucleotide region upstream of purD indicated that a region of 145 bp is enough to give regulated expression of the reporter lacLM genes, which...

  9. EDS1 in tomato is required for resistance mediated by TIR-class R genes and the receptor-like R gene Ve.

    Science.gov (United States)

    Hu, Gongshe; deHart, Amy K A; Li, Yansu; Ustach, Carolyn; Handley, Vanessa; Navarre, Roy; Hwang, Chin-Feng; Aegerter, Brenna J; Williamson, Valerie M; Baker, Barbara

    2005-05-01

    In tobacco and other Solanaceae species, the tobacco N gene confers resistance to tobacco mosaic virus (TMV), and leads to induction of standard defense and resistance responses. Here, we report the use of N-transgenic tomato to identify a fast-neutron mutant, sun1-1 (suppressor of N), that is defective in N-mediated resistance. Induction of salicylic acid (SA) and expression of pathogenesis-related (PR) genes, each signatures of systemic acquired resistance, are both dramatically suppressed in sun1-1 plants after TMV treatment compared to wild-type plants. Application of exogenous SA restores PR gene expression, indicating that SUN1 acts upstream of SA. Upon challenge with additional pathogens, we found that the sun1-1 mutation impairs resistance mediated by certain resistance (R) genes, (Bs4, I, and Ve), but not others (Mi-1). In addition, sun1-1 plants exhibit enhanced susceptibility to TMV, as well as to virulent pathogens. sun1-1 has been identified as an EDS1 homolog present on chromosome 6 of tomato. The discovery of enhanced susceptibility in the sun1-1 (Le_eds1-1) mutant plant, which contrasts to reports in Nicotiana benthamiana using virus-induced gene silencing, provides evidence that the intersection of R gene-mediated pathways with general resistance pathways is conserved in a Solanaceous species. In tomato, EDS1 is important for mediating resistance to a broad range of pathogens (viral, bacterial, and fungal pathogens), yet shows specificity in the class of R genes that it affects (TIR-NBS-LRR as opposed to CC-NBS-LRR). In addition, a requirement for EDS1 for Ve-mediated resistance in tomato exposes that the receptor-like R gene class may also require EDS1.

  10. Inhibitory effect of melatonin on testosterone synthesis is mediated via GATA-4/SF-1 transcription factors.

    Science.gov (United States)

    Qin, Fenju; Zhang, Jie; Zan, Linsen; Guo, Weiqiang; Wang, Jin; Chen, Lili; Cao, Yi; Shen, Ouxi; Tong, Jian

    2015-11-01

    The aim of the present study was to elucidate whether the GATA-4/SF-1 signalling pathway is involved in the inhibitory effects of melatonin on testosterone production in both the TM3 Leydig cell line and in C57BL/6J mice. In-vitro experiments demonstrated that melatonin treatment significantly reduced testosterone levels in cell culture medium (P SF-1 (NR5A1), StAR, P450SCC (CYP11A1) and 3β-HSD (P SF-1 (P SF-1 expression.

  11. A CHROMATIN MODIFYING ENZYME, SDG8, IS REQUIRED FOR MORPHOLOGICAL, GENE EXPRESSION, AND EPIGENETIC RESPONSES TO MECHANICAL STIMULATION

    Directory of Open Access Journals (Sweden)

    Christopher Ian Cazzonelli

    2014-10-01

    Full Text Available Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzyme, SDG8/ASHH2, which can regulate the expression of many touch responsive genes identified in Arabidopsis. SDG8 is required for the permissive expression of touch induced genes; and the loss of function of sdg8 perturbs the maximum levels of induction on selected touch gene targets. SDG8 is required to maintain permissive H3K4 trimethylation marks surrounding the Arabidopsis touch-inducible gene TOUCH 3 (TCH3, which encodes a calmodulin-like protein (CML12. The gene neighbouring was also slightly down regulated, revealing a new target for SDG8 mediated chromatin modification. Finally, sdg8 mutants show perturbed morphological response to wind-agitated mechanical stimuli, implicating an epigenetic memory-forming process in the acclimation response of thigmomorphogenesis.

  12. A novel splice donor site in the gag-pol gene is required for HIV-1 RNA stability

    NARCIS (Netherlands)

    M. Lutzelberger; L.S. Reinert; A.T. Das; B. Berkhout; J. Kjems

    2006-01-01

    Productive infection and successful replication of human immunodeficiency virus 1 (HIV-1) requires the balanced expression of all viral genes. This is achieved by a combination of alternative splicing events and regulated nuclear export of viral RNA. Because viral splicing is incomplete and intron-c

  13. Determination of variants in the 3'-region of the Tyrosinase gene requires locus specific amplification.

    NARCIS (Netherlands)

    Chaki, M.; Mukhopadhyay, A.; Ray, K.

    2005-01-01

    Mutations in the Tyrosinase gene (TYR, 11q14-q21) cause oculocutaneous albinism type 1 (OCA1). The 3'-region of the TYR shows 98.55% sequence identity with a pseudogene, known as Tyrosinase-Like Gene (TYRL, 11p11.2-cen). A large number of publicly available nucleotide variants of TYR in this region

  14. Halomonas indalinina sp. nov., a moderately halophilic bacterium isolated from a solar saltern in Cabo de Gata, Almeria, southern Spain.

    Science.gov (United States)

    Cabrera, Antonio; Aguilera, Margarita; Fuentes, Susana; Incerti, Claudia; Russell, Nick J; Ramos-Cormenzana, Alberto; Monteoliva-Sánchez, Mercedes

    2007-02-01

    A moderately halophilic bacterium, strain CG2.1T, isolated from a solar saltern at Cabo de Gata, a wildlife reserve located in the province of Almería, southern Spain, was subjected to a polyphasic taxonomic study. This organism was an aerobic, motile, Gram-negative rod that produced orange-pigmented colonies. Strain CG2.1T was able to grow at salinities of 3-25 % (w/v) and at temperatures of 15-40 degrees C. The pH range for growth was 5-9. Strain CG2.1T was a heterotroph capable of utilizing various carbohydrates as carbon sources. The organism reduced nitrate and showed phenylalanine deaminase activity. The major fatty acids were C(18 : 1)omega7c, C(16 : 0) and C(19 : 0) cyclo omega8c. The DNA G+C content was 60.9 mol%. On the basis of the phenotypic and phylogenetic data, strain CG2.1T appeared to be a member of the genus Halomonas and clustered closely with Halomonas marisflavi (97.1 % 16S rRNA gene sequence similarity). However, the level of DNA-DNA relatedness between the novel isolate and the most closely related Halomonas species was low. On the basis of these data, strain CG2.1T represents a novel member of the genus Halomonas, for which the name Halomonas indalinina is proposed. The type strain is CG2.1T (=CECT 5902T=LMG 23625T).

  15. Zfy genes are required for efficient meiotic sex chromosome inactivation (MSCI) in spermatocytes.

    Science.gov (United States)

    Vernet, Nadège; Mahadevaiah, Shantha K; de Rooij, Dirk G; Burgoyne, Paul S; Ellis, Peter J I

    2016-10-13

    During spermatogenesis, germ cells that fail to synapse their chromosomes or fail to undergo meiotic sex chromosome inactivation (MSCI) are eliminated via apoptosis during mid-pachytene. Previous work showed that Y-linked genes Zfy1 and Zfy2 act as 'executioners' for this checkpoint, and that wrongful expression of either gene during pachytene triggers germ cell death. Here, we show that in mice, Zfy genes are also necessary for efficient MSCI and the sex chromosomes are not correctly silenced in Zfy-deficient spermatocytes. This unexpectedly reveals a triple role for Zfy at the mid-pachytene checkpoint in which Zfy genes first promote MSCI, then monitor its progress (since if MSCI is achieved, Zfy genes will be silenced), and finally execute cells with MSCI failure. This potentially constitutes a negative feedback loop governing this critical checkpoint mechanism.

  16. Expansion of the Clavulanic Acid Gene Cluster: Identification and In Vivo Functional Analysis of Three New Genes Required for Biosynthesis of Clavulanic Acid by Streptomyces clavuligerus

    Science.gov (United States)

    Li, Rongfeng; Khaleeli, Nusrat; Townsend, Craig A.

    2000-01-01

    Clavulanic acid is a potent inhibitor of β-lactamase enzymes and is of demonstrated value in the treatment of infections by β-lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strain Streptomyces clavuligerus were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219–236, In V. P. Gullo et al., ed., Development in industrial microbiology series, 1993). In contrast, we report the identification of three new genes, orf10 (cyp), orf11 (fd), and orf12, that are required for clavulanic acid biosynthesis as indicated by gene replacement and trans-complementation analysis in S. clavuligerus. These genes are contained within a 3.4-kb DNA fragment located directly downstream of orf9 (cad) in the clavulanic acid cluster. While the orf10 (cyp) and orf11 (fd) proteins show homologies to other known CYP-150 cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by orf12 shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed. PMID:10869089

  17. The nuclear receptor genes HR3 and E75 are required for the circadian rhythm in a primitive insect.

    Directory of Open Access Journals (Sweden)

    Yuichi Kamae

    Full Text Available Insect circadian rhythms are generated by a circadian clock consisting of transcriptional/translational feedback loops, in which CYCLE and CLOCK are the key elements in activating the transcription of various clock genes such as timeless (tim and period (per. Although the transcriptional regulation of Clock (Clk has been profoundly studied, little is known about the regulation of cycle (cyc. Here, we identify the orphan nuclear receptor genes HR3 and E75, which are orthologs of mammalian clock genes, Rorα and Rev-erbα, respectively, as factors involved in the rhythmic expression of the cyc gene in a primitive insect, the firebrat Thermobia domestica. Our results show that HR3 and E75 are rhythmically expressed, and their normal, rhythmic expression is required for the persistence of locomotor rhythms. Their RNAi considerably altered the rhythmic transcription of not only cyc but also tim. Surprisingly, the RNAi of HR3 revealed the rhythmic expression of Clk, suggesting that this ancestral insect species possesses the mechanisms for rhythmic expression of both cyc and Clk genes. When either HR3 or E75 was knocked down, tim, cyc, and Clk or tim and cyc, respectively, oscillated in phase, suggesting that the two genes play an important role in the regulation of the phase relationship among the clock genes. Interestingly, HR3 and E75 were also found to be involved in the regulation of ecdysis, suggesting that they interconnect the circadian clock and developmental processes.

  18. The nuclear receptor genes HR3 and E75 are required for the circadian rhythm in a primitive insect.

    Science.gov (United States)

    Kamae, Yuichi; Uryu, Outa; Miki, Taiki; Tomioka, Kenji

    2014-01-01

    Insect circadian rhythms are generated by a circadian clock consisting of transcriptional/translational feedback loops, in which CYCLE and CLOCK are the key elements in activating the transcription of various clock genes such as timeless (tim) and period (per). Although the transcriptional regulation of Clock (Clk) has been profoundly studied, little is known about the regulation of cycle (cyc). Here, we identify the orphan nuclear receptor genes HR3 and E75, which are orthologs of mammalian clock genes, Rorα and Rev-erbα, respectively, as factors involved in the rhythmic expression of the cyc gene in a primitive insect, the firebrat Thermobia domestica. Our results show that HR3 and E75 are rhythmically expressed, and their normal, rhythmic expression is required for the persistence of locomotor rhythms. Their RNAi considerably altered the rhythmic transcription of not only cyc but also tim. Surprisingly, the RNAi of HR3 revealed the rhythmic expression of Clk, suggesting that this ancestral insect species possesses the mechanisms for rhythmic expression of both cyc and Clk genes. When either HR3 or E75 was knocked down, tim, cyc, and Clk or tim and cyc, respectively, oscillated in phase, suggesting that the two genes play an important role in the regulation of the phase relationship among the clock genes. Interestingly, HR3 and E75 were also found to be involved in the regulation of ecdysis, suggesting that they interconnect the circadian clock and developmental processes.

  19. Construction of two YAC contigs in human xp11.23-p11.22, one encompassing the loci OATL1, GATA, TFE3, and SYP, the other linking DXS255 to DXS146

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, S.E.; Hatchwell, E.; Chand, A.; Ockenden, N.; Craig, I.W. [Univ. of Oxford, Oxford (United Kingdom)] [and others

    1995-09-20

    We have constructed two YAC contigs in the Xp11.23-p11.22 interval of the human X chromosome, a region that was previously poorly characterized. One contig, of at least 1.4 Mb, links the pseudogene OATL1 to the genes GATA1, TFE3, and SYP and also contains loci implicated in Wiskott-Aldrich syndrome and synovial sarcoma. A second contig, mapping proximal to the first, is estimated to be over 2.1 Mb and links the hypervariable locus DXS255 to DXS146, and also contains a chloride channel gene that is responsible for hereditary nephrolithiasis. We have used plasmid rescue, inverse PCR, and Alu-PCR to generate 20 novel markers from this region, 1 of which is polymorphic, and have positioned these relative to one another on the basis of YAC analysis. The order of previously known markers within our contigs, Xpter-OATL1-GATA-TFE3-SYP-DXS255-DXS146-Xcen, agrees with genomic pulsed-field maps of the region. In addition, we have constructed a rare-cutter restriction map for a 710-kb region of the DXS255-DXS146 contig and have identified three CpG islands. These contigs and new markers will provide a useful resource for more detailed analysis of Xp11.23-p11.22, a region implicated in several genetic diseases. 32 refs., 2 figs., 2 tabs.

  20. Functional Characterization of the GATA Transcription Factors GNC and CGA1 Reveals Their Key Role in Chloroplast Development, Growth, and Division in Arabidopsis1[W][OA

    Science.gov (United States)

    Chiang, Yi-Hsuan; Zubo, Yan O.; Tapken, Wiebke; Kim, Hyo Jung; Lavanway, Ann M.; Howard, Louisa; Pilon, Marinus; Kieber, Joseph J.; Schaller, G. Eric

    2012-01-01

    Chloroplasts develop from proplastids in a process that requires the interplay of nuclear and chloroplast genomes, but key steps in this developmental process have yet to be elucidated. Here, we show that the nucleus-localized transcription factors GATA NITRATE-INDUCIBLE CARBON-METABOLISM-INVOLVED (GNC) and CYTOKININ-RESPONSIVE GATA1 (CGA1) regulate chloroplast development, growth, and division in Arabidopsis (Arabidopsis thaliana). GNC and CGA1 are highly expressed in green tissues, and the phytohormone cytokinin regulates their expression. A gnc cga1 mutant exhibits a reduction in overall chlorophyll levels as well as in chloroplast size in the hypocotyl. Ectopic overexpression of either GNC or CGA1 promotes chloroplast biogenesis in hypocotyl cortex and root pericycle cells, based on increases in the number and size of the chloroplasts, and also results in expanded zones of chloroplast production into the epidermis of hypocotyls and cotyledons and into the cortex of roots. Ectopic overexpression also promotes the development of etioplasts from proplastids in dark-grown seedlings, subsequently enhancing the deetiolation process. Inducible expression of GNC demonstrates that GNC-mediated chloroplast biogenesis can be regulated postembryonically, notably so for chloroplast production in cotyledon epidermal cells. Analysis of the gnc cga1 loss-of-function and overexpression lines supports a role for these transcription factors in regulating the effects of cytokinin on chloroplast division. These data support a model in which GNC and CGA1 serve as two of the master transcriptional regulators of chloroplast biogenesis, acting downstream of cytokinin and mediating the development of chloroplasts from proplastids and enhancing chloroplast growth and division in specific tissues. PMID:22811435

  1. Tumor de células da granulosa com metástases numa gata

    Directory of Open Access Journals (Sweden)

    Márcia Regina Giacóia

    1999-01-01

    Full Text Available As características clínicas, macroscópicas e microscópicas de um tumor de células da granulosa no ovário de uma gata de 2 anos de idade são descritas neste trabalho. Essa neoplasia é rara em gatos, principalmente devido à idade apresentada pelo animal. O diagnóstico foi feito clinicamente através de exame ultra-sonográfico. O tumor, uma massa cística na região do ovário esquerdo, metastatizou para o epíploo e para os pulmões. As células tumorais expressaram vimentina e arranjavam-se num padrão sarcomatoso e difuso, sustentado por fino estroma fibrovascular. A presença de sinais clínicos, como perda de pêlos e repetição de estros, é indicativa de síndrome paraneoplásica ortoendócrina, devido à excessiva estimulação estrogênica. A existência desse tipo de tumor deve ser considerada no diagnóstico diferencial de distúrbios comportamentais em gatas.

  2. A Gata3-Mafb transcriptional network directs post-synaptic differentiation in synapses specialized for hearing.

    Science.gov (United States)

    Yu, Wei-Ming; Appler, Jessica M; Kim, Ye-Hyun; Nishitani, Allison M; Holt, Jeffrey R; Goodrich, Lisa V

    2013-12-10

    Information flow through neural circuits is determined by the nature of the synapses linking the subtypes of neurons. How neurons acquire features distinct to each synapse remains unknown. We show that the transcription factor Mafb drives the formation of auditory ribbon synapses, which are specialized for rapid transmission from hair cells to spiral ganglion neurons (SGNs). Mafb acts in SGNs to drive differentiation of the large postsynaptic density (PSD) characteristic of the ribbon synapse. In Mafb mutant mice, SGNs fail to develop normal PSDs, leading to reduced synapse number and impaired auditory responses. Conversely, increased Mafb accelerates synaptogenesis. Moreover, Mafb is responsible for executing one branch of the SGN differentiation program orchestrated by the Gata3 transcriptional network. Remarkably, restoration of Mafb rescues the synapse defect in Gata3 mutants. Hence, Mafb is a powerful regulator of cell-type specific features of auditory synaptogenesis that offers a new entry point for treating hearing loss. DOI: http://dx.doi.org/10.7554/eLife.01341.001.

  3. Neighbourhood continuity is not required for correct testis gene expression in Drosophila.

    Directory of Open Access Journals (Sweden)

    Lisa A Meadows

    Full Text Available It is now widely accepted that gene organisation in eukaryotic genomes is non-random and it is proposed that such organisation may be important for gene expression and genome evolution. In particular, the results of several large-scale gene expression analyses in a range of organisms from yeast to human indicate that sets of genes with similar tissue-specific or temporal expression profiles are clustered within the genome in gene expression neighbourhoods. While the existence of neighbourhoods is clearly established, the underlying reason for this facet of genome organisation is currently unclear and there is little experimental evidence that addresses the genomic requisites for neighbourhood organisation. We report the targeted disruption of three well-defined male-specific gene expression neighbourhoods in the Drosophila genome by the synthesis of precisely mapped chromosomal inversions. We compare gene expression in individuals carrying inverted chromosomes with their non-inverted but otherwise identical progenitors using whole-transcriptome microarray analysis, validating these data with specific quantitative real-time PCR assays. For each neighbourhood we generate and examine multiple inversions. We find no significant differences in the expression of genes that define each of the neighbourhoods. We further show that the inversions spatially separate both halves of a neighbourhood in the nucleus. Thus, models explaining neighbourhood organisation in terms of local sequence interactions, enhancer crosstalk, or short-range chromatin effects are unlikely to account for this facet of genome organisation. Our study challenges the notion that, at least in the case of the testis, expression neighbourhoods are a feature of eukaryotic genome organisation necessary for correct gene expression.

  4. The B-type lamin is required for somatic repression of testis-specific gene clusters

    Science.gov (United States)

    Shevelyov, Y. Y.; Lavrov, S. A.; Mikhaylova, L. M.; Nurminsky, I. D.; Kulathinal, R. J.; Egorova, K. S.; Rozovsky, Y. M.; Nurminsky, D. I.

    2009-01-01

    Large clusters of coexpressed tissue-specific genes are abundant on chromosomes of diverse species. The genes coordinately misexpressed in diverse diseases are also found in similar clusters, suggesting that evolutionarily conserved mechanisms regulate expression of large multigenic regions both in normal development and in its pathological disruptions. Studies on individual loci suggest that silent clusters of coregulated genes are embedded in repressed chromatin domains, often localized to the nuclear periphery. To test this model at the genome-wide scale, we studied transcriptional regulation of large testis-specific gene clusters in somatic tissues of Drosophila. These gene clusters showed a drastic paucity of known expressed transgene insertions, indicating that they indeed are embedded in repressed chromatin. Bioinformatics analysis suggested the major role for the B-type lamin, LamDmo, in repression of large testis-specific gene clusters, showing that in somatic cells as many as three-quarters of these clusters interact with LamDmo. Ablation of LamDmo by using mutants and RNAi led to detachment of testis-specific clusters from nuclear envelope and to their selective transcriptional up-regulation in somatic cells, thus providing the first direct evidence for involvement of the B-type lamin in tissue-specific gene repression. Finally, we found that transcriptional activation of the lamina-bound testis-specific gene cluster in male germ line is coupled with its translocation away from the nuclear envelope. Our studies, which directly link nuclear architecture with coordinated regulation of tissue-specific genes, advance understanding of the mechanisms underlying both normal cell differentiation and developmental disorders caused by lesions in the B-type lamins and interacting proteins. PMID:19218438

  5. Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene

    Institute of Scientific and Technical Information of China (English)

    Chun-Hui HOU; Jian HUANG; Ruo-Lan QIAN

    2004-01-01

    The developmental control of the human ε-globin gene expression is mediated by transcriptional regulatory elements in the 5' flanking DNA of this gene. A previously identified negative regulatory element (-3028 to -2902 bp, termed ε-NRAII) was analyzed and one putative NF-κB site and two GATA sites locate at -3004 bp, -2975 bp and -2948 bp were characterized. Electrophoresis mobility shift assay (EMSA)showed that the putative NF-κB site was specifically bound by nuclear proteins of K562 cells. Data obtained from transient transfection showed that the expression of reporter gene could be upregulated about 50% or 100% respectively when ε-NRAII was inserted upstream of the SV40 promoter or ε-globin gene proximal promoter (-177 bp to +1 bp), suggesting that ε-NRAII might not be a classic silencer. Mutation in the putative NF-κB site or in the GATA site (at-2975 bp) slightly reduced the expression of reporter gene driven by SV40 promoter or ε-globin gene proximal promoter. However, the mutation of GATA site at -2948 bp remarkably reduced the reporter gene activity driven by SV40 promoter, but not by ε-globin gene proximal promoter. Further mutation analysis showed that the negative effect of mutation in GATA site at -2948 bp on SV40 promoter was not affected by the mutation of the putative NF-κB site, whereas it could be abolished by the mutation of GATA site at -2975 bp. Furthermore, the mutation of both GATA sites could synergistically reduce the reporter gene activity driven by ε-globin gene proximal promoter. Those results suggested that ε-NRAII might function differently on the SV40 promoter and ε-globin gene proximal promoter.

  6. Analysis of an ordered, comprehensive STM mutant library in infectious Borrelia burgdorferi: insights into the genes required for mouse infectivity.

    Directory of Open Access Journals (Sweden)

    Tao Lin

    Full Text Available The identification of genes important in the pathogenesis of Lyme disease Borrelia has been hampered by exceedingly low transformation rates in low-passage, infectious organisms. Using the infectious, moderately transformable B. burgdorferi derivative 5A18NP1 and signature-tagged versions of the Himar1 transposon vector pGKT, we have constructed a defined transposon library for the efficient genome-wide investigation of genes required for wild-type pathogenesis, in vitro growth, physiology, morphology, and plasmid replication. To facilitate analysis, the insertion sites of 4,479 transposon mutants were determined by sequencing. The transposon insertions were widely distributed across the entire B. burgdorferi genome, with an average of 2.68 unique insertion sites per kb DNA. The 10 linear plasmids and 9 circular plasmids had insertions in 33 to 100 percent of their predicted genes. In contrast, only 35% of genes in the 910 kb linear chromosome had incapacitating insertions; therefore, the remaining 601 chromosomal genes may represent essential gene candidates. In initial signature-tagged mutagenesis (STM analyses, 434 mutants were examined at multiple tissue sites for infectivity in mice using a semi-quantitative, Luminex-based DNA detection method. Examples of genes found to be important in mouse infectivity included those involved in motility, chemotaxis, the phosphoenolpyruvate phosphotransferase system, and other transporters, as well as putative plasmid maintenance genes. Availability of this ordered STM library and a high-throughput screening method is expected to lead to efficient assessment of the roles of B. burgdorferi genes in the infectious cycle and pathogenesis of Lyme disease.

  7. Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells.

    Directory of Open Access Journals (Sweden)

    Alan M Williams

    Full Text Available The activation induced cytosine deaminase (AID mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM, class switch recombination (CSR, and gene conversion (GCV. SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID.

  8. Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells

    Science.gov (United States)

    Williams, Alan M.; Maman, Yaakov; Alinikula, Jukka; Schatz, David G.

    2016-01-01

    The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID. PMID:26900682

  9. Tescalcin is an essential factor in megakaryocytic differentiation associated with Ets family gene expression.

    Science.gov (United States)

    Levay, Konstantin; Slepak, Vladlen Z

    2007-09-01

    We show here that the process of megakaryocytic differentiation requires the presence of the recently discovered protein tescalcin. Tescalcin is dramatically upregulated during the differentiation and maturation of primary megakaryocytes or upon PMA-induced differentiation of K562 cells. This upregulation requires sustained signaling through the ERK pathway. Overexpression of tescalcin in K562 cells initiates events of spontaneous megakaryocytic differentiation, such as expression of specific cell surface antigens, inhibition of cell proliferation, and polyploidization. Conversely, knockdown of this protein in primary CD34+ hematopoietic progenitors and cell lines by RNA interference suppresses megakaryocytic differentiation. In cells lacking tescalcin, the expression of Fli-1, Ets-1, and Ets-2 transcription factors, but not GATA-1 or MafB, is blocked. Thus, tescalcin is essential for the coupling of ERK cascade activation with the expression of Ets family genes in megakaryocytic differentiation.

  10. Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes

    Directory of Open Access Journals (Sweden)

    Kyung Hoon Lee

    2016-01-01

    Full Text Available Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (<2 months limited their application to further investigations. To develop a culture method that allows for in vitro maintenance of GDCs for long periods, we subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4, which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells.

  11. Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes.

    Science.gov (United States)

    Lee, Kyung Hoon; Lee, Won Young; Kim, Jin Hoi; Park, Chan Kyu; Do, Jeong Tae; Kim, Jae Hwan; Choi, Young Suk; Kim, Nam Hyung; Song, Hyuk

    2016-01-01

    Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs) have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs) and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4), which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells.

  12. Inhibition of interleukin-13 gene expression in T cells through GATA-3 pathway by arsenic trioxide

    Institute of Scientific and Technical Information of China (English)

    YAO Xin; HE Hai-yan; YANG Yan; DAI Shan-lin; SUN Pei-li; YIN Kai-sheng; HUANG Mao

    2008-01-01

    @@ Arsenic trioxide (AT) has a long history of use in both traditional Chinese medicine and in modern medicine in asthma therapy.Recently,Yin et al1 found that AT even at small doses reduced the airway inflammation of sensitized guinea pigs.However the mechanism underlying this is still largely unknown.Interleukin 13 (IL-13),as one of the important TH2 cytokines,plays an important role in asthma pathogenesis through promoting eosinophilic inflammation,mucus secretion and airway hyperresponsiveness.2 To further explore the molecular anti-inflammatory basis of AT,we employed Hut-78 cells,a human T cell line,with activation via CD3/CD28 receptors to mimick in vivo co-stimulation to investigate the effect of AT on IL-13 transcription.

  13. Mutational Analysis of GATA4 and NKX2.5 Genes in Dilated ...

    African Journals Online (AJOL)

    Tropical Journal of Pharmaceutical Research February 2016; 15 (2): 285-292. ISSN: 1596-5996 (print); ... in regulating survival and hypertrophic growth of the adult heart. .... USA), 125 mmol/l of each dNTP (Roche,. Germany), 1.5 mmol/l ...

  14. Intestinal Specific Gene Regulation by Transcription Factors Gata4 and Hnfla in Vivo

    OpenAIRE

    Bosse, Tjalling

    2006-01-01

    textabstractThe mammalian small intestine is responsible for the terminal digestion and absorption of nutrients, water homeostasis, and the elimination of waste products, which in turn, are essential processes for life. These processes however, are easily disrupted by infection, inflammatory processes such as Crohn’s disease, cancer, and resection. The small intestine is equipped with specific proteins, such as enzymes to digest nutrients (digestion) and ‘transporters’ to carry the nutrients ...

  15. Intestinal Specific Gene Regulation by Transcription Factors Gata4 and Hnfla in Vivo

    NARCIS (Netherlands)

    T. Bosse (Tjalling)

    2006-01-01

    textabstractThe mammalian small intestine is responsible for the terminal digestion and absorption of nutrients, water homeostasis, and the elimination of waste products, which in turn, are essential processes for life. These processes however, are easily disrupted by infection, inflammatory process

  16. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.

    Science.gov (United States)

    Si, Lina; Shi, Jin; Gao, Wenqun; Zheng, Min; Liu, Lingjuan; Zhu, Jing; Tian, Jie

    2014-07-18

    BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of GATA4 and Nkx2.5, suggesting that Smad4 mediated BMP2 signaling pathway was essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.

  17. UTERINE PROLAPSE IN QUEEN AND UTERINE PROLAPSO UTERINO EM GATA E RETROFLEXÃO UTERINA EM CADELA

    Directory of Open Access Journals (Sweden)

    Giuliano Queiroz Mostachio

    2008-10-01

    Full Text Available

    Obstetrical emergencies are problem in veterinary clinics and hospital. So, the aim of this report is to describe the clinical-surgical aspect of one of them, the uterine prolapse. Complete protrusion and retroflexion of uterus had been diagnosed in a queen and female dog, respectively. After the stabilization of the animals and reduction of the prolapses followed by ovary-hysterectomy, one of the animals came to death due to septicemia and hypovolemic shock. Rapid assessment and intensive treatments are required to sustain the life of the animal.

    KEY WORDS: Cat, dog, uterine prolapse, uterine retroflexion.

    Emergências obstétricas, como o prolapso uterino, constituem um problema em clínicas e hospitais veterinários. Neste relato descreve-se o aspecto clínico-cirúrgico do prolapso uterino. Diagnosticaram-se, em uma gata e em uma cadela, protrusão completa e retroflexão uterina, respectivamente. Após a estabilização dos animais e redução dos prolapsos seguidos de ovário-histerectomia, um dos animais veio a óbito, em decorrência de septicemia e choque hipovolêmico. Portanto, a rápida avaliação e tratamentos intensivos são requeridos para assegurar a vida do animal.

     

    PALAVRAS-CHAVES: Cão, gato, prolapso uterino, retroflexão uterina

  18. Pseudomonas aeruginosa serA Gene Is Required for Bacterial Translocation through Caco-2 Cell Monolayers

    Science.gov (United States)

    Yasuda, Masashi; Nagata, Syouya; Yamane, Satoshi; Kunikata, Chinami; Kida, Yutaka; Kuwano, Koichi; Suezawa, Chigusa; Okuda, Jun

    2017-01-01

    To specify critical factors responsible for Pseudomonas aeruginosa penetration through the Caco-2 cell epithelial barrier, we analyzed transposon insertion mutants that demonstrated a dramatic reduction in penetration activity relative to P. aeruginosa PAO1 strain. From these strains, mutations could be grouped into five classes, specifically flagellin-associated genes, pili-associated genes, heat-shock protein genes, genes related to the glycolytic pathway, and biosynthesis-related genes. Of these mutants, we here focused on the serA mutant, as the association between this gene and penetration activity is yet unknown. Inactivation of the serA gene caused significant repression of bacterial penetration through Caco-2 cell monolayers with decreased swimming and swarming motilities, bacterial adherence, and fly mortality rate, as well as repression of ExoS secretion; however, twitching motility was not affected. Furthermore, L-serine, which is known to inhibit the D-3-phosphoglycerate dehydrogenase activity of the SerA protein, caused significant reductions in penetration through Caco-2 cell monolayers, swarming and swimming motilities, bacterial adherence to Caco-2 cells, and virulence in flies in the wild-type P. aeruginosa PAO1 strain. Together, these results suggest that serA is associated with bacterial motility and adherence, which are mediated by flagella that play a key role in the penetration of P. aeruginosa through Caco-2 cell monolayers. Oral administration of L-serine to compromised hosts might have the potential to interfere with bacterial translocation and prevent septicemia caused by P. aeruginosa through inhibition of serA function. PMID:28046014

  19. Identification of Brucella melitensis 16M genes required for bacterial survival in the caprine host.

    Science.gov (United States)

    Zygmunt, Michel S; Hagius, Sue D; Walker, Joel V; Elzer, Philip H

    2006-01-01

    Brucella species are gram-negative bacteria which belong to alpha-Proteobacteria family. These organisms are zoonotic pathogens that induce abortion and sterility in domestic mammals and chronic infections in humans known as Malta fever. The virulence of Brucella is dependent upon its ability to enter and colonize the cells in which it multiplies. The genetic basis of this aspect is poorly understood. Signature-tagged mutagenesis (STM) was used to identify potential Brucella virulence factors. PCR amplification has been used in place of DNA hybridization to identify the STM-generated attenuated mutants. A library of 288 Brucella melitensis 16M tagged mini-Tn5 Km2 mutants, in 24 pools, was screened for its ability to colonize spleen, lymph nodes and liver of goats at three weeks post-i.v. infection. This comparative screening identified 7 mutants (approximately 5%) which were not recovered from the output pool in goats. Some genes were known virulence genes involved in biosynthesis of LPS (lpsA gene) or in intracellular survival (the virB operon). Other mutants included ones which had a disrupted gene homologous to flgF, a gene coding for the basal-body rod of the flagellar apparatus, and another with a disruption in a gene homologous to ppk which is involved in the biosynthesis of inorganic polyphosphate (PolyP) from ATP. Other genes identified encoded factors involved in DNA metabolism and oxidoreduction metabolism. Using STM and the caprine host for screening, potential virulence determinants in B. melitensis have been identified.

  20. crumbs and stardust, two genes of Drosophila required for the development of epithelial cell polarity.

    Science.gov (United States)

    Knust, E; Tepass, U; Wodarz, A

    1993-01-01

    Loss-of-function mutations in the Drosophila genes crumbs and stardust are embryonic lethal and cause a breakdown of ectodermally derived epithelia during organogenesis, leading to formation of irregular cell clusters and extensive cell death in some epithelia. The mutant phenotype develops gradually and affects the various epithelia to different extents. crumbs encodes a large transmembrane protein with 30 EGF-like repeats and four laminin A G-domain-like repeats in its extracellular domain, suggesting its participation in protein-protein interactions. The CRUMBS protein is exclusively expressed on the apical membrane of all ectodermally derived epithelia, the tissues affected in crumbs and stardust mutant embryos. The gene function is completely abolished by a crumbs mutation that causes production of a protein with a truncated cytoplasmic domain. Instead of being apically localized as in wild-type, the mutant CRUMBS protein is diffusely distributed in the cytoplasm; this occurs before any morphologically detectable cellular phenotype is visible, suggesting that targeting of proteins is affected in crumbs mutant embryos. Later, the protein can be detected on the apical and basolateral membranes. Mutations in stardust produce a phenotype nearly identical to that associated with crumbs mutations, suggesting that both genes are functionally related. Double mutant combinations and gene dosage studies suggest that both genes are part of a common genetic pathway, in which stardust acts downstream of crumbs.

  1. Emdogain-regulated gene expression in palatal fibroblasts requires TGF-βRI kinase signaling.

    Directory of Open Access Journals (Sweden)

    Alexandra Stähli

    Full Text Available Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB431542. Emdogain caused 39 coding genes to be differentially expressed in palatal fibroblasts by microarray analysis (p10-fold. Importantly, in the presence of the TGF-βRI kinase inhibitor SB431542, Emdogain failed to cause any significant changes in gene expression. Consistent with this mechanism, three independent TGF-βRI kinase inhibitors and a TGF-β neutralizing antibody abrogated the increased expression of IL-11, a selected Emdogain target gene. The MAPK inhibitors SB203580 and U0126 lowered the impact of Emdogain on IL-11 expression. The data support that TGF-βRI kinase activity is necessary to mediate the effects of Emdogain on gene expression in vitro.

  2. Emdogain-regulated gene expression in palatal fibroblasts requires TGF-βRI kinase signaling.

    Science.gov (United States)

    Stähli, Alexandra; Bosshardt, Dieter; Sculean, Anton; Gruber, Reinhard

    2014-01-01

    Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB431542. Emdogain caused 39 coding genes to be differentially expressed in palatal fibroblasts by microarray analysis (p10-fold). Importantly, in the presence of the TGF-βRI kinase inhibitor SB431542, Emdogain failed to cause any significant changes in gene expression. Consistent with this mechanism, three independent TGF-βRI kinase inhibitors and a TGF-β neutralizing antibody abrogated the increased expression of IL-11, a selected Emdogain target gene. The MAPK inhibitors SB203580 and U0126 lowered the impact of Emdogain on IL-11 expression. The data support that TGF-βRI kinase activity is necessary to mediate the effects of Emdogain on gene expression in vitro.

  3. MCTP2 is a dosage-sensitive gene required for cardiac outflow tract development

    Science.gov (United States)

    Lalani, Seema R.; Ware, Stephanie M.; Wang, Xueqing; Zapata, Gladys; Tian, Qi; Franco, Luis M.; Jiang, Zhengxin; Bucasas, Kristine; Scott, Daryl A.; Campeau, Philippe M.; Hanchard, Neil; Umaña, Luis; Cast, Ashley; Patel, Ankita; Cheung, Sau W.; McBride, Kim L.; Bray, Molly; Craig Chinault, A.; Boggs, Barbara A.; Huang, Miao; Baker, Mariah R.; Hamilton, Susan; Towbin, Jeff; Jefferies, John L.; Fernbach, Susan D.; Potocki, Lorraine; Belmont, John W.

    2013-01-01

    Coarctation of the aorta (CoA) and hypoplastic left heart syndrome (HLHS) have been reported in rare individuals with large terminal deletions of chromosome 15q26. However, no single gene important for left ventricular outflow tract (LVOT) development has been identified in this region. Using array-comparative genomic hybridization, we identified two half-siblings with CoA with a 2.2 Mb deletion on 15q26.2, inherited from their mother, who was mosaic for this deletion. This interval contains an evolutionary conserved, protein-coding gene, MCTP2 (multiple C2-domains with two transmembrane regions 2). Using gene-specific array screening in 146 individuals with non-syndromic LVOT obstructive defects, another individual with HLHS and CoA was found to have a de novo 41 kb intragenic duplication within MCTP2, predicted to result in premature truncation, p.F697X. Alteration of Mctp2 gene expression in Xenopus laevis embryos by morpholino knockdown and mRNA overexpression resulted in the failure of proper OT development, confirming the functional importance of this dosage-sensitive gene for cardiogenesis. Our results identify MCTP2 as a novel genetic cause of CoA and related cardiac malformations. PMID:23773997

  4. Multiple BiP genes of Arabidopsis thaliana are required for male gametogenesis and pollen competitiveness.

    Science.gov (United States)

    Maruyama, Daisuke; Sugiyama, Tomoyuki; Endo, Toshiya; Nishikawa, Shuh-Ichi

    2014-04-01

    Immunoglobulin-binding protein (BiP) is a molecular chaperone of the heat shock protein 70 (Hsp70) family. BiP is localized in the endoplasmic reticulum (ER) and plays key roles in protein translocation, protein folding and quality control in the ER. The genomes of flowering plants contain multiple BiP genes. Arabidopsis thaliana has three BiP genes. BIP1 and BIP2 are ubiquitously expressed. BIP3 encodes a less well conserved BiP paralog, and it is expressed only under ER stress conditions in the majority of organs. Here, we report that all BiP genes are expressed and functional in pollen and pollen tubes. Although the bip1 bip2 double mutation does not affect pollen viability, the bip1 bip2 bip3 triple mutation is lethal in pollen. This result indicates that lethality of the bip1 bip2 double mutation is rescued by BiP3 expression. A decrease in the copy number of the ubiquitously expressed BiP genes correlates well with a decrease in pollen tube growth, which leads to reduced fitness of mutant pollen during fertilization. Because an increased protein secretion activity is expected to increase the protein folding demand in the ER, the multiple BiP genes probably cooperate with each other to ensure ER homeostasis in cells with active secretion such as rapidly growing pollen tubes.

  5. Genome-wide screening of Saccharomyces cerevisiae genes required to foster tolerance towards industrial wheat straw hydrolysates.

    Science.gov (United States)

    Pereira, Francisco B; Teixeira, Miguel C; Mira, Nuno P; Sá-Correia, Isabel; Domingues, Lucília

    2014-12-01

    The presence of toxic compounds derived from biomass pre-treatment in fermentation media represents an important drawback in second-generation bio-ethanol production technology and overcoming this inhibitory effect is one of the fundamental challenges to its industrial production. The aim of this study was to systematically identify, in industrial medium and at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors of lignocellulosic fermentations. Based on the screening of EUROSCARF haploid mutant collection, 242 and 216 determinants of tolerance to inhibitory compounds present in industrial wheat straw hydrolysate (WSH) and in inhibitor-supplemented synthetic hydrolysate were identified, respectively. Genes associated to vitamin metabolism, mitochondrial and peroxisomal functions, ribosome biogenesis and microtubule biogenesis and dynamics are among the newly found determinants of WSH resistance. Moreover, PRS3, VMA8, ERG2, RAV1 and RPB4 were confirmed as key genes on yeast tolerance and fermentation of industrial WSH.

  6. Requirements for Clinical Trials with Gene Therapy and Transplant Products in Switzerland.

    Science.gov (United States)

    Marti, Andreas

    2015-01-01

    This chapter aims to describe and summarize the regulation of gene and cell therapy products in Switzerland and its legal basis. Product types are briefly described, as are Swiss-specific terminologies such as the term "transplant product," which means products manufactured from cells, tissues, or even whole organs. Although some parts of this chapter may show a guideline character, they are not legally binding, but represent the current thinking of Swissmedic, the Swiss Agency for Therapeutic Products. As so far the experience with marketing approval of gene therapy and cell therapy products in Switzerland is limited, this chapter focuses on the regulation of clinical trials conducted with these products. Quality, nonclinical, and clinical aspects are summarized separately for gene therapy products and transplant products.

  7. Genome-wide screen for salmonella genes required for long-term systemic infection of the mouse.

    Directory of Open Access Journals (Sweden)

    2006-02-01

    Full Text Available A microarray-based negative selection screen was performed to identify Salmonella enterica serovar Typhimurium (serovar Typhimurium genes that contribute to long-term systemic infection in 129X1/SvJ (Nramp1(r mice. A high-complexity transposon-mutagenized library was used to infect mice intraperitoneally, and the selective disappearance of mutants was monitored after 7, 14, 21, and 28 d postinfection. One hundred and eighteen genes were identified to contribute to serovar Typhimurium infection of the spleens of mice by 28 d postinfection. The negatively selected mutants represent many known aspects of Salmonella physiology and pathogenesis, although the majority of the identified genes are of putative or unknown function. Approximately 30% of the negatively selected genes correspond to horizontally acquired regions such as those within Salmonella pathogenicity islands (SPI 1-5, prophages (Gifsy-1 and -2 and remnant, and the pSLT virulence plasmid. In addition, mutations in genes responsible for outer membrane structure and remodeling, such as LPS- and PhoP-regulated and fimbrial genes, were also selected against. Competitive index experiments demonstrated that the secreted SPI2 effectors SseK2 and SseJ as well as the SPI4 locus are attenuated relative to wild-type bacteria during systemic infection. Interestingly, several SPI1-encoded type III secretion system effectors/translocases are required by serovar Typhimurium to establish and, unexpectedly, to persist systemically, challenging the present description of Salmonella pathogenesis. Moreover, we observed a progressive selection against serovar Typhimurium mutants based upon the duration of the infection, suggesting that different classes of genes may be required at distinct stages of infection. Overall, these data indicate that Salmonella long-term systemic infection in the mouse requires a diverse repertoire of virulence factors. This diversity of genes presumably reflects the fact that

  8. Late Neogene-Recent uplift of the Cabo de Gata volcanic province, Almerı´a, SE Spain

    Science.gov (United States)

    Martín, José M.; Braga, Juan C.; Betzler, Christian

    2003-02-01

    Cabo de Gata is a Miocene volcanic province in the Betic Cordillera in southeastern Spain. The distribution of coastal deposits in the successive marine sedimentary units overlying the last volcanic episode (about 7.5 Ma old) has been used to reconstruct the post-volcanic palaeogeographic evolution of the region during the Late Neogene. The current elevation of well-dated shoreline marker rocks has been used to estimate uplift amount and rates. Since the late Tortonian, a N45°E-aligned (the strike of the Carboneras fault system) topographic relief was emergent in the Cabo de Gata region. The extension and height of this island increased throughout the late Neogene. Smaller, independent islands were emergent and finally became connected to the main island during the Messinian. The Carboneras and Agua Amarga Pliocene sub-basins were the last two marine basins prior to the final emergence of the region. Since the last volcanic eruption (ca. 7.5 Ma), maximum uplift of sedimentary rocks in Cabo de Gata has taken place on the western margin of the N45°E-aligned palaeorelief. The altitude of the shoreline marker rocks in the successive sedimentary units decreases eastwards to the present-day coast and northwards of the Rambla del Plomo. Uplift rates since deposition remain nearly constant for the successive Messinian rocks and decrease slightly for the lower Pliocene outcrops. Most of the uplift took place before the Pliocene while the main island enlarged. Uplift amounts and rates since deposition of the upper Neogene sedimentary units in the Cabo de Gata area are similar to the ones estimated for laterally equivalent units in the eastern Betic basins (approximately 50 m/Ma). Despite its volcanic nature and the occurrence of the Carboneras fault system, the Cabo de Gata probably became elevated as a consequence of regional uplift in connection with the rest of the Betic Cordillera.

  9. Protective effect of curcumin on acute airway inflammation of allergic asthma in mice through Notch1-GATA3 signaling pathway.

    Science.gov (United States)

    Chong, Lei; Zhang, Weixi; Nie, Ying; Yu, Gang; Liu, Liu; Lin, Li; Wen, Shunhang; Zhu, Lili; Li, Changchong

    2014-10-01

    Curcumin, a natural product derived from the plant Curcuma longa, has been found to have anti-inflammatory, antineoplastic and antifibrosis effects. It has been reported that curcumin attenuates allergic airway inflammation in mice through inhibiting NF-κB and its downstream transcription factor GATA3. It also has been proved the antineoplastic effect of curcumin through down-regulating Notch1 receptor and its downstream nuclear transcription factor NF-κB levels. In this study, we aimed to investigate the anti-inflammatory effect of curcumin on acute allergic asthma and its underlying mechanisms. 36 male BALB/c mice were randomly divided into four groups (normal, asthma, asthma+budesonide and asthma+curcumin groups). BALF (bronchoalveolar lavage fluid) and lung tissues were analyzed for airway inflammation and the expression of Notch1, Notch2, Notch3, Notch4 and the downstream transcription factor GATA3. Our findings showed that the levels of Notch1 and Notch2 receptors were up-regulated in asthma group, accompanied by the increased expression of GATA3. But the expression of Notch2 receptor was lower than Notch1 receptor. Curcumin pretreatment improved the airway inflammatory cells infiltration and reversed the increasing levels of Notch1/2 receptors and GATA3. Notch3 receptor was not expressed in all of the four groups. Notch4 receptor protein and mRNA expression level in the four groups had no significant differences. The results of the present study suggested that Notch1 and Notch2 receptor, major Notch1 receptor, played an important role in the development of allergic airway inflammation and the inhibition of Notch1-GATA3 signaling pathway by curcumin can prevent the development and deterioration of the allergic airway inflammation. This may be a possible therapeutic option of allergic asthma.

  10. No control genes required: Bayesian analysis of qRT-PCR data.

    Directory of Open Access Journals (Sweden)

    Mikhail V Matz

    Full Text Available BACKGROUND: Model-based analysis of data from quantitative reverse-transcription PCR (qRT-PCR is potentially more powerful and versatile than traditional methods. Yet existing model-based approaches cannot properly deal with the higher sampling variances associated with low-abundant targets, nor do they provide a natural way to incorporate assumptions about the stability of control genes directly into the model-fitting process. RESULTS: In our method, raw qPCR data are represented as molecule counts, and described using generalized linear mixed models under Poisson-lognormal error. A Markov Chain Monte Carlo (MCMC algorithm is used to sample from the joint posterior distribution over all model parameters, thereby estimating the effects of all experimental factors on the expression of every gene. The Poisson-based model allows for the correct specification of the mean-variance relationship of the PCR amplification process, and can also glean information from instances of no amplification (zero counts. Our method is very flexible with respect to control genes: any prior knowledge about the expected degree of their stability can be directly incorporated into the model. Yet the method provides sensible answers without such assumptions, or even in the complete absence of control genes. We also present a natural Bayesian analogue of the "classic" analysis, which uses standard data pre-processing steps (logarithmic transformation and multi-gene normalization but estimates all gene expression changes jointly within a single model. The new methods are considerably more flexible and powerful than the standard delta-delta Ct analysis based on pairwise t-tests. CONCLUSIONS: Our methodology expands the applicability of the relative-quantification analysis protocol all the way to the lowest-abundance targets, and provides a novel opportunity to analyze qRT-PCR data without making any assumptions concerning target stability. These procedures have been

  11. The ChIP-seq-defined networks of Bcl-3 gene binding support its required role in skeletal muscle atrophy.

    Directory of Open Access Journals (Sweden)

    Robert W Jackman

    Full Text Available NF-kappaB transcriptional activation is required for skeletal muscle disuse atrophy. We are continuing to study how the activation of NF-kB regulates the genes that encode the protein products that cause atrophy. Using ChIP-sequencing we found that Bcl-3, an NF-kB transcriptional activator required for atrophy, binds to the promoters of a number of genes whose collective function describes two major aspects of muscle wasting. By means of bioinformatics analysis of ChIP-sequencing data we found Bcl-3 to be directing transcription networks of proteolysis and energy metabolism. The proteolytic arm of the Bcl-3 networks includes many E3 ligases associated with proteasomal protein degradation, including that of the N-end rule pathway. The metabolic arm appears to be involved in organizing the change from oxidative phosphorylation to glycolysis in atrophying muscle. For one gene, MuRF1, ChIP-sequencing data identified the location of Bcl-3 and p50 binding in the promoter region which directed the creation of deletant and base-substitution mutations of MuRF1 promoter constructs to determine the effect on gene transcription. The results provide the first direct confirmation that the NF-kB binding site is involved in the muscle unloading regulation of MuRF1. Finally, we have combined the ChIP-sequencing results with gene expression microarray data from unloaded muscle to map several direct targets of Bcl-3 that are transcription factors whose own targets describe a set of indirect targets for NF-kB in atrophy. ChIP-sequencing provides the first molecular explanation for the finding that Bcl3 knockout mice are resistant to disuse muscle atrophy. Mapping the transcriptional regulation of muscle atrophy requires an unbiased analysis of the whole genome, which we show is now possible with ChIP-sequencing.

  12. Zinc finger protein Loz1 is required for zinc-responsive regulation of gene expression in fission yeast

    Science.gov (United States)

    Corkins, Mark E.; May, Margot; Ehrensberger, Kate M.; Hu, Ya-Mei; Liu, Yi-Hsuan; Bloor, Sean D.; Jenkins, Blair; Runge, Kurt W.; Bird, Amanda J.

    2013-01-01

    In Schizosaccharomyces pombe, alcohol dehydrogenase 1 (Adh1) is an abundant zinc-requiring enzyme that catalyses the conversion of acetaldehyde to ethanol during fermentation. In a zinc-replete cell, adh1 is highly expressed. However, in zinc-limited cells, adh1 gene expression is repressed, and cells induce the expression of an alternative alcohol dehydrogenase encoded by the adh4 gene. In our studies examining this zinc-dependent switch in alcohol dehydrogenase gene expression, we isolated an adh1Δ strain containing a partial loss of function mutation that resulted in higher levels of adh4 transcripts in zinc-replete cells. This mutation also led to the aberrant expression of other genes that are typically regulated by zinc. Using linkage analysis, we have mapped the position of this mutation to a single gene called Loss Of Zinc sensing 1 (loz1). Loz1 is a 55-kDa protein that contains a double C2H2-type zinc finger domain. The mapped mutation that disrupts Loz1 function leads to an arginine to glycine substitution in the second zinc finger domain, suggesting that the double zinc finger domain is important for Loz1 function. We show that loz1Δ cells hyperaccumulate zinc and that Loz1 is required for gene repression in zinc-replete cells. We also have found that Loz1 negatively autoregulates its own expression. We propose that Loz1 is a unique metalloregulatory factor that plays a central role in zinc homeostasis in S. pombe. PMID:24003116

  13. Virus-induced gene silencing reveals signal transduction components required for the Pvr9-mediated hypersensitive response in Nicotiana benthamiana.

    Science.gov (United States)

    Tran, Phu-Tri; Choi, Hoseong; Choi, Doil; Kim, Kook-Hyung

    2016-08-01

    Resistance to pathogens mediated by plant resistance (R) proteins requires different signaling transduction components and pathways. Our previous studies revealed that a potyvirus resistance gene in pepper, Pvr9, confers a hypersensitive response (HR) to pepper mottle virus in Nicotiana benthamiana. Our results show that the Pvr9-mediated HR against pepper mottle virus infection requires HSP90, SGT1, NDR1, but not EDS1. These results suggest that the Pvr9-mediated HR is possibly related to the SA pathway but not the ET, JA, ROS or NO pathways.

  14. The AFT1 transcriptional factor is differentially required for expression of high-affinity iron uptake genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Casas, C; Aldea, M; Espinet, C; Gallego, C; Gil, R; Herrero, E

    1997-06-15

    High-affinity iron uptake in Saccharomyces cerevisiae involves the extracytoplasmic reduction of ferric ions by FRE1 and FRE2 reductases. Ferrous ions are then transported across the plasma membrane through the FET3 oxidase-FTR1 permease complex. Expression of the high-affinity iron uptake genes is induced upon iron deprivation. We demonstrate that AFT1 is differentially involved in such regulation. Aft1 protein is required for maintaining detectable non-induced level of FET3 expression and for induction of FRE2 in iron starvation conditions. On the contrary, FRE1 mRNA induction is normal in the absence of Aft1, although the existence of AFT1 point mutations causing constitutive expression of FRE1 (Yamaguchi-Iwai et al., EMBO J. 14: 1231-1239, 1995) indicates that Aft1 may also participate in FRE1 expression in a dispensable way. The alterations in the basal levels of expression of the high-affinity iron uptake genes may explain why the AFT1 mutant is unable to grow on respirable carbon sources. Overexpression of AFT1 leads to growth arrest of the G1 stage of the cell cycle. Aft1 is a transcriptional activator that would be part of the different transcriptional complexes interacting with the promoter of the high-affinity iron uptake genes. Aft1 displays phosphorylation modifications depending on the growth stage of the cells, and it might link induction of genes for iron uptake to other metabolically dominant requirement for cell growth.

  15. Microarray Analysis of Transposon Insertion Mutants in Bacillus Anthracis: Global Identification of Genes Required for Sporulation and Germination

    Science.gov (United States)

    2007-02-01

    Gilois, M. Rose, and D. Lereclus. 2001. Oligopep- tide permease is required for expression of the Bacillus thuringiensis plcR regulon and for...non- toxin gene expression in Bacillus anthracis. Infect. Immun. 65:3091–3099. 10. Ikeda, R. A., C. M. Ligman, and S. Warshamana. 1992. T7 promoter con...nontoxi- genic Bacillus anthracis spore vaccines based on strains expressing mutant vari- ants of lethal toxin components. Vaccine 23:5688–5697. 17. Read

  16. Identification of genes required for neural-specific glycosylation using functional genomics.

    Directory of Open Access Journals (Sweden)

    Miki Yamamoto-Hino

    Full Text Available Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. For example, α1,3-fucosylation, the addition of a fucose moiety abundant in Drosophila neural cells, is essential for neural development, function, and behavior. However, it remains largely unknown how neural-specific α1,3-fucosylation is regulated. In the present study, we searched for genes involved in the glycosylation of a neural-specific protein using a Drosophila RNAi library. We obtained 109 genes affecting glycosylation that clustered into nine functional groups. Among them, members of the RNA regulation group were enriched by a secondary screen that identified genes specifically regulating α1,3-fucosylation. Further analyses revealed that an RNA-binding protein, second mitotic wave missing (Swm, upregulates expression of the neural-specific glycosyltransferase FucTA and facilitates its mRNA export from the nucleus. This first large-scale genetic screen for glycosylation-related genes has revealed novel regulation of fucTA mRNA in neural cells.

  17. The HOBBIT gene is required for formation of the root meristem in the Arabidopsis embryo

    NARCIS (Netherlands)

    Scheres, B.J.G.; Willemsen, V.; Wolkenfelt, H.; Vrieze, G. de; Weisbeek, P.

    1998-01-01

    In Arabidopsis, the root meristem originates from the hypophyseal cell and from an adjoining cell tier that is distinct at the heart stage of embryogenesis. We have analysed mutations in the HOBBIT (HBT) gene that is essential for root meristem formation. hbt embryos display incorrect

  18. W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity

    DEFF Research Database (Denmark)

    Huizinga, J D; Thuneberg, L; Klüppel, M;

    1995-01-01

    that the interstitial cells of Cajal express the Kit receptor tyrosine kinase. Furthermore, mice with mutations in the dominant white spotting (W) locus, which have cellular defects in haematopoiesis, melanogenesis and gametogenesis as a result of mutations in the Kit gene, also lack the network of interstitial cells...

  19. Regulation of general amino acid permeases Gap1p, GATA transcription factors Gln3p and Gat1p on 2-phenylethanol biosynthesis via Ehrlich pathway.

    Science.gov (United States)

    Chen, Xianrui; Wang, Zhaoyue; Guo, Xuena; Liu, Sha; He, Xiuping

    2017-01-20

    In Saccharomyces cerevisiae, when l-phenylalanine (l-Phe) is used as the sole nitrogen source, 2-phenylethanol (PE) is mainly synthesized via the Ehrlich pathway. General amino acid permease Gap1p is response of aromatic amino acids transportation, and GATA transcription factors Gln3p and Gat1p regulate the transcription of permease gene and catabolic enzyme genes for nitrogen sources and aromatic amino acids utilization. In this study, it was demonstrated that over-expressing GLN3 gene from industrial yeast strain MT2 or S. cerevisiae haploid strain YS58, 2-PE synthesis levels of recombinant strains increased 54% or 40% than that of the control strain, which suggested that higher Gln3p activity in yeast has positive regulation effect on 2-PE biosynthesis via Ehrlich pathway. The recombinant strains with over-expression of GAT1 gene from MT2 or YS58 also up-regulated Ehrlich pathway for 2-PE biosynthesis and increased 2-PE production. Similarly, when GAP1 gene respectively from MT2 or YS58 was over-expressed, 2-PE yield was improved obviously, suggesting that GAP1 over-expressing in yeast also promoted Ehrlich pathway to produce 2-PE. The synergistic regulation of GLN3/GAT1 or GLN3/GAP1 over-expression was similar to that of single factor over-expression. Among these regulatory factors, Gln3p of industrial yeast strain MT2 caused stronger regulation on target genes than that of haploid strain YS58, which might be due to the differences in translational efficiency or nuclear localization of each Gln3p, or due to their different spatial structures and binding domains. Further results showed that efficient Gln3p expression in MT2 brought about higher 2-PE, 3.59gL(-1), which was of potential significant for commercial exploitation. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. The rgg0182 gene encodes a transcriptional regulator required for the full Streptococcus thermophilus LMG18311 thermal adaptation

    Directory of Open Access Journals (Sweden)

    Bertin Stéphane

    2011-10-01

    Full Text Available Abstract Background Streptococcus thermophilus is an important starter strain for the production of yogurt and cheeses. The analysis of sequenced genomes of four strains of S. thermophilus indicates that they contain several genes of the rgg familly potentially encoding transcriptional regulators. Some of the Rgg proteins are known to be involved in bacterial stress adaptation. Results In this study, we demonstrated that Streptococcus thermophilus thermal stress adaptation required the rgg0182 gene which transcription depends on the culture medium and the growth temperature. This gene encoded a protein showing similarity with members of the Rgg family transcriptional regulator. Our data confirmed that Rgg0182 is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a Δrgg0182 mutant revealed that this protein played a role in the heat shock adaptation of Streptococcus thermophilus LMG18311. Conclusions These data showed the importance of the Rgg0182 transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.

  1. The structure-specific endonuclease Ercc1–Xpf is required for targeted gene replacement in embryonic stem cells

    Science.gov (United States)

    Niedernhofer, Laura J.; Essers, Jeroen; Weeda, Geert; Beverloo, Berna; de Wit, Jan; Muijtjens, Manja; Odijk, Hanny; Hoeijmakers, Jan H.J.; Kanaar, Roland

    2001-01-01

    The Ercc1–Xpf heterodimer, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair and homologous recombination. Ercc1–Xpf incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here we demonstrate that although Ercc1 is dispensable for recombination between sister chromatids, it is essential for targeted gene replacement in mouse embryonic stem cells. Surprisingly, the role of Ercc1–Xpf in gene targeting is distinct from its previously identified role in removing nonhomologous termini from recombination intermediates because it was required irrespective of whether the ends of the DNA targeting constructs were heterologous or homologous to the genomic locus. Our observations have implications for the mechanism of gene targeting in mammalian cells and define a new role for Ercc1–Xpf in mammalian homologous recombination. We propose a model for the mechanism of targeted gene replacement that invokes a role for Ercc1–Xpf in making the recipient genomic locus receptive for gene replacement. PMID:11707424

  2. Targeted and random mutagenesis of Ehrlichia chaffeensis for the identification of genes required for in vivo infection.

    Science.gov (United States)

    Cheng, Chuanmin; Nair, Arathy D S; Indukuri, Vijaya V; Gong, Shanzhong; Felsheim, Roderick F; Jaworski, Deborah; Munderloh, Ulrike G; Ganta, Roman R

    2013-02-01

    Ehrlichia chaffeensis is a tick transmitted pathogen responsible for the disease human monocytic ehrlichiosis. Research to elucidate gene function in rickettsial pathogens is limited by the lack of genetic manipulation methods. Mutational analysis was performed, targeting to specific and random insertion sites within the bacterium's genome. Targeted mutagenesis at six genomic locations by homologous recombination and mobile group II intron-based methods led to the consistent identification of mutants in two genes and in one intergenic site; the mutants persisted in culture for 8 days. Three independent experiments using Himar1 transposon mutagenesis of E. chaffeensis resulted in the identification of multiple mutants; these mutants grew continuously in macrophage and tick cell lines. Nine mutations were confirmed by sequence analysis. Six insertions were located within non-coding regions and three were present in the coding regions of three transcriptionally active genes. The intragenic mutations prevented transcription of all three genes. Transposon mutants containing a pool of five different insertions were assessed for their ability to infect deer and subsequent acquisition by Amblyomma americanum ticks, the natural reservoir and vector, respectively. Three of the five mutants with insertions into non-coding regions grew well in deer. Transposition into a differentially expressed hypothetical gene, Ech_0379, and at 18 nucleotides downstream to Ech_0230 gene coding sequence resulted in the inhibition of growth in deer, which is further evidenced by their failed acquisition by ticks. Similarly, a mutation into the coding region of ECH_0660 gene inhibited the in vivo growth in deer. This is the first study evaluating targeted and random mutagenesis in E. chaffeensis, and the first to report the generation of stable mutants in this obligate intracellular bacterium. We further demonstrate that in vitro mutagenesis coupled with in vivo infection assessment is a

  3. Targeted and random mutagenesis of Ehrlichia chaffeensis for the identification of genes required for in vivo infection.

    Directory of Open Access Journals (Sweden)

    Chuanmin Cheng

    2013-02-01

    Full Text Available Ehrlichia chaffeensis is a tick transmitted pathogen responsible for the disease human monocytic ehrlichiosis. Research to elucidate gene function in rickettsial pathogens is limited by the lack of genetic manipulation methods. Mutational analysis was performed, targeting to specific and random insertion sites within the bacterium's genome. Targeted mutagenesis at six genomic locations by homologous recombination and mobile group II intron-based methods led to the consistent identification of mutants in two genes and in one intergenic site; the mutants persisted in culture for 8 days. Three independent experiments using Himar1 transposon mutagenesis of E. chaffeensis resulted in the identification of multiple mutants; these mutants grew continuously in macrophage and tick cell lines. Nine mutations were confirmed by sequence analysis. Six insertions were located within non-coding regions and three were present in the coding regions of three transcriptionally active genes. The intragenic mutations prevented transcription of all three genes. Transposon mutants containing a pool of five different insertions were assessed for their ability to infect deer and subsequent acquisition by Amblyomma americanum ticks, the natural reservoir and vector, respectively. Three of the five mutants with insertions into non-coding regions grew well in deer. Transposition into a differentially expressed hypothetical gene, Ech_0379, and at 18 nucleotides downstream to Ech_0230 gene coding sequence resulted in the inhibition of growth in deer, which is further evidenced by their failed acquisition by ticks. Similarly, a mutation into the coding region of ECH_0660 gene inhibited the in vivo growth in deer. This is the first study evaluating targeted and random mutagenesis in E. chaffeensis, and the first to report the generation of stable mutants in this obligate intracellular bacterium. We further demonstrate that in vitro mutagenesis coupled with in vivo infection

  4. Estudio inmunocitoquímico de los tumores de mama de la gata.

    OpenAIRE

    Peñafiel Verdú, Cristina

    2013-01-01

    En el presente trabajo se plantea analizar en tumores mamarios de gata la expresión de marcadores establecidos en el cáncer de mama de la mujer y observar su relación con metástasis, clasificación molecular y modelos de oncogénesis. Se aplican técnicas inmunocitoquímicas sobre 160 muestras (21 adenomas, 66 carcinomas metastáticos y 73 no metastáticos). La expresión de diversos marcadores se asocia con la metástasis y podría relacionarse con los dos modelos de carcinogénesis propuestos, ...

  5. Genome-wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid

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    Sá-Correia Isabel

    2010-10-01

    Full Text Available Abstract Background Acetic acid is a byproduct of Saccharomyces cerevisiae alcoholic fermentation. Together with high concentrations of ethanol and other toxic metabolites, acetic acid may contribute to fermentation arrest and reduced ethanol productivity. This weak acid is also a present in lignocellulosic hydrolysates, a highly interesting non-feedstock substrate in industrial biotechnology. Therefore, the better understanding of the molecular mechanisms underlying S. cerevisiae tolerance to acetic acid is essential for the rational selection of optimal fermentation conditions and the engineering of more robust industrial strains to be used in processes in which yeast is explored as cell factory. Results The yeast genes conferring protection against acetic acid were identified in this study at a genome-wide scale, based on the screening of the EUROSCARF haploid mutant collection for susceptibility phenotypes to this weak acid (concentrations in the range 70-110 mM, at pH 4.5. Approximately 650 determinants of tolerance to acetic acid were identified. Clustering of these acetic acid-resistance genes based on their biological function indicated an enrichment of genes involved in transcription, internal pH homeostasis, carbohydrate metabolism, cell wall assembly, biogenesis of mitochondria, ribosome and vacuole, and in the sensing, signalling and uptake of various nutrients in particular iron, potassium, glucose and amino acids. A correlation between increased resistance to acetic acid and the level of potassium in the growth medium was found. The activation of the Snf1p signalling pathway, involved in yeast response to glucose starvation, is demonstrated to occur in response to acetic acid stress but no evidence was obtained supporting the acetic acid-induced inhibition of glucose uptake. Conclusions Approximately 490 of the 650 determinants of tolerance to acetic acid identified in this work are implicated, for the first time, in tolerance to

  6. Genome-wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid

    Science.gov (United States)

    2010-01-01

    Background Acetic acid is a byproduct of Saccharomyces cerevisiae alcoholic fermentation. Together with high concentrations of ethanol and other toxic metabolites, acetic acid may contribute to fermentation arrest and reduced ethanol productivity. This weak acid is also a present in lignocellulosic hydrolysates, a highly interesting non-feedstock substrate in industrial biotechnology. Therefore, the better understanding of the molecular mechanisms underlying S. cerevisiae tolerance to acetic acid is essential for the rational selection of optimal fermentation conditions and the engineering of more robust industrial strains to be used in processes in which yeast is explored as cell factory. Results The yeast genes conferring protection against acetic acid were identified in this study at a genome-wide scale, based on the screening of the EUROSCARF haploid mutant collection for susceptibility phenotypes to this weak acid (concentrations in the range 70-110 mM, at pH 4.5). Approximately 650 determinants of tolerance to acetic acid were identified. Clustering of these acetic acid-resistance genes based on their biological function indicated an enrichment of genes involved in transcription, internal pH homeostasis, carbohydrate metabolism, cell wall assembly, biogenesis of mitochondria, ribosome and vacuole, and in the sensing, signalling and uptake of various nutrients in particular iron, potassium, glucose and amino acids. A correlation between increased resistance to acetic acid and the level of potassium in the growth medium was found. The activation of the Snf1p signalling pathway, involved in yeast response to glucose starvation, is demonstrated to occur in response to acetic acid stress but no evidence was obtained supporting the acetic acid-induced inhibition of glucose uptake. Conclusions Approximately 490 of the 650 determinants of tolerance to acetic acid identified in this work are implicated, for the first time, in tolerance to this weak acid. These are

  7. Roles of Candida albicans Gat2, a GATA-type zinc finger transcription factor, in biofilm formation, filamentous growth and virulence.

    Science.gov (United States)

    Du, Han; Guan, Guobo; Xie, Jing; Sun, Yuan; Tong, Yaojun; Zhang, Lixin; Huang, Guanghua

    2012-01-01

    Candida albicans is the most common human fungal pathogen, causing not only superficial infections, but also life-threatening systemic disease. C. albicans can grow in several morphological forms including unicellular yeast-form, elongated hyphae and pseudohyphae. In certain natural environments, C. albicans also exists as biofilms, which are structured and surface-attached microbial communities. Transcription factors play a critical role in morphogenesis and biofilm development. In this study, we identified four adhesion-promoting transcription factors (Tec1, Cph1, Ume6 and Gat2) by screening a transcription factor overexpression library. Sequence analysis indicates that Gat2 is a GATA-type zinc finger transcription factor. Here we showed that the gat2/gat2 mutant failed to form biofilms on the plastic and silicone surfaces. Overexpression of GAT2 gene promoted filamentous and invasive growth on agar containing Lee's medium, while deletion of this gene had an opposite effect. However, inactivation of Gat2 had no obvious effect on N-acetyl-glucosamine (GlcNAc) induced hyphal development. In a mouse model of systemic infection, the gat2/gat2 mutant showed strongly attenuated virulence. Our results suggest that Gat2 plays a critical role in C. albicans biofilm formation, filamentous growth and virulence.

  8. Reconstruction of the gene regulatory network involved in the sonic hedgehog pathway with a potential role in early development of the mouse brain.

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    Jinhua Liu

    2014-10-01

    Full Text Available The Sonic hedgehog (Shh signaling pathway is crucial for pattern formation in early central nervous system development. By systematically analyzing high-throughput in situ hybridization data of E11.5 mouse brain, we found that Shh and its receptor Ptch1 define two adjacent mutually exclusive gene expression domains: Shh+Ptch1- and Shh-Ptch1+. These two domains are associated respectively with Foxa2 and Gata3, two transcription factors that play key roles in specifying them. Gata3 ChIP-seq experiments and RNA-seq assays on Gata3-knockdown cells revealed that Gata3 up-regulates the genes that are enriched in the Shh-Ptch1+ domain. Important Gata3 targets include Slit2 and Slit3, which are involved in the process of axon guidance, as well as Slc18a1, Th and Qdpr, which are associated with neurotransmitter synthesis and release. By contrast, Foxa2 both up-regulates the genes expressed in the Shh+Ptch1- domain and down-regulates the genes characteristic of the Shh-Ptch1+ domain. From these and other data, we were able to reconstruct a gene regulatory network governing both domains. Our work provides the first genome-wide characterization of the gene regulatory network involved in the Shh pathway that underlies pattern formation in the early mouse brain.

  9. Resistance to organic hydroperoxides requires ohr and ohrR genes in Sinorhizobium meliloti

    Directory of Open Access Journals (Sweden)

    Dufour Virginie

    2011-05-01

    Full Text Available Abstract Background Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Medicago sativa. During nodule formation bacteria have to withstand oxygen radicals produced by the plant. Resistance to H2O2 and superoxides has been extensively studied in S. meliloti. In contrast resistance to organic peroxides has not been investigated while S. meliloti genome encodes putative organic peroxidases. Organic peroxides are produced by plants and are highly toxic. The resistance to these oxygen radicals has been studied in various bacteria but never in plant nodulating bacteria. Results In this study we report the characterisation of organic hydroperoxide resistance gene ohr and its regulator ohrR in S. meliloti. The inactivation of ohr affects resistance to cumene and ter-butyl hydroperoxides but not to hydrogen peroxide or menadione in vitro. The expression of ohr and ohrR genes is specifically induced by organic peroxides. OhrR binds to the intergenic region between the divergent genes ohr and ohrR. Two binding sites were characterised. Binding to the operator is prevented by OhrR oxidation that promotes OhrR dimerisation. The inactivation of ohr did not affect symbiosis and nitrogen fixation, suggesting that redundant enzymatic activity exists in this strain. Both ohr and ohrR are expressed in nodules suggesting that they play a role during nitrogen fixation. Conclusions This report demonstrates the significant role Ohr and OhrR proteins play in bacterial stress resistance against organic peroxides in S. meliloti. The ohr and ohrR genes are expressed in nodule-inhabiting bacteroids suggesting a role during nodulation.

  10. A forkhead gene related to HNF-3beta is required for gastrulation and axis formation in the ascidian embryo.

    Science.gov (United States)

    Olsen, C L; Jeffery, W R

    1997-09-01

    We have isolated a member of the HNF-3/forkhead gene family in ascidians as a means to determine the role of winged-helix genes in chordate development. The MocuFH1 gene, isolated from a Molgula oculata cDNA library, exhibits a forkhead DNA-binding domain most similar to zebrafish axial and rodent HNF-3beta. MocuFH1 is a single copy gene but there is at least one other related forkhead gene in the M. oculata genome. The MocuFH1 gene is expressed in the presumptive endoderm, mesenchyme and notochord cells beginning during the late cleavage stages. During gastrulation, MocuFH1 expression occurs in the prospective endoderm cells, which invaginate at the vegetal pole, and in the presumptive notochord and mesenchyme cells, which involute over the anterior and lateral lips of the blastopore, respectively. However, this gene is not expressed in the presumptive muscle cells, which involute over the posterior lip of the blastopore. MocuFH1 expression continues in the same cell lineages during neurulation and axis formation, however, during the tailbud stage, MocuFH1 is also expressed in ventral cells of the brain and spinal cord. The functional role of the MocuFH1 gene was studied using antisense oligodeoxynucleotides (ODNs), which transiently reduce MocuFH1 transcript levels during gastrulation. Embryos treated with antisense ODNs cleave normally and initiate gastrulation. However, gastrulation is incomplete, some of the endoderm and notochord cells do not enter the embryo and undergo subsequent movements, and axis formation is abnormal. In contrast, the prospective muscle cells, which do not express MocuFH1, undergo involution and later express muscle actin and acetylcholinesterase, markers of muscle cell differentiation. The results suggest that MocuFH1 is required for morphogenetic movements of the endoderm and notochord precursor cells during gastrulation and axis formation. The effects of inhibiting MocuFH1 expression on embryonic axis formation in ascidians are

  11. Utility of GATA3 immunohistochemistry in differentiating urothelial carcinoma from prostate adenocarcinoma and squamous cell carcinomas of the uterine cervix, anus, and lung.

    Science.gov (United States)

    Chang, Alex; Amin, Ali; Gabrielson, Edward; Illei, Peter; Roden, Richard B; Sharma, Rajni; Epstein, Jonathan I

    2012-10-01

    Distinguishing invasive high-grade urothelial carcinoma (UC) from other carcinomas occurring in the genitourinary tract may be difficult. The differential diagnosis includes high-grade prostatic adenocarcinoma, spread from an anal squamous cell carcinoma (SCC), or spread from a uterine cervical SCC. In terms of metastatic UC, the most common problem is differentiating spread of UC to the lung from a primary pulmonary SCC. Immunohistochemical analysis (IHC) for GATA binding protein 3 (GATA3), thrombomodulin (THROMBO), and uroplakin III was performed on a tissue microarray (TMA) containing 35 cases of invasive high-grade UC. GATA3 IHC was also performed on TMAs containing 38 high-grade (Gleason score ≥8) prostatic adenocarcinomas, representative tissue sections from 15 invasive anal SCCs, representative tissue sections from 19 invasive cervical SCCs, and TMAs with 12 invasive cervical carcinomas of the cervix [SCC (n=10), SCC with neuroendocrine features (n=1), and adenosquamous carcinoma (n=1)]. In addition, GATA3 IHC was performed on representative tissue sections from 15 pulmonary UC metastases and a TMA with 25 SCCs of the lung and 5 pulmonary non-small cell carcinomas with squamous features. GATA3, THROMBO, and uroplakin III were positive in 28 (80%), 22 (63%), and 21 (60%) cases of high-grade UC, respectively. All cases of GATA3-positive staining were nonfocal; 25 (89%) cases demonstrated moderate to strong staining, and 3 (11%) demonstrated weak staining. Of the 7 cases that failed to express GATA3, 5 were positive for THROMBO and/or uroplakin III, whereas 2 were negative for all 3 markers. None of the 38 high-grade prostatic adenocarcinomas was positive for GATA3. Weak GATA3 staining was present in occasional basal cells of benign prostate glands, in a few benign atrophic glands, and in urothelial metaplasia. Of the 15 cases of anal SCCs, 2 (7%) cases showed focal weak staining, and 1 (3%) showed focal moderate staining. Weak staining was also rarely

  12. Efficient construction of homozygous diploid strains identifies genes required for the hyper-filamentous phenotype in Saccharomyces cerevisiae.

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    Kentaro Furukawa

    Full Text Available Yeast cells undergo diploid-specific developments such as spore formation via meiosis and pseudohyphal development under certain nutrient-limited conditions. Studies on these aspects require homozygous diploid mutants, which are generally constructed by crossing strains of opposite mating-type with the same genetic mutation. So far, there has been no direct way to generate and select diploids from haploid cells. Here, we developed a method for efficient construction of homozygous diploids using a PGAL1-HO gene (galactose-inducible mating-type switch and a PSTE18-URA3 gene (counter selection marker for diploids. Diploids are generated by transient induction of the HO endonuclease, which is followed by mating of part of the haploid population. Since the STE18 promoter is repressed in diploids, diploids carrying PSTE18-URA3 can be selected on 5-fluoroorotic acid (5-FOA plates where the uracil prototrophic haploids cannot grow. To demonstrate that this method is useful for genetic studies, we screened suppressor mutations of the complex colony morphology, strong agar invasion and/or hyper-filamentous growth caused by lack of the Hog1 MAPK in the diploid Σ1278b strain background. Following this approach, we identified 49 suppressor mutations. Those include well-known positive regulator genes for filamentous growth signaling pathways, genes involved in mitochondrial function, DNA damage checkpoint, chromatin remodeling, and cell cycle, and also previously uncharacterized genes. Our results indicate that combinatorial use of the PGAL1-HO and PSTE18-URA3 genes is suitable to efficiently construct and select diploids and that this approach is useful for genetic studies especially when combined with large-scale screening.

  13. Dissection of the beta-globin replication-initiation region reveals specific requirements for replicator elements during gene amplification.

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    Naoya Okada

    Full Text Available Gene amplification plays a pivotal role in malignant transformation of human cells. A plasmid with both a mammalian replication-initiation region (IR/origin/replicator and a nuclear matrix-attachment region (MAR is spontaneously amplified in transfected cells by a mechanism that involves amplification at the extrachromosomal site, followed by amplification at the chromosomal arm, ultimately generating a long homogeneously staining region (HSR. Several observations suggest that replication initiation from IR sequences might mediate amplification. To test this idea, we previously dissected c-myc and DHFR IRs to identify the minimum sequence required to support amplification. In this study, we applied an improved analysis that discriminates between two amplification steps to the ß-globin RepP IR, which contains separate elements already known to be essential for initiation on the chromosome arm. The IR sequence was required at least for the extrachromosomal amplification step. In addition to the vector-encoded MAR, amplification also required an AT-rich region and a MAR-like element, consistent with the results regarding replicator activity on the chromosome. However, amplification did not require the AG-rich tract necessary for replicator activity, but instead required a novel sequence containing another AG-rich tract. The differential sequence requirement might be a consequence of extrachromosomal replication.

  14. The Drosophila Medea gene is required downstream of dpp and encodes a functional homolog of human Smad4.

    Science.gov (United States)

    Hudson, J B; Podos, S D; Keith, K; Simpson, S L; Ferguson, E L

    1998-04-01

    The Transforming Growth Factor-beta superfamily member decapentaplegic (dpp) acts as an extracellular morphogen to pattern the embryonic ectoderm of the Drosophila embryo. To identify components of the dpp signaling pathway, we screened for mutations that act as dominant maternal enhancers of a weak allele of the dpp target gene zerknŁllt. In this screen, we recovered new alleles of the Mothers against dpp (Mad) and Medea genes. Phenotypic analysis of the new Medea mutations indicates that Medea, like Mad, is required for both embryonic and imaginal disc patterning. Genetic analysis suggests that Medea may have two independently mutable functions in patterning the embryonic ectoderm. Complete elimination of maternal and zygotic Medea activity in the early embryo results in a ventralized phenotype identical to that of null dpp mutants, indicating that Medea is required for all dpp-dependent signaling in embryonic dorsal-ventral patterning. Injection of mRNAs encoding DPP or a constitutively activated form of the DPP receptor, Thick veins, into embryos lacking all Medea activity failed to induce formation of any dorsal cell fates, demonstrating that Medea acts downstream of the thick veins receptor. We cloned Medea and found that it encodes a protein with striking sequence similarity to human SMAD4. Moreover, injection of human SMAD4 mRNA into embryos lacking all Medea activity conferred phenotypic rescue of the dorsal-ventral pattern, demonstrating conservation of function between the two gene products.

  15. pRB is required for interferon-gamma-induction of the MHC class II abeta gene.

    Science.gov (United States)

    Zhu, X; Pattenden, S; Bremner, R

    1999-09-02

    pRB is required for IFN-gamma-induction of MHC class II in human tumor cell lines, providing a potential link between tumor suppressors and the immune system. However, other genes, such as cyclin D1, show pRB-dependency only in tumor cells, so by analogy, pRB may not be necessary for cII-regulation in normal cells. Here, we demonstrate that induction of the mouse MHC class II I-A heterodimer is normal in RB+/+ mouse embryonic fibroblasts (MEFs), but deficient in RB-/- MEFs. Inducibility is restored in RB-/- MEFs stably transfected with wild type RB cDNA or infected with an adenovirus expressing pRB. Thus, involvement of pRB in MHC class II expression is conserved in the mouse and is not an aberrant feature of tumorigenic, aneuploid, human tumor cells. Although cII genes are generally induced in a coordinate fashion, suggesting a common mechanism, we found that pRB was specifically required for induction of the Abeta, but not Aalpha or other MHC cII genes including Ebeta, Ii and H2-Malpha. Finally, IFN-gamma-induction of class II transactivator (CIITA), was pRB-independent, suggesting that pRB works downstream of this master-regulator of MHC class II expression.

  16. Genes required for growth at high hydrostatic pressure in Escherichia coli K-12 identified by genome-wide screening.

    Science.gov (United States)

    Black, S Lucas; Dawson, Angela; Ward, F Bruce; Allen, Rosalind J

    2013-01-01

    Despite the fact that much of the global microbial biosphere is believed to exist in high pressure environments, the effects of hydrostatic pressure on microbial physiology remain poorly understood. We use a genome-wide screening approach, combined with a novel high-throughput high-pressure cell culture method, to investigate the effects of hydrostatic pressure on microbial physiology in vivo. The Keio collection of single-gene deletion mutants in Escherichia coli K-12 was screened for growth at a range of pressures from 0.1 MPa to 60 MPa. This led to the identification of 6 genes, rodZ, holC, priA, dnaT, dedD and tatC, whose products were required for growth at 30 MPa and a further 3 genes, tolB, rffT and iscS, whose products were required for growth at 40 MPa. Our results support the view that the effects of pressure on cell physiology are pleiotropic, with DNA replication, cell division, the cytoskeleton and cell envelope physiology all being potential failure points for cell physiology during growth at elevated pressure.

  17. The fire blight pathogen Erwinia amylovora requires the rpoN gene for pathogenicity in apple.

    Science.gov (United States)

    Ramos, Laura S; Lehman, Brian L; Sinn, Judith P; Pfeufer, Emily E; Halbrendt, Noemi O; McNellis, Timothy W

    2013-10-01

    RpoN is a σ(54) factor regulating essential virulence gene expression in several plant pathogenic bacteria, including Pseudomonas syringae and Pectobacterium carotovorum. In this study, we found that mutation of rpoN in the fire blight pathogen Erwinia amylovora caused a nonpathogenic phenotype. The E. amylovora rpoN Tn5 transposon mutant rpoN1250::Tn5 did not cause fire blight disease symptoms on shoots of mature apple trees. In detached immature apple fruits, the rpoN1250::Tn5 mutant failed to cause fire blight disease symptoms and grew to population levels 12 orders of magnitude lower than the wild-type. In addition, the rpoN1250::Tn5 mutant failed to elicit a hypersensitive response when infiltrated into nonhost tobacco plant leaves, and rpoN1250::Tn5 cells failed to express HrpN protein when grown in hrp (hypersensitive response and pathogenicity)-inducing liquid medium. A plasmid-borne copy of the wild-type rpoN gene complemented all the rpoN1250::Tn5 mutant phenotypes tested. The rpoN1250::Tn5 mutant was prototrophic on minimal solid and liquid media, indicating that the rpoN1250::Tn5 nonpathogenic phenotype was not caused by a defect in basic metabolism or growth. This study provides clear genetic evidence that rpoN is an essential virulence gene of E. amylovora, suggesting that rpoN has the same function in E. amylovora as in P. syringae and Pe. carotovorum.

  18. Genome-wide identification of Saccharomyces cerevisiae genes required for maximal tolerance to ethanol.

    Science.gov (United States)

    Teixeira, Miguel C; Raposo, Luís R; Mira, Nuno P; Lourenço, Artur B; Sá-Correia, Isabel

    2009-09-01

    The understanding of the molecular basis of yeast resistance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. In this study, the yeast disruptome was screened for mutants with differential susceptibility to stress induced by high ethanol concentrations in minimal growth medium. Over 250 determinants of resistance to ethanol were identified. The most significant gene ontology terms enriched in this data set are those associated with intracellular organization, biogenesis, and transport, in particular, regarding the vacuole, the peroxisome, the endosome, and the cytoskeleton, and those associated with the transcriptional machinery. Clustering the proteins encoded by the identified determinants of ethanol resistance by their known physical and genetic interactions highlighted the importance of the vacuolar protein sorting machinery, the vacuolar H(+)-ATPase complex, and the peroxisome protein import machinery. Evidence showing that vacuolar acidification and increased resistance to the cell wall lytic enzyme beta-glucanase occur in response to ethanol-induced stress was obtained. Based on the genome-wide results, the particular role of the FPS1 gene, encoding a plasma membrane aquaglyceroporin which mediates controlled glycerol efflux, in ethanol stress resistance was further investigated. FPS1 expression contributes to decreased [(3)H]ethanol accumulation in yeast cells, suggesting that Fps1p may also play a role in maintaining the intracellular ethanol level during active fermentation. The increased expression of FPS1 confirmed the important role of this gene in alcoholic fermentation, leading to increased final ethanol concentration under conditions that lead to high ethanol production.

  19. Emdogain-regulated gene expression in palatal fibroblasts requires TGF-βRI kinase signaling.

    OpenAIRE

    Stähli, Alexandra Beatrice; Bosshardt, Dieter; Sculean, Anton; Gruber, Reinhard

    2014-01-01

    Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB43...

  20. Emdogain-Regulated Gene Expression in Palatal Fibroblasts Requires TGF-βRI Kinase Signaling

    OpenAIRE

    Alexandra Stähli; Dieter Bosshardt; Anton Sculean; Reinhard Gruber

    2014-01-01

    Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB43...

  1. Expression and Characterization of Streptococcal rgp Genes Required for Rhamnan Synthesis in Escherichia coli†

    OpenAIRE

    Shibata, Yukie; Yamashita, Yoshihisa; Ozaki, Kazuhisa; Nakano, Yoshio; Koga, Toshihiko

    2002-01-01

    Six genes (rgpA through rgpF) that were involved in assembling the rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans were previously identified (Y. Yamashita, Y. Tsukioka, K. Tomihisa, Y. Nakano, and T. Koga, J. Bacteriol. 180:5803-5807, 1998). The group-specific antigens of Lancefield group A, C, and E streptococci and the polysaccharide antigen of Streptococcus sobrinus have the same rhamnan backbone as the RGP of S. mutans. Escherichia coli harboring plasmid pRGP1 containing al...

  2. The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression.

    Science.gov (United States)

    Streatfield, S J; Weber, A; Kinsman, E A; Häusler, R E; Li, J; Post-Beittenmiller, D; Kaiser, W M; Pyke, K A; Flügge, U I; Chory, J

    1999-09-01

    The Arabidopsis chlorophyll a/b binding protein (CAB) gene underexpressed 1 (cue1) mutant underexpresses light-regulated nuclear genes encoding chloroplast-localized proteins. cue1 also exhibits mesophyll-specific chloroplast and cellular defects, resulting in reticulate leaves. Both the gene underexpression and the leaf cell morphology phenotypes are dependent on light intensity. In this study, we determine that CUE1 encodes the plastid inner envelope phosphoenolpyruvate/phosphate translocator (PPT) and define amino acid residues that are critical for translocator function. The biosynthesis of aromatics is compromised in cue1, and the reticulate phenotype can be rescued by feeding aromatic amino acids. Determining that CUE1 encodes PPT indicates the in vivo role of the translocator in metabolic partitioning and reveals a mesophyll cell-specific requirement for the translocator in Arabidopsis leaves. The nuclear gene expression defects in cue1 suggest that a light intensity-dependent interorganellar signal is modulated through metabolites dependent on a plastid supply of phosphoenolpyruvate.

  3. The Popeye domain containing 2 (popdc2) gene in zebrafish is required for heart and skeletal muscle development.

    Science.gov (United States)

    Kirchmaier, Bettina C; Poon, Kar Lai; Schwerte, Thorsten; Huisken, Jan; Winkler, Christoph; Jungblut, Benno; Stainier, Didier Y; Brand, Thomas

    2012-03-15

    The Popeye domain containing (Popdc) genes encode a family of transmembrane proteins with an evolutionary conserved Popeye domain. These genes are abundantly expressed in striated muscle tissue, however their function is not well understood. In this study we have investigated the role of the popdc2 gene in zebrafish. Popdc2 transcripts were detected in the embryonic myocardium and transiently in the craniofacial and tail musculature. Morpholino oligonucleotide-mediated knockdown of popdc2 resulted in aberrant development of skeletal muscle and heart. Muscle segments in the trunk were irregularly shaped and craniofacial muscles were severely reduced or even missing. In the heart, pericardial edema was prevalent in the morphants and heart chambers were elongated and looping was abnormal. These pathologies in muscle and heart were alleviated after reducing the morpholino concentration. However the heart still was abnormal displaying cardiac arrhythmia at later stages of development. Optical recordings of cardiac contractility revealed irregular ventricular contractions with a 2:1, or 3:1 atrial/ventricular conduction ratio, which caused a significant reduction in heart frequency. Recordings of calcium transients with high spatiotemporal resolution using a transgenic calcium indicator line (Tg(cmlc2:gCaMP)(s878)) and SPIM microscopy confirmed the presence of a severe arrhythmia phenotype. Our results identify popdc2 as a gene important for striated muscle differentiation and cardiac morphogenesis. In addition it is required for the development of the cardiac conduction system.

  4. Regulation of the New Arabidopsis Imprinted Gene AtBMI1C Requires the Interplay of Different Epigenetic Mechanisms

    Institute of Scientific and Technical Information of China (English)

    Fabian Bratzel; ChaoYang; Alexandra Ancelova; Gema López-Torrejón; Marcus Koch; Juan Carlos del Pozo; Myriam Calonje

    2012-01-01

    Recently,it has been shown that plants contain homologs to the animal Polycomb repressive complex 1 (PRC1)components BMI1 and RING1A/B.In Arabidopsis,there are three BMI1-like genes,two of which,AtBMI1A and B,are required during post-embryonic plant growth to repress embryonic traits and allow cell differentiation.However,little is known about the third BMI1-like gene,AtBMI1C.In this work,we show that AtBMI1C is only expressed during endosperm and stamen development.AtBMI1C is an imprinted gene expressed from the maternal allele in the endosperm but biallelically expressed in stamen.We found that the characteristic expression pattern of AtBMI1C is the result of a complex epigenetic regulation that involves CG DNA methylation,RNA-directed non-CG DNA methylation (RdDM),and PcG activity.Our results show the orchestrated interplay of different epigenetic mechanisms in regulating gene expression throughout development,shedding light on the current hypotheses for the origin and mechanism of imprinting in plant endosperm.

  5. DELAYED FLOWERING, an Arabidopsis Gene That Acts in the Autonomous Flowering Promotion Pathway and Is Required for Normal Development

    Institute of Scientific and Technical Information of China (English)

    Ming-Jie Chen; Zheng Yuan; Hai Huang

    2006-01-01

    The control of flowering time in higher plants is one of the most important physiological processes and is critical for their reproductive success. To investigate the mechanisms controlling flowering time, we screened for Arabidopsis mutants with late-flowering phenotypes. One mutant, designated delayed flowering (dfr) in the Landsberg erecta (Ler) ecotype, was identified with delayed flowering time. Genetic analysis revealed that dfr is a single gene recessive nuclear mutant and the mutation was mapped to a locus tightly linked to UFO on chromosome 1. To our knowledge, no gene regulating flowering time has been reported yet in this region. The dfr mutant plant showed a delayed flowering time under the different growth conditions examined,including long- and short-day photoperiods and gibberellic acid GA3 treatments, suggesting that DFR is a gene involved in the autonomous flowering promotion pathway. The Arabidopsis gene FLOWERING LOCUS C (FLC) plays a central role in repressing flowering and its transcripts are undetectable in wild-type Ler.However, FLCexpression was upregulated in the dfrmutant, suggesting that DFR is a negative regulator of FLC. In addition, the dfr mutant plant displayed altered valve shapes of the silique and the number of trichomes and branches of each trichome were both reduced, indicating that the DRFgene is also required for normal plant development. Moreover, dfr leafy-5 (Ify-5) double mutant plants showed a much later flowering time than either dfr or Ify-5 single mutants, indicating that DFR and LFYact synergistically to promote flowering in Arabidopsis.

  6. A gene (ccmA) required for carboxysome formation in the cyanobacterium Synechocystis sp. strain PCC6803.

    Science.gov (United States)

    Ogawa, T; Marco, E; Orus, M I

    1994-04-01

    A high-CO2-requiring mutant, G7, of Synechocystis sp. strain PCC6803 capable of inorganic carbon transport but unable to utilize the intracellular inorganic carbon pool for photosynthesis was isolated. Transmission electron micrographs of the mutant indicated that the mutant does not have any carboxysomes. A clone (pHPG7) with a 7.5-kbp DNA insert that transforms the G7 mutant to the wild-type phenotype was isolated from a genomic library of wild-type Synechocystis sp. strain PCC6803. Complementation tests with subclones identified the mutation site in G7 within 208 bp. Sequencing of nucleotides in this region elucidated an open reading frame, designated ccmA, encoding a protein of 302 amino acids. Cloning and sequence analysis of the respective G7 gene revealed an A-to-G substitution that results in an Asp-to-Gly substitution in the deduced amino acid. The result indicated that the ccmA gene encodes a protein essential for the formation of carboxysomes. An open reading frame encoding a proline-rich protein of 271 amino acids was found downstream of the ccmA gene, but no ccm-like genes or rbc operon was found in this region.

  7. GATA1 mutation negative acute megakaryoblastic leukemia with acquired trisomy 21 presenting with extensive bone marrow necrosis in an adult: A case report and review of the literature

    Directory of Open Access Journals (Sweden)

    Todd P. Williams

    2016-03-01

    Conclusions: To our knowledge, this is the first reported case of an adult with AMKL with acquired trisomy 21 in which the GATA1 mutation was investigated and the second reported case of AMKL presenting with extensive bone marrow necrosis. We will present a diagnostic approach to AMKL in which extensive bone marrow necrosis renders examination of the bone marrow difficult. Furthermore, we will examine the absence of the GATA1 mutation in a case of AMKL with trisomy 21 in an adult.

  8. The Transcription Factor GATA-6 is Overexpressed in Vivo and Contributes to Silencing 15-LOX-1 in Vitro in Human Colon Cancer

    OpenAIRE

    Shureiqi, Imad; Zuo, Xiangsheng; Broaddus, Russell; Wu, Yuanqing; Guan, Baoxiang; Morris, Jeffrey S.; Lippman, Scott M.

    2006-01-01

    Transcriptional suppression of 15-lipoxygenase-1 (15-LOX-1) helps enable human colorectal cancer cells escape apoptosis, a critical mechanism for colonic tumorigenesis. GATA-6 is strongly expressed in vitro in cancer cells; its downregulation by pharmaceuticals is associated with reversal of 15-LOX-1 transcriptional suppression. The mechanistic contribution of GATA-6 overexpression to colonic tumorigenesis, especially concerning 15-LOX-1 transcriptional suppression, remains unknown. We tested...

  9. B-lineage transcription factors and cooperating gene lesions required for leukemia development

    NARCIS (Netherlands)

    Tijchon, E.J.H.; Havinga, J.; Leeuwen, F.N. van; Scheijen, B.

    2013-01-01

    Differentiation of hematopoietic stem cells into B lymphocytes requires the concerted action of specific transcription factors, such as RUNX1, IKZF1, E2A, EBF1 and PAX5. As key determinants of normal B-cell development, B-lineage transcription factors are frequently deregulated in hematological

  10. Identification of Genes Required for the Survival of BRCA 1-/- Cells

    Science.gov (United States)

    2010-02-01

    homologous recombination26, 27. The numerous functions of BRCA1 complexes highlight the importance of this protein in normal cellular function, and the ways...to double-strand breaks. Science 286, 1162-6 (1999). 36. Smogorzewska, A. et al. Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair. Cell 129, 289-301 (2007).

  11. The arrest gene is required for germline cyst formation during Drosophila oogenesis.

    Science.gov (United States)

    Parisi, M J; Deng, W; Wang, Z; Lin, H

    2001-04-01

    In Drosophila, oogenesis is initiated when a germline stem cell produces a differentiating daughter cell called the cystoblast. The cystoblast undergoes four rounds of synchronous divisions with incomplete cytokinesis to generate a syncytial cyst of 16 interconnected cystocytes, in which one cystocyte differentiates into an oocyte. Strong mutations of the arrest (aret) gene disrupt cyst formation and cause the production of clusters of ill-differentiated germline cells that retain cellular and molecular characteristics of cystoblasts. These mutant germ cells express high levels of BAM-C and SXL proteins in the cytoplasm but do not accumulate markers for advanced cystocytes or differentiating oocytes, such as the nuclear localization of SXL or the accumulation of osk mRNA, orb mRNA, and cytoplasmic dynein. However, the mutant germ cells do not contain spectrosomes, the cytoplasmic structure that objectifies the divisional asymmetry of the cystoblast. The aret mutant germ cells undergo active mitosis with complete cytokinesis. Their mitosis is accompanied by massive necrosis, so that the number of germ cells in a stem cell-derived cluster ranges from one to greater than 70. These defects of aret mutants reveal a novel function of aret as the first gene with a defined function in the cystoblast to cyst transition during early oogenesis.

  12. The tumor suppressor gene retinoblastoma-1 is required for retinotectal development and visual function in zebrafish.

    Directory of Open Access Journals (Sweden)

    Michael Gyda

    Full Text Available Mutations in the retinoblastoma tumor suppressor gene (rb1 cause both sporadic and familial forms of childhood retinoblastoma. Despite its clinical relevance, the roles of rb1 during normal retinotectal development and function are not well understood. We have identified mutations in the zebrafish space cadet locus that lead to a premature truncation of the rb1 gene, identical to known mutations in sporadic and familial forms of retinoblastoma. In wild-type embryos, axons of early born retinal ganglion cells (RGC pioneer the retinotectal tract to guide later born RGC axons. In rb1 deficient embryos, these early born RGCs show a delay in cell cycle exit, causing a transient deficit of differentiated RGCs. As a result, later born mutant RGC axons initially fail to exit the retina, resulting in optic nerve hypoplasia. A significant fraction of mutant RGC axons eventually exit the retina, but then frequently project to the incorrect optic tectum. Although rb1 mutants eventually establish basic retinotectal connectivity, behavioral analysis reveals that mutants exhibit deficits in distinct, visually guided behaviors. Thus, our analysis of zebrafish rb1 mutants reveals a previously unknown yet critical role for rb1 during retinotectal tract development and visual function.

  13. CTCF Is Required for Neural Development and Stochastic Expression of Clustered Pcdh Genes in Neurons

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    Teruyoshi Hirayama

    2012-08-01

    Full Text Available The CCCTC-binding factor (CTCF is a key molecule for chromatin conformational changes that promote cellular diversity, but nothing is known about its role in neurons. Here, we produced mice with a conditional knockout (cKO of CTCF in postmitotic projection neurons, mostly in the dorsal telencephalon. The CTCF-cKO mice exhibited postnatal growth retardation and abnormal behavior and had defects in functional somatosensory mapping in the brain. In terms of gene expression, 390 transcripts were expressed at significantly different levels between CTCF-deficient and control cortex and hippocampus. In particular, the levels of 53 isoforms of the clustered protocadherin (Pcdh genes, which are stochastically expressed in each neuron, declined markedly. Each CTCF-deficient neuron showed defects in dendritic arborization and spine density during brain development. Their excitatory postsynaptic currents showed normal amplitude but occurred with low frequency. Our results indicate that CTCF regulates functional neural development and neuronal diversity by controlling clustered Pcdh expression.

  14. glpx Gene in Mycobacterium tuberculosis Is Required for In Vitro Gluconeogenic Growth and In Vivo Survival.

    Directory of Open Access Journals (Sweden)

    Hiten J Gutka

    Full Text Available Several enzymes involved in central carbon metabolism and gluconeogenesis play a critical role in survival and pathogenesis of Mycobacterium tuberculosis (Mtb. The only known functional fructose 1,6-bisphosphatase (FBPase in Mtb is encoded by the glpX gene and belongs to the Class II sub-family of FBPase. We describe herein the generation of a ΔglpX strain using homologous recombination. Although the growth profile of ΔglpX is comparable to that of wild type Mtb when grown on the standard enrichment media, its growth is dysgonic with individual gluconeogenic substrates such as oleic acid, glycerol and acetate. In mice lung CFU titers of ΔglpX were 2-3 log10 lower than the wild-type Mtb strain. The results indicate that glpX gene encodes a functional FBPase and is essential for both in vitro and in vivo growth and survival of Mtb. Loss of glpX results in significant reduction of FBPase activity but not complete abolition. These findings verify that the glpX encoded FBPase II in Mtb can be a potential target for drug discovery.

  15. Drosophila convoluted/dALS is an essential gene required for tracheal tube morphogenesis and apical matrix organization.

    Science.gov (United States)

    Swanson, Lianna E; Yu, Marcus; Nelson, Kevin S; Laprise, Patrick; Tepass, Ulrich; Beitel, Greg J

    2009-04-01

    Insulin-like growth factors (IGFs) control cell and organism growth through evolutionarily conserved signaling pathways. The mammalian acid-labile subunit (ALS) is a secreted protein that complexes with IGFs to modulate their activity. Recent work has shown that a Drosophila homolog of ALS, dALS, can also complex with and modulate the activity of a Drosophila IGF. Here we report the first mutations in the gene encoding dALS. Unexpectedly, we find that these mutations are allelic to a previously described mutation in convoluted (conv), a gene required for epithelial morphogenesis. In conv mutants, the tubes of the Drosophila tracheal system become abnormally elongated without altering tracheal cell number. conv null mutations cause larval lethality, but do not disrupt several processes required for tracheal tube size control, including septate junction formation, deposition of a lumenal/apical extracellular matrix, and lumenal secretion of Vermiform and Serpentine, two putative matrix-modifying proteins. Clearance of lumenal matrix and subcellular localization of clathrin also appear normal in conv mutants. However, we show that Conv/dALS is required for the dynamic organization of the transient lumenal matrix and normal structure of the cuticle that lines the tracheal lumen. These and other data suggest that the Conv/dALS-dependent tube size control mechanism is distinct from other known processes involved in tracheal tube size regulation. Moreover, we present evidence indicating that Conv/dALS has a novel, IGF-signaling independent function in tracheal morphogenesis.

  16. A new PKLR gene mutation in the R-type promoter region affects the gene transcription causing pyruvate kinase deficiency.

    Science.gov (United States)

    Manco, L; Ribeiro, M L; Máximo, V; Almeida, H; Costa, A; Freitas, O; Barbot, J; Abade, A; Tamagnini, G

    2000-09-01

    Mutations in the PKLR gene responsible for pyruvate kinase (PK)-deficient anaemia are mainly located in the coding regions: 11 are in the splicing sites and, recently, three mutations have been described in the promoter region. We now report a novel point mutation A-->G on nucleotide 72, upstream from the initiation codon of the PKLR gene, in four Portuguese PK-deficient patients. This new regulatory mutation occurs within the most proximal of the four GATA motifs (GATA-A element) in the R-type promoter region. In two patients who were homozygous for this mutation, a semiquantitative reverse transcription polymerase chain reaction (PCR) procedure was used to evaluate the amount of R-PK mRNA transcript in the reticulocytes. The mRNA level was about five times lower than in normal controls, demonstrating that the PKLR gene transcription is severely affected, most probably because the -72A-->G point mutation disables the binding of the erythroid transcription factor GATA-1 to the GATA-A element. Supporting these data, the two patients homozygous for the -72A-->G mutation had severe haemolytic anaemia and were transfusion dependent until splenectomy. Two other patients who were compound heterozygous for this mutation and the previously described missense mutation 1456C-->T had a mild condition.

  17. ChREBP, but not LXRs, is required for the induction of glucose-regulated genes in mouse liver

    Science.gov (United States)

    Denechaud, Pierre-Damien; Bossard, Pascale; Lobaccaro, Jean-Marc A.; Millatt, Lesley; Staels, Bart; Girard, Jean; Postic, Catherine

    2008-01-01

    The transcription factor carbohydrate-responsive element–binding protein (ChREBP) has emerged as a central regulator of lipid synthesis in liver because it is required for glucose-induced expression of the glycolytic enzyme liver–pyruvate kinase (L-PK) and acts in synergy with SREBP to induce lipogenic genes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). Liver X receptors (LXRs) are also important regulators of the lipogenic pathway, and the recent finding that ChREBP is a direct target of LXRs and that glucose itself can bind and activate LXRs prompted us to study the role of LXRs in the induction of glucose-regulated genes in liver. Using an LXR agonist in wild-type mice, we found that LXR stimulation did not promote ChREBP phosphorylation or nuclear localization in the absence of an increased intrahepatic glucose flux. Furthermore, the induction of ChREBP, L-PK, and ACC by glucose or high-carbohydrate diet was similar in LXRα/β knockout compared with wild-type mice, suggesting that the activation of these genes by glucose occurs by an LXR-independent mechanism. We used fluorescence resonance energy transfer analysis to demonstrate that glucose failed to promote the interaction of LXRα/β with specific cofactors. Finally, siRNA silencing of ChREBP in LXRα/β knockout hepatocytes abrogated glucose-induced expression of L-PK and ACC, further demonstrating the central role of ChREBP in glucose signaling. Taken together, our results demonstrate that glucose is required for ChREBP functional activity and that LXRs are not necessary for the induction of glucose-regulated genes in liver. PMID:18292813

  18. Division genes in Escherichia coli are expressed coordinately to cell septum requirements by gearbox promoters.

    Science.gov (United States)

    Aldea, M; Garrido, T; Pla, J; Vicente, M

    1990-11-01

    The cell division ftsQAZ cluster and the ftsZ-dependent bolA morphogene of Escherichia coli are found to be driven by gearboxes, a distinct class of promoters characterized by showing an activity that is inversely dependent on growth rate. These promoters contain specific sequences upstream from the mRNA start point, and their -10 region is essential for the inverse growth rate dependence. Gearbox promoters are essential for driving ftsQAZ and bolA gene expression so that the encoded products are synthesized at constant amounts per cell independently of cell size. This mode of regulation would be expected for the expression of proteins that either play a regulatory role in cell division or form a stoichiometric component of the septum, a structure that, independently of cell size and growth rate, is produced once per cell cycle.

  19. Successful Reconstruction of Tooth Germ with Cell Lines Requires Coordinated Gene Expressions from the Initiation Stage

    Directory of Open Access Journals (Sweden)

    Yasuhiro Tomooka

    2012-10-01

    Full Text Available Tooth morphogenesis is carried out by a series of reciprocal interactions between the epithelium and mesenchyme in embryonic germs. Previously clonal dental epithelial cell (epithelium of molar tooth germ (emtg lines were established from an embryonic germ. They were odontogenic when combined with a dental mesenchymal tissue, although the odontogenesis was quantitatively imperfect. To improve the microenvironment in the germs, freshly isolated dental epithelial cells were mixed with cells of lines, and germs were reconstructed in various combinations. The results demonstrated that successful tooth construction depends on the mixing ratio, the age of dental epithelial cells and the combination with cell lines. Analyses of gene expression in these germs suggest that some signal(s from dental epithelial cells makes emtg cells competent to communicate with mesenchymal cells and the epithelial and mesenchymal compartments are able to progress  odontogenesis from the initiation stage.

  20. Regulation of extracellular matrix genes by arecoline in primary gingival fibroblasts requires epithelial factors.

    Science.gov (United States)

    Thangjam, G S; Agarwal, P; Balapure, A K; Rao, S Girish; Kondaiah, P

    2009-12-01

    Oral submucous fibrosis, a disease of collagen disorder, has been attributed to arecoline present in the saliva of betel quid chewers. However, the molecular basis of the action of arecoline in the pathogenesis of oral submucous fibrosis is poorly understood. The basic aim of our study was to elucidate the mechanism underlying the action of arecoline on the expression of genes in oral fibroblasts. Human keratinocytes (HaCaT cells) and primary human gingival fibroblasts were treated with arecoline in combination with various pathway inhibitors, and the expression of transforming growth factor-beta isoform genes and of collagen isoforms was assessed using reverse transcription-polymerase chain reaction analysis. We observed the induction of transforming growth factor-beta2 by arecoline in HaCaT cells and this induction was found to be caused by activation of the M-3 muscarinic acid receptor via the induction of calcium and the protein kinase C pathway. Most importantly, we showed that transforming growth factor-beta2 was significantly overexpressed in oral submucous fibrosis tissues (p = 0.008), with a median of 2.13 (n = 21) compared with 0.75 (n = 18) in normal buccal mucosal tissues. Furthermore, arecoline down-regulated the expression of collagens 1A1 and 3A1 in human primary gingival fibroblasts; however these collagens were induced by arecoline in the presence of spent medium of cultured human keratinocytes. Treatment with a transforming growth factor-beta blocker, transforming growth factor-beta1 latency-associated peptide, reversed this up-regulation of collagen, suggesting a role for profibrotic cytokines, such as transforming growth factor-beta, in the induction of collagens. Taken together, our data highlight the importance of arecolineinduced epithelial changes in the pathogenesis of oral submucous fibrosis.

  1. Mutations in SRY and WT1 genes required for gonadal development are not responsible for XY partial gonadal dysgenesis

    Directory of Open Access Journals (Sweden)

    E.B. Tagliarini

    2005-01-01

    Full Text Available The WT1 transcription factor regulates SRY expression during the initial steps of the sex determination process in humans, activating a gene cascade leading to testis differentiation. In addition to causing Wilms' tumor, mutations in WT1 are often responsible for urogenital defects in men, while SRY mutations are mainly related to 46,XY pure gonadal dysgenesis. In order to evaluate their role in abnormal testicular organogenesis, we screened for SRY and WT1 gene mutations in 10 children with XY partial gonadal dysgenesis, 2 of whom with a history of Wilms' tumor. The open reading frame and 360 bp of the 5' flanking sequence of the SRY gene, and the ten exons and intron boundaries of the WT1 gene were amplified by PCR of genomic DNA. Single-strand conformation polymorphism was initially used for WT1 mutation screening. Since shifts in fragment migration were only observed for intron/exon 4, the ten WT1 exons from all patients were sequenced manually. No mutations were detected in the SRY 5' untranslated region or within SRY open-reading frame sequences. WT1 sequencing revealed one missense mutation (D396N in the ninth exon of a patient who also had Wilms' tumor. In addition, two silent point mutations were found in the first exon including one described here for the first time. Some non-coding sequence variations were detected, representing one new (IVS4+85A>G and two already described (-7ATG T>G, IVS9-49 T>C single nucleotide polymorphisms. Therefore, mutations in two major genes required for gonadal development, SRY and WT1, are not responsible for XY partial gonadal dysgenesis.

  2. The cobY gene of the archaeon Halobacterium sp. strain NRC-1 is required for de novo cobamide synthesis.

    Science.gov (United States)

    Woodson, J D; Peck, R F; Krebs, M P; Escalante-Semerena, J C

    2003-01-01

    Genetic and nutritional analyses of mutants of the extremely halophilic archaeon Halobacterium sp. strain NRC-1 showed that open reading frame (ORF) Vng1581C encodes a protein with nucleoside triphosphate:adenosylcobinamide-phosphate nucleotidyltransferase enzyme activity. This activity was previously associated with the cobY gene of the methanogenic archaeon Methanobacterium thermoautotrophicum strain DeltaH, but no evidence was obtained to demonstrate the direct involvement of this protein in cobamide biosynthesis in archaea. Computer analysis of the Halobacterium sp. strain NRC-1 ORF Vng1581C gene and the cobY gene of M. thermoautotrophicum strain DeltaH showed the primary amino acid sequence of the proteins encoded by these two genes to be 35% identical and 48% similar. A strain of Halobacterium sp. strain NRC-1 carrying a null allele of the cobY gene was auxotrophic for cobinamide-GDP, a known intermediate of the late steps of cobamide biosynthesis. The auxotrophic requirement for cobinamide-GDP was corrected when a wild-type allele of cobY was introduced into the mutant strain, demonstrating that the lack of cobY function was solely responsible for the observed block in cobamide biosynthesis in this archaeon. The data also show that Halobacterium sp. strain NRC-1 possesses a high-affinity transport system for corrinoids and that this archaeon can synthesize cobamides de novo under aerobic growth conditions. To the best of our knowledge this is the first genetic and nutritional analysis of cobalamin biosynthetic mutants in archaea.

  3. Bioenergetics-based modeling of Plasmodium falciparum metabolism reveals its essential genes, nutritional requirements, and thermodynamic bottlenecks.

    Science.gov (United States)

    Chiappino-Pepe, Anush; Tymoshenko, Stepan; Ataman, Meriç; Soldati-Favre, Dominique; Hatzimanikatis, Vassily

    2017-03-01

    Novel antimalarial therapies are urgently needed for the fight against drug-resistant parasites. The metabolism of malaria parasites in infected cells is an attractive source of drug targets but is rather complex. Computational methods can handle this complexity and allow integrative analyses of cell metabolism. In this study, we present a genome-scale metabolic model (iPfa) of the deadliest malaria parasite, Plasmodium falciparum, and its thermodynamics-based flux analysis (TFA). Using previous absolute concentration data of the intraerythrocytic parasite, we applied TFA to iPfa and predicted up to 63 essential genes and 26 essential pairs of genes. Of the 63 genes, 35 have been experimentally validated and reported in the literature, and 28 have not been experimentally tested and include previously hypothesized or novel predictions of essential metabolic capabilities. Without metabolomics data, four of the genes would have been incorrectly predicted to be non-essential. TFA also indicated that substrate channeling should exist in two metabolic pathways to ensure the thermodynamic feasibility of the flux. Finally, analysis of the metabolic capabilities of P. falciparum led to the identification of both the minimal nutritional requirements and the genes that can become indispensable upon substrate inaccessibility. This model provides novel insight into the metabolic needs and capabilities of the malaria parasite and highlights metabolites and pathways that should be measured and characterized to identify potential thermodynamic bottlenecks and substrate channeling. The hypotheses presented seek to guide experimental studies to facilitate a better understanding of the parasite metabolism and the identification of targets for more efficient intervention.

  4. Bioenergetics-based modeling of Plasmodium falciparum metabolism reveals its essential genes, nutritional requirements, and thermodynamic bottlenecks.

    Directory of Open Access Journals (Sweden)

    Anush Chiappino-Pepe

    2017-03-01

    Full Text Available Novel antimalarial therapies are urgently needed for the fight against drug-resistant parasites. The metabolism of malaria parasites in infected cells is an attractive source of drug targets but is rather complex. Computational methods can handle this complexity and allow integrative analyses of cell metabolism. In this study, we present a genome-scale metabolic model (iPfa of the deadliest malaria parasite, Plasmodium falciparum, and its thermodynamics-based flux analysis (TFA. Using previous absolute concentration data of the intraerythrocytic parasite, we applied TFA to iPfa and pre