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Sample records for satb1 targets chromatin

  1. p63 regulates Satb1 to control tissue-specific chromatin remodeling during development of the epidermis

    Science.gov (United States)

    Fessing, Michael Y.; Mardaryev, Andrei N.; Gdula, Michal R.; Sharov, Andrey A.; Sharova, Tatyana Y.; Rapisarda, Valentina; Gordon, Konstantin B.; Smorodchenko, Anna D.; Poterlowicz, Krzysztof; Ferone, Giustina; Kohwi, Yoshinori; Missero, Caterina

    2011-01-01

    During development, multipotent progenitor cells establish tissue-specific programs of gene expression. In this paper, we show that p63 transcription factor, a master regulator of epidermal morphogenesis, executes its function in part by directly regulating expression of the genome organizer Satb1 in progenitor cells. p63 binds to a proximal regulatory region of the Satb1 gene, and p63 ablation results in marked reduction in the Satb1 expression levels in the epidermis. Satb1−/− mice show impaired epidermal morphology. In Satb1-null epidermis, chromatin architecture of the epidermal differentiation complex locus containing genes associated with epidermal differentiation is altered primarily at its central domain, where Satb1 binding was confirmed by chromatin immunoprecipitation–on-chip analysis. Furthermore, genes within this domain fail to be properly activated upon terminal differentiation. Satb1 expression in p63+/− skin explants treated with p63 small interfering ribonucleic acid partially restored the epidermal phenotype of p63-deficient mice. These data provide a novel mechanism by which Satb1, a direct downstream target of p63, contributes in epidermal morphogenesis via establishing tissue-specific chromatin organization and gene expression in epidermal progenitor cells. PMID:21930775

  2. N-terminal PDZ-like domain of chromatin organizer SATB1 ...

    Indian Academy of Sciences (India)

    and high affinity binding of MARs (Dickinson et al. 1997;. Purbey et al. 2008). Cleavage of SATB1 during T cell apoptosis at position 254 by caspase 6 causes detachment of. SATB1 from the chromatin (Galande et al. 2001). This observation resulted in the identification of an oligomerization domain in the N-terminal of ...

  3. N-terminal PDZ-like domain of chromatin organizer SATB1 ...

    Indian Academy of Sciences (India)

    2011-07-08

    Jul 8, 2011 ... Home; Journals; Journal of Biosciences; Volume 36; Issue 3. N-terminal PDZ-like domain of chromatin ... In the present study, we set out to address whether the PDZ-domain-mediated interactions of SATB1 are critical for its in vivo function as a global repressor. We reasoned that since the N-terminal ...

  4. Global regulator SATB1 recruits beta-catenin and regulates T(H2 differentiation in Wnt-dependent manner.

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    Dimple Notani

    2010-01-01

    Full Text Available In vertebrates, the conserved Wnt signalling cascade promotes the stabilization and nuclear accumulation of beta-catenin, which then associates with the lymphoid enhancer factor/T cell factor proteins (LEF/TCFs to activate target genes. Wnt/beta -catenin signalling is essential for T cell development and differentiation. Here we show that special AT-rich binding protein 1 (SATB1, the T lineage-enriched chromatin organizer and global regulator, interacts with beta-catenin and recruits it to SATB1's genomic binding sites. Gene expression profiling revealed that the genes repressed by SATB1 are upregulated upon Wnt signalling. Competition between SATB1 and TCF affects the transcription of TCF-regulated genes upon beta-catenin signalling. GATA-3 is a T helper type 2 (T(H2 specific transcription factor that regulates production of T(H2 cytokines and functions as T(H2 lineage determinant. SATB1 positively regulated GATA-3 and siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4(+ T cells, suggesting that SATB1 influences T(H2 lineage commitment by reprogramming gene expression. In the presence of Dickkopf 1 (Dkk1, an inhibitor of Wnt signalling, GATA-3 is downregulated and the expression of signature T(H2 cytokines such as IL-4, IL-10, and IL-13 is reduced, indicating that Wnt signalling is essential for T(H2 differentiation. Knockdown of beta-catenin also produced similar results, confirming the role of Wnt/beta-catenin signalling in T(H2 differentiation. Furthermore, chromatin immunoprecipitation analysis revealed that SATB1 recruits beta-catenin and p300 acetyltransferase on GATA-3 promoter in differentiating T(H2 cells in a Wnt-dependent manner. SATB1 coordinates T(H2 lineage commitment by reprogramming gene expression. The SATB1:beta-catenin complex activates a number of SATB1 regulated genes, and hence this study has potential to find novel Wnt responsive genes. These results demonstrate that SATB1

  5. SIRT1 deacetylates SATB1 to facilitate MAR HS2-MAR ε interaction and promote ε-globin expression.

    Science.gov (United States)

    Xue, Zheng; Lv, Xiang; Song, Wei; Wang, Xing; Zhao, Guang-Nian; Wang, Wen-Tian; Xiong, Jian; Mao, Bei-Bei; Yu, Wei; Yang, Ben; Wu, Jie; Zhou, Li-Quan; Hao, De-Long; Dong, Wen-Ji; Liu, De-Pei; Liang, Chih-Chuan

    2012-06-01

    The higher order chromatin structure has recently been revealed as a critical new layer of gene transcriptional control. Changes in higher order chromatin structures were shown to correlate with the availability of transcriptional factors and/or MAR (matrix attachment region) binding proteins, which tether genomic DNA to the nuclear matrix. How posttranslational modification to these protein organizers may affect higher order chromatin structure still pending experimental investigation. The type III histone deacetylase silent mating type information regulator 2, S. cerevisiae, homolog 1 (SIRT1) participates in many physiological processes through targeting both histone and transcriptional factors. We show that MAR binding protein SATB1, which mediates chromatin looping in cytokine, MHC-I and β-globin gene loci, as a new type of SIRT1 substrate. SIRT1 expression increased accompanying erythroid differentiation and the strengthening of β-globin cluster higher order chromatin structure, while knockdown of SIRT1 in erythroid k562 cells weakened the long-range interaction between two SATB1 binding sites in the β-globin locus, MAR(HS2) and MAR(ε). We also show that SIRT1 activity significantly affects ε-globin gene expression in a SATB1-dependent manner and that knockdown of SIRT1 largely blocks ε-globin gene activation during erythroid differentiation. Our work proposes that SIRT1 orchestrates changes in higher order chromatin structure during erythropoiesis, and reveals the dynamic higher order chromatin structure regulation at posttranslational modification level.

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

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    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. SATB1 tethers multiple gene loci to reprogram expression profiledriving breast cancer metastasis

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    Han, Hye-Jung; Kohwi, Yoshinori; Kohwi-Shigematsu, Terumi

    2006-07-13

    Global changes in gene expression occur during tumor progression, as indicated by expression profiling of metastatic tumors. How this occurs is poorly understood. SATB1 functions as a genome organizer by folding chromatin via tethering multiple genomic loci and recruiting chromatin remodeling enzymes to regulate chromatin structure and expression of a large number of genes. Here we show that SATB1 is expressed at high levels in aggressive breast cancer cells, and is undetectable in non-malignant breast epithelial cells. Importantly, RNAi-mediated removal of SATB1 from highly-aggressive MDA-MB-231 cells altered the expression levels of over 1200 genes, restored breast-like acinar polarity in three-dimensional cultures, and prevented the metastastic phenotype in vivo. Conversely, overexpression of SATB1 in the less-aggressive breast cancer cell line Hs578T altered the gene expression profile and increased metastasis dramatically in vivo. Thus, SATB1 is a global regulator of gene expression in breast cancer cells, directly regulating crucial metastasis-associated genes, including ERRB2 (HER2/NEU), TGF-{beta}1, matrix metalloproteinase 3, and metastasin. The identification of SATB1 as a protein that re-programs chromatin organization and transcription profiles to promote breast cancer metastasis suggests a new model for metastasis and may provide means of therapeutic intervention.

  8. Effect of SATB1 silencing on the proliferation, invasion and apoptosis of TE-1 esophageal cancer cells.

    Science.gov (United States)

    Huang, Bo; Xiong, Fei; Wang, Siwang; Lang, Xianping; Wang, Xiaodong; Zhou, Hongli

    2017-05-01

    The aim of the present study was to investigate the effect of special AT-rich sequence-binding protein-1 (SATB1)-targeted small interfering RNA (siRNA) on the proliferation, invasion and apoptosis of TE-1 human esophageal cancer cells. SATB1 has been correlated with the metastasis and poor prognosis of colon and breast cancer, but the role of SATB1 in esophageal cancer remains unknown. Therefore, the present study constructed and transfected SATB1-siRNA into TE-1 cells in order to knockdown the expression of the SATB1 gene. Western blot analysis, a cell counting kit, transwell chamber assays and flow cytometry were used to assess the effect of SATB1-siRNA on the proliferation, invasion and apoptosis of cells. The results demonstrated that the expression of the SATB1 gene was efficiently knocked down by SATB1-siRNA, and that SATB1-siRNA inhibited the proliferation, invasion and apoptosis of TE-1 cells. Therefore, it was concluded that the SATB1 gene is important in the pathogenesis of human esophageal cancer, and may present a novel therapeutic target for esophageal cancer.

  9. Multiple effects of the special AT-rich binding protein 1 (SATB1) in colon carcinoma.

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    Frömberg, Anja; Rabe, Michael; Aigner, Achim

    2014-12-01

    SATB1 (special AT-rich binding protein 1) is a global chromatin organizer regulating the expression of a large number of genes. Overexpression has been found in various solid tumors and positively correlated with prognostic and clinicopathological properties. In colorectal cancer (CRC), SATB1 overexpression and its correlation with poor differentiation, invasive depth, TNM (tumor, nodes, metastases) stage and prognosis have been demonstrated. However, more detailed studies on the SATB1 functions in CRC are warranted. In this article, we comprehensively analyze the cellular and molecular role of SATB1 in CRC cell lines with different SATB1 expression levels by using RNAi-mediated knockdown. Using siRNAs with different knockdown efficacies, we demonstrate antiproliferative, cell cycle-inhibitory and proapoptotic effects of SATB1 knockdown in a SATB1 gene dose-dependent manner. Tumor growth inhibition is confirmed in vivo in a subcutaneous tumor xenograft mouse model using stable knockdown cells. The in-depth analysis of cellular effects reveals increased activities of caspases-3, -7, -8, -9 and other mediators of apoptotic pathways. Similarly, the analysis of E- and N-cadherin, slug, twist, β-catenin and MMP7 indicates SATB1 effects on epithelial-mesenchymal transition (EMT) and matrix breakdown. Our results also establish SATB1 effects on receptor tyrosine kinases and (proto-)oncogenes such as HER receptors and Pim-1. Taken together, this suggests a more complex molecular interplay between tumor-promoting and possible inhibitory effects in CRC by affecting multiple pathways and molecules involved in proliferation, cell cycle, EMT, invasion and cell survival. © 2014 UICC.

  10. Fluorosed mouse ameloblasts have increased SATB1 retention and Gαq activity.

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    Yan Zhang

    Full Text Available Dental fluorosis is characterized by subsurface hypomineralization and increased porosity of enamel, associated with a delay in the removal of enamel matrix proteins. To investigate the effects of fluoride on ameloblasts, A/J mice were given 50 ppm sodium fluoride in drinking water for four weeks, resulting serum fluoride levels of 4.5 µM, a four-fold increase over control mice with no fluoride added to drinking water. MicroCT analyses showed delayed and incomplete mineralization of fluorosed incisor enamel as compared to control enamel. A microarray analysis of secretory and maturation stage ameloblasts microdissected from control and fluorosed mouse incisors showed that genes clustered with Mmp20 appeared to be less downregulated in maturation stage ameloblasts of fluorosed incisors as compared to control maturation ameloblasts. One of these Mmp20 co-regulated genes was the global chromatin organizer, special AT-rich sequence-binding protein-1 (SATB1. Immunohistochemical analysis showed increased SATB1 protein present in fluorosed ameloblasts compared to controls. In vitro, exposure of human ameloblast-lineage cells to micromolar levels of both NaF and AlF3 led to a significantly increase in SATB1 protein content, but not levels of Satb1 mRNA, suggesting a fluoride-induced mechanism protecting SABT1 from degradation. Consistent with this possibility, we used immunohistochemistry and Western blot to show that fluoride exposed ameloblasts had increased phosphorylated PKCα both in vivo and in vitro. This kinase is known to phosphorylate SATB1, and phosphorylation is known to protect SATB1 from degradation by caspase-6. In addition, production of cellular diacylglycerol (DAG was significantly increased in fluorosed ameloblasts, suggesting that the increased phosphorylation of SATB1 may be related to an effect of fluoride to enhance Gαq activity of secretory ameloblasts.

  11. Fluorosed mouse ameloblasts have increased SATB1 retention and Gαq activity.

    Science.gov (United States)

    Zhang, Yan; Kim, Ji-Yeon; Horst, Orapin; Nakano, Yukiko; Zhu, Li; Radlanski, Ralf J; Ho, Sunita; Den Besten, Pamela K

    2014-01-01

    Dental fluorosis is characterized by subsurface hypomineralization and increased porosity of enamel, associated with a delay in the removal of enamel matrix proteins. To investigate the effects of fluoride on ameloblasts, A/J mice were given 50 ppm sodium fluoride in drinking water for four weeks, resulting serum fluoride levels of 4.5 µM, a four-fold increase over control mice with no fluoride added to drinking water. MicroCT analyses showed delayed and incomplete mineralization of fluorosed incisor enamel as compared to control enamel. A microarray analysis of secretory and maturation stage ameloblasts microdissected from control and fluorosed mouse incisors showed that genes clustered with Mmp20 appeared to be less downregulated in maturation stage ameloblasts of fluorosed incisors as compared to control maturation ameloblasts. One of these Mmp20 co-regulated genes was the global chromatin organizer, special AT-rich sequence-binding protein-1 (SATB1). Immunohistochemical analysis showed increased SATB1 protein present in fluorosed ameloblasts compared to controls. In vitro, exposure of human ameloblast-lineage cells to micromolar levels of both NaF and AlF3 led to a significantly increase in SATB1 protein content, but not levels of Satb1 mRNA, suggesting a fluoride-induced mechanism protecting SABT1 from degradation. Consistent with this possibility, we used immunohistochemistry and Western blot to show that fluoride exposed ameloblasts had increased phosphorylated PKCα both in vivo and in vitro. This kinase is known to phosphorylate SATB1, and phosphorylation is known to protect SATB1 from degradation by caspase-6. In addition, production of cellular diacylglycerol (DAG) was significantly increased in fluorosed ameloblasts, suggesting that the increased phosphorylation of SATB1 may be related to an effect of fluoride to enhance Gαq activity of secretory ameloblasts.

  12. MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation

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    Lena, A.M.; Mancini, M.; Rivetti di Val Cervo, P. [University of ' Tor Vergata' , Department of Experimental Medicine and Biochemical Sciences, Via Montpellier 1, Rome 00133 (Italy); Istituto Dermopatico dell' Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico (IDI-IRCCS), Laboratory of Biochemistry c/o Department of Experimental Medicine and Biochemical Sciences, University of Rome ' Tor Vergata' , Rome 00133 (Italy); Saintigny, G.; Mahe, C. [CHANEL Parfums Beaute, 135 av. Charles de Gaulle, F 92521, Neuilly/Seine (France); Melino, G., E-mail: gerry.melino@uniroma2.it [University of ' Tor Vergata' , Department of Experimental Medicine and Biochemical Sciences, Via Montpellier 1, Rome 00133 (Italy); Istituto Dermopatico dell' Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico (IDI-IRCCS), Laboratory of Biochemistry c/o Department of Experimental Medicine and Biochemical Sciences, University of Rome ' Tor Vergata' , Rome 00133 (Italy); Association Cell Death and Differentiation c/o Department of Experimental Medicine and Biochemical Sciences, University of Rome ' Tor Vergata' , Rome 00133 (Italy); and others

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer miR-191 expression is upregulated in senescencent human epidermal keratinocytes. Black-Right-Pointing-Pointer miR-191 overexpression is sufficient per se to induce senescence in keratinocytes. Black-Right-Pointing-Pointer SATB1 and CDK6 are downregulated in senescence and are direct miR-191 targets. Black-Right-Pointing-Pointer SATB1 and CDK6 silencing by siRNA triggers senescence in HEKn cells. -- Abstract: Keratinocyte replicative senescence has an important role in time-dependent changes of the epidermis, a tissue with high turnover. Senescence encompasses growth arrest during which cells remain metabolically active but acquire a typical enlarged, vacuolar and flattened morphology. It is also accompanied by the expression of endogenous senescence-associated-{beta}-galactosidase and specific gene expression profiles. MicroRNAs levels have been shown to be modulated during keratinocytes senescence, playing key roles in inhibiting proliferation and in the acquisition of senescent markers. Here, we identify miR-191 as an anti-proliferative and replicative senescence-associated miRNA in primary human keratinocytes. Its overexpression is sufficient per se to induce senescence, as evaluated by induction of several senescence-associated markers. We show that SATB1 and CDK6 3 Prime UTRs are two miR-191 direct targets involved in this pathway. Cdk6 and Satb1 protein levels decrease during keratinocytes replicative senescence and their silencing by siRNA is able to induce a G1 block in cell cycle, accompanied by an increase in senescence-associated markers.

  13. The mRNA expression of SATB1 and SATB2 in human breast cancer

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    Mansel Robert

    2009-07-01

    Full Text Available Abstract Background SATB1 is a nuclear protein that has been recently reported to be a 'genome organizer' which delineates specific epigenetic modifications at target gene loci, directly up-regulating metastasis-associated genes while down-regulating tumor-suppressor genes. In this study, the level of mRNA expression of SATB1 and SATB2 were assessed in normal and malignant breast tissue in a cohort of women with breast cancer and correlated to conventional clinico-pathological parameters. Materials and methods Breast cancer tissues (n = 115 and normal background tissues (n = 31 were collected immediately after excision during surgery. Following RNA extraction, reverse transcription was carried out and transcript levels were determined using real-time quantitative PCR and normalized against β-actin expression. Transcript levels within the breast cancer specimens were compared to the normal background tissues and analyzed against TNM stage, nodal involvement, tumour grade and clinical outcome over a 10 year follow-up period. Results The levels of SATB1 were higher in malignant compared with normal breast tissue (p = 0.0167. SATB1 expression increased with increasing TNM stage (TNM1 vs. TNM2 p = 0.0264, increasing tumour grade (grade1 vs. grade 3 p = 0.017; grade 2 vs. grade 3 p = 0.0437; grade 1 vs. grade 2&3 p = 0.021 and Nottingham Prognostic Index (NPI (NPI-1 vs. NPI-3 p = 0.0614; NPI-2 vs. NPI-3 p = 0.0495. Transcript levels were associated with oestrogen receptor (ER positivity (ER(- vs. ER(+ p = 0.046. SABT1 expression was also significantly correlated with downstream regulated genes IL-4 and MAF-1 (Pearson's correlation coefficient r = 0.21 and r = 0.162 and SATB2 (r = 0.506. After a median follow up of 10 years, there was a trend for higher SATB1 expression to be associated with shorter overall survival (OS. Higher levels of SATB2 were also found in malignant compared to background tissue (p = 0.049. SATB2 expression increased with

  14. Molecular correlates and prognostic significance of SATB1 expression in colorectal cancer

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    Nodin Björn

    2012-08-01

    Full Text Available Abstract Background Special AT-rich sequence-binding protein 1 (SATB1 is a global gene regulator that has been reported to confer malignant behavior and associate with poor prognosis in several cancer forms. SATB1 expression has been demonstrated to correlate with unfavourable tumour characteristics in rectal cancer, but its association with clinical outcome in colorectal cancer (CRC remains unclear. In this study, we examined the prognostic impact of SATB1 expression in CRC, and its association with important molecular characteristics; i.e. beta-catenin overexpression, microsatellite instability (MSI screening status, and SATB2 expression. Methods Immunohistochemical expression of SATB1 and beta-catenin was assessed in tissue microarrays with tumours from 529 incident CRC cases in the prospective population-based Malmö Diet and Cancer Study, previously analysed for SATB2 expression and MSI screening status. Spearmans Rho and Chi-Square tests were used to explore correlations between SATB1 expression, clinicopathological and investigative parameters. Kaplan Meier analysis and Cox proportional hazards modelling were used to explore the impact of SATB1 expression on cancer specific survival (CSS and overall survival (OS. Results SATB1 was expressed in 222 (42% CRC cases and negative, or sparsely expressed, in adjacent colorectal mucosa (n = 16. SATB1 expression was significantly associated with microsatellite stable tumours (p interaction = 0.011 for CSS and HR = 2.31; 95% CI 1.32-4.04; pinteraction = 0.015 for OS, remaining significant in multivariable analysis. Conclusions The results of this study demonstrate that SATB1 expression in CRC is significantly associated with beta-catenin overexpression, microsatellite stability and SATB2 expression. Furthermore, SATB1 expression is a factor of poor prognosis in SATB2 negative tumours. Altogether, these data indicate an important role for SATB1 in colorectal carcinogenesis and

  15. SATB1 is Correlated with Progression and Metastasis of Breast Cancers: A Meta-Analysis

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    Zhongya Pan

    2016-05-01

    Full Text Available Background/Aims: Several researches have evaluated the significance of SATB1 (Special AT-rich sequence binding protein 1 expression in breast cancers (BCs, but the results have been disputed, especially in the aspects of clinicopathological features and prognosis. Therefore, our study aimed to use a meta-analysis to summarize the clinical and prognostic relevance of SATB1 gene expression in BCs. Methods: A literature search of PubMed, EMBASE, Cochrane library, Chinese Wanfang and CNKI was performed to identify eligible studies. Ten studies total, comprising 5,185 patients (1,699 SATB1-positive and 3,486 SATB1-negative, were enrolled in our study, which was performed using Revman5.3 Software and Stata11.0 Software. Results: This meta-analysis showed that the expression of SATB1 was significantly higher in breast cancer than in normal tissues (OR = 12.28; 95%CI = 6.01-25.09, and was statistically related to several clinicopathological parameters, including lymph node metastasis (OR = 1.55, 95%CI = 1.01-2.39 and Tumor Node Metastasis(TNM stage (OR = 0.35, 95%CI = 0.22-0.56. However, the level of SATB1 was not statistically associated with the age (OR = 1.13, 95%CI = 0.87-1.46, tumour size (OR = 0.72, 95%CI = 0.44-1.19, estrogen receptor (OR = 0.78, 95%CI = 0.55-1.09, progesterone receptor (OR = 0.64, 95%CI = 0.32-1.29, HER2 status (OR=1.98, 95%CI = 0.74-5.30, and histological type (OR = 0.49, 95%CI = 0.22-1.11. Conclusion: High expression of SATB1 was significantly correlated with tumourigenesis and metastasis of BCs, indicating poor prognosis for patients. SATB1 could serve as a potential marker for detection and prognosis evaluation of breast cancers.

  16. Chromatin proteins and modifications as drug targets

    DEFF Research Database (Denmark)

    Helin, Kristian; Dhanak, Dashyant

    2013-01-01

    is a frequent event in disease, and the first epigenetic-based therapies for cancer treatment have been approved. A generation of new classes of potent and specific inhibitors for several chromatin-associated proteins have shown promise in preclinical trials. Although the biology of epigenetic regulation......A plethora of groundbreaking studies have demonstrated the importance of chromatin-associated proteins and post-translational modifications of histones, proteins and DNA (so-called epigenetic modifications) for transcriptional control and normal development. Disruption of epigenetic control...

  17. Baicalein suppresses metastasis of breast cancer cells by inhibiting EMT via downregulation of SATB1 and Wnt/β-catenin pathway.

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    Ma, Xingcong; Yan, Wanjun; Dai, Zhijun; Gao, Xiaoyan; Ma, Yinan; Xu, Quntao; Jiang, Jiantao; Zhang, Shuqun

    2016-01-01

    The flavonoid baicalein, a historically used Chinese herbal medicine, shows a wide range of biological and pharmaceutical effects, among which its potent antitumor activity has raised great interest in recent years. However, the molecular mechanism involved in the antimetastatic effect of baicalein remains poorly understood. This study aimed to verify the inhibitory effects of baicalein on metastasis of MDA-MB-231 human breast cancer cells both in vitro and in vivo, as well as to investigate the related mechanisms. MTT assay was used to examine the inhibition of baicalein on proliferation of MDA-MB-231 cells. Wound healing assay and the in vitro invasion assay was carried out to investigate the effects of baicalein on migration and invasion of MDA-MB-231 cells, respectively. In order to explore the effects of baicalein on tumor metastasis in vivo, xenograft nude mouse model of MDA-MB-231 cells was established. Animals were randomly divided into four groups (control, therapy group, and low-dose and high-dose prevention group, n=6), and treated with baicalein as designed. Following sacrifice, their lungs and livers were collected to examine the presence of metastases. qRT-PCR and Western blot were performed to study the effects of baicalein on expression of SATB1, EMT-related molecules, and Wnt/β-catenin signaling components of MDA-MB-231 cells as well as the metastatic tissue. Effects of baicalein on the expression of target proteins in vivo were also analyzed by immunohistochemistry. Our results indicated that baicalein suppressed proliferation, migration, and invasion of MDA-MB-231 cells in a time- and dose-dependent manner. Based on assays carried out in xenograft nude mouse model, we found that baicalein inhibited tumor metastasis in vivo. Furthermore, baicalein significantly decreased the expression of SATB1 in MDA-MB-231 cells. It suppressed the expression of vimentin and SNAIL while enhancing the expression of E-cadherin. Baicalein also downregulated the

  18. Analysis of Myc-induced histone modifications on target chromatin.

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    Francesca Martinato

    Full Text Available The c-myc proto-oncogene is induced by mitogens and is a central regulator of cell growth and differentiation. The c-myc product, Myc, is a transcription factor that binds a multitude of genomic sites, estimated to be over 10-15% of all promoter regions. Target promoters generally pre-exist in an active or poised chromatin state that is further modified by Myc, contributing to fine transcriptional regulation (activation or repression of the afferent gene. Among other mechanisms, Myc recruits histone acetyl-transferases to target chromatin and locally promotes hyper-acetylation of multiple lysines on histones H3 and H4, although the identity and combination of the modified lysines is unknown. Whether Myc dynamically regulates other histone modifications (or marks at its binding sites also remains to be addressed. Here, we used quantitative chromatin immunoprecipitation (qChIP to profile a total of 24 lysine-acetylation and -methylation marks modulated by Myc at target promoters in a human B-cell line with a regulatable c-myc transgene. Myc binding promoted acetylation of multiple lysines, primarily of H3K9, H3K14, H3K18, H4K5 and H4K12, but significantly also of H4K8, H4K91 and H2AK5. Dimethylation of H3K79 was also selectively induced at target promoters. A majority of target promoters showed co-induction of multiple marks - in various combinations - correlating with recruitment of the two HATs tested (Tip60 and HBO1, incorporation of the histone variant H2A.Z and transcriptional activation. Based on this and previous findings, we surmise that Myc recruits the Tip60/p400 complex to achieve a coordinated histone acetylation/exchange reaction at activated promoters. Our data are also consistent with the additive and redundant role of multiple acetylation events in transcriptional activation.

  19. Role of CBP and SATB-1 in aging, dietary restriction, and insulin-like signaling.

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    Minhua Zhang

    2009-11-01

    Full Text Available How dietary restriction (DR increases lifespan and decreases disease burden are questions of major interest in biomedical research. Here we report that hypothalamic expression of CREB-binding protein (CBP and CBP-binding partner Special AT-rich sequence binding protein 1 (SATB-1 is highly correlated with lifespan across five strains of mice, and expression of these genes decreases with age and diabetes in mice. Furthermore, in Caenorhabditis elegans, cbp-1 is induced by bacterial dilution DR (bDR and the daf-2 mutation, and cbp-1 RNAi specifically in adults completely blocks lifespan extension by three distinct protocols of DR, partially blocks lifespan extension by the daf-2 mutation but not of cold, and blocks delay of other age-related pathologies by bDR. Inhibiting the C. elegans ortholog of SATB-1 and CBP-binding partners daf-16 and hsf-1 also attenuates lifespan extension by bDR, but not other protocols of DR. In a transgenic Abeta42 model of Alzheimer's disease, cbp-1 RNAi prevents protective effects of bDR and accelerates Abeta42-related pathology. Furthermore, consistent with the function of CBP as a histone acetyltransferase, drugs that enhance histone acetylation increase lifespan and reduce Abeta42-related pathology, protective effects completely blocked by cbp-1 RNAi. Other factors implicated in lifespan extension are also CBP-binding partners, suggesting that CBP constitutes a common factor in the modulation of lifespan and disease burden by DR and the insulin/IGF1 signaling pathway.

  20. Baicalein suppresses metastasis of breast cancer cells by inhibiting EMT via downregulation of SATB1 and Wnt/β-catenin pathway

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    Ma X

    2016-04-01

    Full Text Available Xingcong Ma,1 Wanjun Yan,1 Zhijun Dai,1 Xiaoyan Gao,1 Yinan Ma,1 Quntao Xu,2 Jiantao Jiang,3 Shuqun Zhang11Department of Oncology, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, People's Republic of China, 2Department of Oncology, Institute of Health, China North Industries Group Corporation, Xi'an, Shaanxi, People's Republic of China, 3Department of Thoracic Surgery, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, People's Republic of China Background: The flavonoid baicalein, a historically used Chinese herbal medicine, shows a wide range of biological and pharmaceutical effects, among which its potent antitumor activity has raised great interest in recent years. However, the molecular mechanism involved in the antimetastatic effect of baicalein remains poorly understood. This study aimed to verify the inhibitory effects of baicalein on metastasis of MDA-MB-231 human breast cancer cells both in vitro and in vivo, as well as to investigate the related mechanisms.Methods: MTT assay was used to examine the inhibition of baicalein on proliferation of MDA-MB-231 cells. Wound healing assay and the in vitro invasion assay was carried out to investigate the effects of baicalein on migration and invasion of MDA-MB-231 cells, respectively. In order to explore the effects of baicalein on tumor metastasis in vivo, xenograft nude mouse model of MDA-MB-231 cells was established. Animals were randomly divided into four groups (control, therapy group, and low-dose and high-dose prevention group, n=6, and treated with baicalein as designed. Following sacrifice, their lungs and livers were collected to examine the presence of metastases. qRT-PCR and Western blot were performed to study the effects of baicalein on expression of SATB1, EMT-related molecules, and Wnt/β-catenin signaling components of MDA-MB-231 cells as well as the metastatic tissue. Effects of baicalein on the expression of target

  1. Mapping Long Noncoding RNA Chromatin Occupancy Using Capture Hybridization Analysis of RNA Targets (CHART).

    Science.gov (United States)

    Vance, Keith W

    2017-01-01

    Capture Hybridization Analysis of RNA Targets (CHART) has recently been developed to map the genome-wide binding profile of chromatin-associated RNAs. This protocol uses a small number of 22-28 nucleotide biotinylated antisense oligonucleotides, complementary to regions of the target RNA that are accessible for hybridization, to purify RNAs from a cross-linked chromatin extract. RNA-chromatin complexes are next immobilized on beads, washed, and specifically eluted using RNase H. Associated genomic DNA is then sequenced using high-throughput sequencing technologies and mapped to the genome to identify RNA-chromatin associations on a large scale. CHART-based strategies can be applied to determine the nature and extent of long noncoding RNA (long ncRNA) association with chromatin genome-wide and identify direct long ncRNA transcriptional targets.

  2. Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put

    Science.gov (United States)

    Simon, Jeffrey A.; Kingston, Robert E.

    2013-01-01

    Summary Polycomb repressive complexes are conserved chromatin regulators with key roles in multicellular development, stem cell biology, and cancer. New findings advance molecular understanding of how they target to sites of action, interact with and alter local chromatin to silence genes, and maintain silencing in successive generations of proliferating cells. Chromatin modification by Polycomb proteins provides an essential strategy for gene silencing in higher eukaryotes. Polycomb repressive complexes (PRCs) silence many key developmental regulators and are centrally integrated in the transcriptional circuitry of embryonic and adult stem cells. PRC2 trimethylates histone H3 on lysine-27 (H3-K27me3) and PRC1-type complexes ubiquitylate histone H2A and compact polynucleosomes. How PRCs and these signature activities are deployed to select and silence genomic targets is the subject of intense current investigation. We review recent advances on targeting, modulation, and functions of PRC1 and PRC2, and we consider progress on defining transcriptional steps impacted in Polycomb silencing. Key recent findings demonstrate PRC1 targeting independent of H3-K27me3 and emphasize nonenzymatic PRC1-mediated compaction. We also evaluate expanding connections between Polycomb machinery and non-coding RNAs. Exciting new studies supply the first systematic analyses of what happens to Polycomb complexes, and associated histone modifications, during the wholesale chromatin reorganizations that accompany DNA replication and mitosis. The stage is now set to reveal fundamental epigenetic mechanisms that determine how Polycomb target genes are silenced and how Polycomb silence is preserved through cell cycle progression. PMID:23473600

  3. Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context.

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    Artyom A Alekseyenko

    Full Text Available The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at "entry sites" that contain a consensus sequence motif ("MSL recognition element" or MRE. However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex and female Kc cells (which lack the complex, we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.

  4. Osa-containing Brahma chromatin remodeling complexes are required for the repression of Wingless target genes

    Science.gov (United States)

    Collins, Russell T.; Treisman, Jessica E.

    2000-01-01

    The Wingless signaling pathway directs many developmental processes in Drosophila by regulating the expression of specific downstream target genes. We report here that the product of the trithorax group gene osa is required to repress such genes in the absence of the Wingless signal. The Wingless-regulated genes nubbin, Distal-less, and decapentaplegic and a minimal enhancer from the Ultrabithorax gene are misexpressed in osa mutants and repressed by ectopic Osa. Osa-mediated repression occurs downstream of the up-regulation of Armadillo but is sensitive both to the relative levels of activating Armadillo/Pangolin and repressing Groucho/Pangolin complexes present and to the responsiveness of the promoter to Wingless. Osa functions as a component of the Brahma chromatin-remodeling complex; other components of this complex are likewise required to repress Wingless target genes. These results suggest that altering the conformation of chromatin is an important mechanism by which Wingless signaling activates gene expression. PMID:11124806

  5. DNA breaks and chromatin structural changes enhance the transcription of autoimmune regulator target genes.

    Science.gov (United States)

    Guha, Mithu; Saare, Mario; Maslovskaja, Julia; Kisand, Kai; Liiv, Ingrid; Haljasorg, Uku; Tasa, Tõnis; Metspalu, Andres; Milani, Lili; Peterson, Pärt

    2017-04-21

    The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Osa associates with the Brahma chromatin remodeling complex and promotes the activation of some target genes.

    Science.gov (United States)

    Collins, R T; Furukawa, T; Tanese, N; Treisman, J E

    1999-01-01

    The yeast SWI/SNF complex and its Drosophila and mammalian homologs are thought to control gene expression by altering chromatin structure, but the mechanism and specificity of this process are not fully understood. The Drosophila osa gene, like yeast SWI1, encodes an AT-rich interaction (ARID) domain protein. We present genetic and biochemical evidence that Osa is a component of the Brahma complex, the Drosophila homolog of SWI/SNF. The ARID domain of Osa binds DNA without sequence specificity in vitro, but it is sufficient to direct transcriptional regulatory domains to specific target genes in vivo. Endogenous Osa appears to promote the activation of some of these genes. We show evidence that some Brahma-containing complexes do not contain Osa and that Osa is not required to localize Brahma to chromatin. These data suggest that Osa modulates the function of the Brahma complex. PMID:10601025

  7. Targeting chromatin defects in selected solid tumors based on oncogene addiction, synthetic lethality and epigenetic antagonism.

    Science.gov (United States)

    Morel, D; Almouzni, G; Soria, J-C; Postel-Vinay, S

    2017-02-01

    Although the role of epigenetic abnormalities has been studied for several years in cancer genesis and development, epigenetic-targeting drugs have historically failed to demonstrate efficacy in solid malignancies. However, successful targeting of chromatin remodeling deficiencies, histone writers and histone reader alterations has been achieved very recently using biomarker-driven and mechanism-based approaches. Epigenetic targeting is now one of the most active areas in drug development and could represent novel therapeutic opportunity for up to 25% of all solid tumors. We reviewed preclinical and clinical studies that described epigenetic oncogenic addictions, synthetic lethal relationships or epigenetic antagonisms in chromatin regulators. Experimental approaches, their clinical relevance and applicability, as well as corresponding on-going studies are described. The most successful approaches that have been clinically validated so far include the targeting of the BRD4-NUT fusion transcript in NUT-midline carcinoma by BET (Bromodomain Extra-Terminal) inhibitors, and the use of EZH2 (Enhancer of Zest Homolog 2) inhibitors in SMARCB1-deficient malignant rhabdoid tumors and SMARCA4-deficient ovarian small cell carcinomas. Clinical validation is still required for other synthetic lethal relationships or epigenetic antagonisms, including those described between EZH2 inhibitors and deficiencies in components of the Polycomb or SWI/SNF chromatin-remodeling complexes (including BAP1, ARID1A and PBRM1 subunits), as well as between the CREBBP and EP300 histone acetylases. Further, interplays between epigenetic modifiers and non-epigenetic cellular processes might be therapeutically exploited, and combinatorial strategies could be envisioned to overcome resistance or to sensitize cells to already approved drugs. Epigenetic-targeting drugs have historically failed proving efficacy in solid malignancies when used broadly, but novel mechanism-based approaches in molecularly

  8. Inactivation of a Human Kinetochore by Specific Targeting of Chromatin Modifiers

    Science.gov (United States)

    Nakano, Megumi; Cardinale, Stefano; Noskov, Vladimir N.; Gassmann, Reto; Vagnarelli, Paola; Kandels-Lewis, Stefanie; Larionov, Vladimir; Earnshaw, William C.; Masumoto, Hiroshi

    2008-01-01

    Summary We have used a human artificial chromosome (HAC) to manipulate the epigenetic state of chromatin within an active kinetochore. The HAC has a dimeric α-satellite repeat containing one natural monomer with a CENP-B binding site, and one completely artificial synthetic monomer with the CENP-B box replaced by a tetracycline operator (tetO). This HAC exhibits normal kinetochore protein composition and mitotic stability. Targeting of several tet-repressor (tetR) fusions into the centromere had no effect on kinetochore function. However, altering the chromatin state to a more open configuration with the tTA transcriptional activator or to a more closed state with the tTS transcription silencer caused missegregation and loss of the HAC. tTS binding caused the loss of CENP-A, CENP-B, CENP-C, and H3K4me2 from the centromere accompanied by an accumulation of histone H3K9me3. Our results reveal that a dynamic balance between centromeric chromatin and heterochromatin is essential for vertebrate kinetochore activity. PMID:18410728

  9. Pharmacologic Targeting of Chromatin Modulators As Therapeutics of Acute Myeloid Leukemia

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    Rui Lu

    2017-10-01

    Full Text Available Acute myeloid leukemia (AML, a common hematological cancer of myeloid lineage cells, generally exhibits poor prognosis in the clinic and demands new treatment options. Recently, direct sequencing of samples from human AMLs and pre-leukemic diseases has unveiled their mutational landscapes and significantly advanced the molecular understanding of AML pathogenesis. The newly identified recurrent mutations frequently “hit” genes encoding epigenetic modulators, a wide range of chromatin-modifying enzymes and regulatory factors involved in gene expression regulation, supporting aberration of chromatin structure and epigenetic modification as a main oncogenic mechanism and cancer-initiating event. Increasing body of evidence demonstrates that chromatin modification aberrations underlying the formation of blood cancer can be reversed by pharmacological targeting of the responsible epigenetic modulators, thus providing new mechanism-based treatment strategies. Here, we summarize recent advances in development of small-molecule inhibitors specific to chromatin factors and their potential applications in the treatment of genetically defined AMLs. These compounds selectively inhibit various subclasses of “epigenetic writers” (such as histone methyltransferases MLL/KMT2A, G9A/KMT1C, EZH2/KMT6A, DOT1L/KMT4, and PRMT1, “epigenetic readers” (such as BRD4 and plant homeodomain finger proteins, and “epigenetic erasers” (such as histone demethylases LSD1/KDM1A and JMJD2C/KDM4C. We also discuss about the molecular mechanisms underpinning therapeutic effect of these epigenetic compounds in AML and favor their potential usage for combinational therapy and treatment of pre-leukemia diseases.

  10. The regulatory element 3' to the A gamma-globin gene binds to the nuclear matrix and interacts with special A-T-rich binding protein 1 (SATB1), an SAR/MAR-associating region DNA binding protein.

    Science.gov (United States)

    Cunningham, J M; Purucker, M E; Jane, S M; Safer, B; Vanin, E F; Ney, P A; Lowrey, C H; Nienhuis, A W

    1994-08-15

    A cis-acting DNA regulatory element 3' to the A gamma-globin gene contains eight distinct regions of DNA-protein interaction distributed over 750 bp of DNA. The sequences of two foot-printed regions (sites I and IV) are A-T rich and generate a highly retarded complex on gel shift analysis with nuclear extract from human erythroleukemia (K562) cells. We have purified a 98-kD protein that reproduces this gel shift. Tryptic cleavage and peptide sequence analysis demonstrated that the 98-kD protein is identical to a recently cloned protein, special A-T-rich binding protein 1 (SATB1), that binds selectively to nuclear matrix/scaffold-associated regions of DNA (MARs/SARs). We have shown by functional analysis that the 3' A gamma regulatory element associates with the nuclear matrix. SATB1 mRNA was identified in K562 cells, and reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated its transcript in several other hematopoietic lines. Antisera to SATB1 caused ablation of the gel shift complex generated by both the crude nuclear extract and the purified 98-kD protein with the site I oligonucleotide. Furthermore, oligonucleotides that bind SATB1 inhibited formation of the site I gel shift complex when added as excess unlabeled competitor. An immunoblot analysis of the site I gel shift complex documented the presence of SATB1. Binding of SATB1 to two sites within the 3' A gamma regulatory element and its MAR/SAR activity suggests that this element may influence gene expression through interaction with the nuclear matrix.

  11. Dynamics of 1α,25-dihydroxyvitamin D3-dependent chromatin accessibility of early vitamin D receptor target genes.

    Science.gov (United States)

    Seuter, Sabine; Pehkonen, Petri; Heikkinen, Sami; Carlberg, Carsten

    2013-12-01

    The signaling cascade of the transcription factor vitamin D receptor (VDR) is triggered by its specific ligand 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study we demonstrate that in THP-1 human monocytic leukemia cells 87.4% of the 1034 most prominent genome-wide VDR binding sites co-localize with loci of open chromatin. At 165 of them 1α,25(OH)2D3 strongly increases chromatin accessibility and has at further 217 sites weaker effects. Interestingly, VDR binding sites in 1α,25(OH)2D3-responsive chromatin regions are far more often composed of direct repeats with 3 intervening nucleotides (DR3s) than those in ligand insensitive regions. DR3-containing VDR sites are enriched in the neighborhood of genes that are involved in controling cellular growth, while non-DR3 VDR binding is often found close to genes related to immunity. At the example of six early VDR target genes we show that the slope of their 1α,25(OH)2D3-induced transcription correlates with the basal chromatin accessibility of their major VDR binding regions. However, the chromatin loci controlling these genes are indistinguishable in their VDR association kinetics. Taken together, ligand responsive chromatin loci represent dynamically regulated contact points of VDR with the genome, from where it controls early 1α,25(OH)2D3 target genes. © 2013.

  12. Flightless I (Drosophila) homolog facilitates chromatin accessibility of the estrogen receptor α target genes in MCF-7 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Kwang Won, E-mail: kwjeong@gachon.ac.kr

    2014-04-04

    Highlights: • H3K4me3 and Pol II binding at TFF1 promoter were reduced in FLII-depleted MCF-7 cells. • FLII is required for chromatin accessibility of the enhancer of ERalpha target genes. • Depletion of FLII causes inhibition of proliferation of MCF-7 cells. - Abstract: The coordinated activities of multiple protein complexes are essential to the remodeling of chromatin structure and for the recruitment of RNA polymerase II (Pol II) to the promoter in order to facilitate the initiation of transcription in nuclear receptor-mediated gene expression. Flightless I (Drosophila) homolog (FLII), a nuclear receptor coactivator, is associated with the SWI/SNF-chromatin remodeling complex during estrogen receptor (ER)α-mediated transcription. However, the function of FLII in estrogen-induced chromatin opening has not been fully explored. Here, we show that FLII plays a critical role in establishing active histone modification marks and generating the open chromatin structure of ERα target genes. We observed that the enhancer regions of ERα target genes are heavily occupied by FLII, and histone H3K4me3 and Pol II binding induced by estrogen are decreased in FLII-depleted MCF-7 cells. Furthermore, formaldehyde-assisted isolation of regulatory elements (FAIRE)-quantitative polymerase chain reaction (qPCR) experiments showed that depletion of FLII resulted in reduced chromatin accessibility of multiple ERα target genes. These data suggest FLII as a key regulator of ERα-mediated transcription through its role in regulating chromatin accessibility for the binding of RNA Polymerase II and possibly other transcriptional coactivators.

  13. Disparate chromatin landscapes and kinetics of inactivation impact differential regulation of p53 target genes.

    Science.gov (United States)

    Gomes, Nathan P; Espinosa, Joaquín M

    2010-09-01

    The p53 transcription factor regulates the expression of genes involved in cellular responses to stress, including cell cycle arrest and apoptosis. The p53 transcriptional program is extremely malleable, with target gene expression varying in a stress- and cell type-specific fashion. The molecular mechanisms underlying differential p53 target gene expression remain elusive. Here we provide evidence for gene-specific mechanisms affecting expression of three important p53 target genes. First we show that transcription of the apoptotic gene PUMA is regulated through intragenic chromatin boundaries, as revealed by distinct histone modification territories that correlate with binding of the insulator factors CTCF, Cohesins and USF1/2. Interestingly, this mode of regulation produces an evolutionary conserved long non-coding RNA of unknown function. Second, we demonstrate that the kinetics of transcriptional competence of the cell cycle arrest gene p21 and the apoptotic gene FAS are markedly different in vivo, as predicted by recent biochemical dissection of their core promoter elements in vitro. After a pulse of p53 activity in cells, assembly of the transcriptional apparatus on p21 is rapidly reversed, while FAS transcriptional activation is more sustained. Collectively these data add to a growing list of p53-autonomous mechanisms that impact differential regulation of p53 target genes.

  14. Identification of several potential chromatin binding sites of HOXB7 and its downstream target genes in breast cancer.

    Science.gov (United States)

    Heinonen, Henna; Lepikhova, Tatiana; Sahu, Biswajyoti; Pehkonen, Henna; Pihlajamaa, Päivi; Louhimo, Riku; Gao, Ping; Wei, Gong-Hong; Hautaniemi, Sampsa; Jänne, Olli A; Monni, Outi

    2015-11-15

    HOXB7 encodes a transcription factor that is overexpressed in a number of cancers and encompasses many oncogenic functions. Previous results have shown it to promote cell proliferation, angiogenesis, epithelial-mesenchymal transition, DNA repair and cell survival. Because of its role in many cancers and tumorigenic processes, HOXB7 has been suggested to be a potential drug target. However, HOXB7 binding sites on chromatin and its targets are poorly known. The aim of our study was to identify HOXB7 binding sites on breast cancer cell chromatin and to delineate direct target genes located nearby these binding sites. We found 1,504 HOXB7 chromatin binding sites in BT-474 breast cancer cell line that overexpresses HOXB7. Seventeen selected binding sites were validated by ChIP-qPCR in several breast cancer cell lines. Furthermore, we analyzed expression of a large number of genes located nearby HOXB7 binding sites and found several new direct targets, such as CTNND2 and SCGB1D2. Identification of HOXB7 chromatin binding sites and target genes is essential to understand better the role of HOXB7 in breast cancer and mechanisms by which it regulates tumorigenic processes. © 2015 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.

  15. Differential interactions of the autonomous pathway RRM proteins and chromatin regulators in the silencing of Arabidopsis targets.

    Directory of Open Access Journals (Sweden)

    Isabel Bäurle

    2008-07-01

    Full Text Available We have recently shown that two proteins containing RRM-type RNA-binding domains, FCA and FPA, originally identified through their role in flowering time control in Arabidopsis, silence transposons and other repeated sequences in the Arabidopsis genome. In flowering control, FCA and FPA function in the autonomous pathway with conserved chromatin regulators, the histone demethylase FLD and the MSI1-homologue FVE, a conserved WD-repeat protein found in many chromatin complexes. Here, we investigate how the RRM proteins interact genetically with these chromatin regulators at a range of loci in the Arabidopsis genome. We also investigate their interaction with the DNA methylation pathway. In several cases the RRM protein activity at least partially required a chromatin regulator to effect silencing. However, the interactions of the autonomous pathway components differed at each target analysed, most likely determined by certain properties of the target loci and/or other silencing pathways. We speculate that the RNA-binding proteins FCA and FPA function as part of a transcriptome surveillance mechanism linking RNA recognition with chromatin silencing mechanisms.

  16. Five friends of methylated chromatin target of protein-arginine- methyltransferase[Prmt]-1 (Chtop), a complex linking arginine methylation to desumoylation

    NARCIS (Netherlands)

    P. Fanis (Pavlos); N. Gillemans (Nynke); A. Aghajanirefah (Ali); F. Pourfarzad, F. (Farzin); J.A.A. Demmers (Jeroen); F. Esteghamat (Fatemehsadat); R.K. Vadlamudi (Ratna); F.G. Grosveld (Frank); J.N.J. Philipsen (Sjaak); T.B. van Dijk (Thamar)

    2012-01-01

    textabstractChromatin target of Prmt1 (Chtop) is a vertebrate-specific chromatin-bound protein that plays an important role in transcriptional regulation. As its mechanism of action remains unclear, we identified Chtop-interacting proteins using a biotinylation-proteomics approach. Here we describe

  17. The Groucho co-repressor is primarily recruited to local target sites in active chromatin to attenuate transcription.

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    Aamna Kaul

    2014-08-01

    Full Text Available Gene expression is regulated by the complex interaction between transcriptional activators and repressors, which function in part by recruiting histone-modifying enzymes to control accessibility of DNA to RNA polymerase. The evolutionarily conserved family of Groucho/Transducin-Like Enhancer of split (Gro/TLE proteins act as co-repressors for numerous transcription factors. Gro/TLE proteins act in several key pathways during development (including Notch and Wnt signaling, and are implicated in the pathogenesis of several human cancers. Gro/TLE proteins form oligomers and it has been proposed that their ability to exert long-range repression on target genes involves oligomerization over broad regions of chromatin. However, analysis of an endogenous gro mutation in Drosophila revealed that oligomerization of Gro is not always obligatory for repression in vivo. We have used chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq to profile Gro recruitment in two Drosophila cell lines. We find that Gro predominantly binds at discrete peaks (<1 kilobase. We also demonstrate that blocking Gro oligomerization does not reduce peak width as would be expected if Gro oligomerization induced spreading along the chromatin from the site of recruitment. Gro recruitment is enriched in "active" chromatin containing developmentally regulated genes. However, Gro binding is associated with local regions containing hypoacetylated histones H3 and H4, which is indicative of chromatin that is not fully open for efficient transcription. We also find that peaks of Gro binding frequently overlap the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing and that depletion of Gro leads to release of polymerase pausing and increased transcription at a bona fide target gene. Our results demonstrate that Gro is recruited to local sites by transcription factors to attenuate rather than silence gene expression by promoting histone

  18. Arabidopsis chromatin-associated HMGA and HMGB use different nuclear targeting signals and display highly dynamic localization within the nucleus

    DEFF Research Database (Denmark)

    Launholt, Dorte; Merkle, Thomas; Houben, Andreas

    2006-01-01

    HMGproteins appear to be involved in the regulation of transcription and other DNA-dependent processes. We have examined the subcellular localization of Arabidopsis thaliana HMGA, HMGB1, and HMGB5, revealing that they localize to the cell nucleus. They display a speckled distribution pattern throughout the chromatin...... of interphase nuclei, whereas none of the proteins associate with condensed mitotic chromosomes. HMGA is targeted to the nucleus by a monopartite nuclear localization signal, while efficient nuclear accumulation of HMGB1/5 requires large portions of the basic N-terminal part of the proteins. The acidic C......-terminal domain interferes with nucleolar targeting of HMGB1. Fluorescence recovery after photobleaching experiments revealed that HMGA and HMGB proteins are extremely dynamic in the nucleus, indicating that they bind chromatin only transiently before moving on to the next site, thereby continuously scanning...

  19. Identification of potential target genes for the tomato fruit-ripening regulator RIN by chromatin immunoprecipitation

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    Nakano Toshitsugu

    2011-01-01

    Full Text Available Abstract Background During ripening, climacteric fruits increase their ethylene level and subsequently undergo various physiological changes, such as softening, pigmentation and development of aroma and flavor. These changes occur simultaneously and are caused by the highly synchronized expression of numerous genes at the onset of ripening. In tomatoes, the MADS-box transcription factor RIN has been regarded as a key regulator responsible for the onset of ripening by acting upstream of both ethylene- and non-ethylene-mediated controls. However, except for LeACS2, direct targets of RIN have not been clarified, and little is known about the transcriptional cascade for ripening. Results Using immunoprecipitated (IPed DNA fragments recovered by chromatin immunoprecipitation (ChIP with anti-RIN antibody from ripening tomato fruit, we analyzed potential binding sites for RIN (CArG-box sites in the promoters of representative ripening-induced genes by quantitative PCR. Results revealed nearly a 5- to 20-fold enrichment of CArG boxes in the promoters of LeACS2, LeACS4, PG, TBG4, LeEXP1, and LeMAN4 and of RIN itself, indicating direct interaction of RIN with their promoters in vivo. Moreover, sequence analysis and genome mapping of 51 cloned IPed DNAs revealed potential RIN binding sites. Quantitative PCR revealed that four of the potential binding sites were enriched 4- to 17-fold in the IPed DNA pools compared with the controls, indicating direct interaction of RIN with these sites in vivo. Near one of the four CArG boxes we found a gene encoding a protein similar to thioredoxin y1. An increase in the transcript level of this gene was observed with ripening in normal fruit but not in the rin mutant, suggesting that RIN possibly induces its expression. Conclusions The presented results suggest that RIN controls fruit softening and ethylene production by the direct transcriptional regulation of cell-wall-modifying genes and ethylene biosynthesis genes

  20. A RECQ5-RNA polymerase II association identified by targeted proteomic analysis of human chromatin.

    Science.gov (United States)

    Aygün, Ozan; Svejstrup, Jesper; Liu, Yilun

    2008-06-24

    Although the active forms of factors involved in DNA-related processes such as DNA replication, repair, and transcription are associated with chromatin, proteins are rarely purified from this source. Here, we describe a protocol for the isolation of chromatin-associated factors and use it to identify proteins interacting with human RNA polymerase II (RNAPII). Our data establish RECQ5 helicase as a bona fide RNAPII-associated protein. The RECQ5-RNAPII interaction is direct and is mediated by the RPB1 subunit of RNAPII, and RECQ5 appears to be the only member of the human RECQ family of helicases that associates with RNAPII. These data suggest an unexpected role for RECQ5 helicase at the interface of transcription and genomic stability.

  1. Targeting of chromatin readers: a novel strategy used by the Shigella flexneri virulence effector OspF to reprogram transcription

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    Habiba Harouz

    2014-12-01

    Full Text Available Shigella flexneri, a gram-negative bacterium responsible of bacillary dysentery, uses multiple strategies to overcome host immune defense. We have decrypted how this bacterium manipulates host-cell chromatin binders to take control of immune gene expression. We found that OspF, an injected virulence factor previously identified as a repressor of immune gene expression, targets the chromatin reader HP1γ. Heterochromatin Protein 1 family members specifically recognize and bind histone H3 methylated at Lys 9. Although initially identified as chromatin-associated transcriptional silencers in heterochromatin, their location in euchromatin indicates an active role in gene expression. Notably, HP1γ phosphorylation at Serine 83 defines a subpopulation exclusively located to euchromatin, targeted to the site of transcriptional elongation. We showed that OspF directly interacts with HP1γ, and causes HP1 dephosphorylation, suggesting a model in which this virulence effector “uses” HP1 proteins as beacons to target and repress immune gene expression (Harouz, et al. EMBO J (2014. OspF alters HP1γ phosphorylation mainly by inactivating the Erk-activated kinase MSK1, spotlighting it as a new HP1 kinase. In vivo, infectious stresses trigger HP1γ phosphorylation in the colon, principally in the lamina propria and the intestinal crypts. Several lines of evidence suggest that HP1 proteins are modified as extensively as histones, and decrypting the impact of these HP1 post-translational modifications on their transcriptional activities in vivo will be the next challenges to be taken up.

  2. Targeting of chromatin readers: a novel strategy used by the Shigella flexneri virulence effector OspF to reprogram transcription.

    Science.gov (United States)

    Harouz, Habiba; Rachez, Christophe; Meijer, Benoit; Muchardt, Christian; Arbibe, Laurence

    2014-12-28

    Shigella flexneri, a gram-negative bacterium responsible of bacillary dysentery, uses multiple strategies to overcome host immune defense. We have decrypted how this bacterium manipulates host-cell chromatin binders to take control of immune gene expression. We found that OspF, an injected virulence factor previously identified as a repressor of immune gene expression, targets the chromatin reader HP1γ. Heterochromatin Protein 1 family members specifically recognize and bind histone H3 methylated at Lys 9. Although initially identified as chromatin-associated transcriptional silencers in heterochromatin, their location in euchromatin indicates an active role in gene expression. Notably, HP1γ phosphorylation at Serine 83 defines a subpopulation exclusively located to euchromatin, targeted to the site of transcriptional elongation. We showed that OspF directly interacts with HP1γ, and causes HP1 dephosphorylation, suggesting a model in which this virulence effector "uses" HP1 proteins as beacons to target and repress immune gene expression (Harouz, et al. EMBO J (2014)). OspF alters HP1γ phosphorylation mainly by inactivating the Erk-activated kinase MSK1, spotlighting it as a new HP1 kinase. In vivo, infectious stresses trigger HP1γ phosphorylation in the colon, principally in the lamina propria and the intestinal crypts. Several lines of evidence suggest that HP1 proteins are modified as extensively as histones, and decrypting the impact of these HP1 post-translational modifications on their transcriptional activities in vivo will be the next challenges to be taken up.

  3. Polycomb repressive complex 2 targets murine cytomegalovirus chromatin for modification and associates with viral replication centers.

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    Christopher G Abraham

    Full Text Available Regulation of viral transcription by chromatin structure has emerged as a fundamental determinant in the establishment of lytic and latent herpesvirus infections. The Polycomb group (PcG of epigenetic repressors promotes heterochromatin formation by trimethylating histone H3 on lysine-27 (H3K27me3 and regulates development, stem cell renewal and differentiation and the cell cycle. These cellular processes are tightly coupled to the molecular switch between lytic and latent herpesvirus infections. Using chromatin immunoprecipitation analysis, we observed enrichment of H3K27me3 at the major immediate-early (MIE locus of murine cytomegalovirus (MCMV very early following infection of permissive fibroblasts. As lytic replication progressed, we observed a loss of H3K27me3 enrichment concomitant with the appearance of H3K4me3. However, late during infection, as viral replication centers are established, we observed a significant increase in PcG protein association with chromatin. Additionally, in co-immunofluorescence assays using confocal microscopy, we detected strong enrichments for PcG protein within the viral replication compartment, suggesting an association between viral DNA synthesis machinery and PcG proteins. Together, our results suggest a novel, dynamic interaction between PcG epigenetic repressors and MCMV genomes.

  4. An Allosteric Interaction Links USP7 to Deubiquitination and Chromatin Targeting of UHRF1

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    Zhi-Min Zhang

    2015-09-01

    Full Text Available The protein stability and chromatin functions of UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1 are regulated in a cell-cycle-dependent manner. We report a structural characterization of the complex between UHRF1 and the deubiquitinase USP7. The first two UBL domains of USP7 bind to the polybasic region (PBR of UHRF1, and this interaction is required for the USP7-mediated deubiquitination of UHRF1. Importantly, we find that the USP7-binding site of the UHRF1 PBR overlaps with the region engaging in an intramolecular interaction with the N-terminal tandem Tudor domain (TTD. We show that the USP7-UHRF1 interaction perturbs the TTD-PBR interaction of UHRF1, thereby shifting the conformation of UHRF1 from a TTD-“occluded” state to a state open for multivalent histone binding. Consistently, introduction of a USP7-interaction-defective mutation to UHRF1 significantly reduces its chromatin association. Together, these results link USP7 interaction to the dynamic deubiquitination and chromatin association of UHRF1.

  5. Five Friends of Methylated Chromatin Target of Protein-Arginine-Methyltransferase[Prmt]-1 (Chtop), a Complex Linking Arginine Methylation to Desumoylation*

    OpenAIRE

    Fanis, Pavlos; Gillemans, Nynke; Aghajanirefah, Ali; Pourfarzad, Farzin; Demmers, Jeroen; Esteghamat, Fatemehsadat; Vadlamudi, Ratna K.; Grosveld, Frank; Philipsen, Sjaak; van Dijk, Thamar B.

    2012-01-01

    Chromatin target of Prmt1 (Chtop) is a vertebrate-specific chromatin-bound protein that plays an important role in transcriptional regulation. As its mechanism of action remains unclear, we identified Chtop-interacting proteins using a biotinylation-proteomics approach. Here we describe the identification and initial characterization of Five Friends of Methylated Chtop (5FMC). 5FMC is a nuclear complex that can only be recruited by Chtop when the latter is arginine-methylated by Prmt1. It con...

  6. Pax6 interactions with chromatin and identification of its novel direct target genes in lens and forebrain.

    Directory of Open Access Journals (Sweden)

    Qing Xie

    Full Text Available Pax6 encodes a specific DNA-binding transcription factor that regulates the development of multiple organs, including the eye, brain and pancreas. Previous studies have shown that Pax6 regulates the entire process of ocular lens development. In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific populations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification in telencephalon. In the pancreas, Pax6 is essential for the differentiation of α-, β- and δ-islet cells. To elucidate molecular roles of Pax6, chromatin immunoprecipitation experiments combined with high-density oligonucleotide array hybridizations (ChIP-chip were performed using three distinct sources of chromatin (lens, forebrain and β-cells. ChIP-chip studies, performed as biological triplicates, identified a total of 5,260 promoters occupied by Pax6. 1,001 (133 of these promoter regions were shared between at least two (three distinct chromatin sources, respectively. In lens chromatin, 2,335 promoters were bound by Pax6. RNA expression profiling from Pax6⁺/⁻ lenses combined with in vivo Pax6-binding data yielded 76 putative Pax6-direct targets, including the Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca genes. RNA and ChIP data were validated for all these genes. In lens cells, reporter assays established Kib1b and Snca as Pax6 activated and repressed genes, respectively. In situ hybridization revealed reduced expression of these genes in E14 cerebral cortex. Moreover, we examined differentially expressed transcripts between E9.5 wild type and Pax6⁻/⁻ lens placodes that suggested Efnb2, Fat4, Has2, Nav1, and Trpm3 as novel Pax6-direct targets. Collectively, the present studies, through the identification of Pax6-direct target genes, provide novel insights into the molecular mechanisms of Pax6 gene control during mouse embryonic development. In addition, the present data demonstrate that Pax6

  7. Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin.

    Science.gov (United States)

    Zhen, Chao Yu; Tatavosian, Roubina; Huynh, Thao Ngoc; Duc, Huy Nguyen; Das, Raibatak; Kokotovic, Marko; Grimm, Jonathan B; Lavis, Luke D; Lee, Jun; Mejia, Frances J; Li, Yang; Yao, Tingting; Ren, Xiaojun

    2016-10-10

    The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes.

  8. Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein.

    Science.gov (United States)

    Collas, P; Courvalin, J C; Poccia, D

    1996-12-01

    We have identified an integral membrane protein of sea urchin gametes with an apparent molecular mass of 56 kD that cross-reacts with an antibody against the nucleoplasmic NH2-terminal domain of human lamin B receptor (LBR). In mature sperm, p56 is located at the tip and base of the nucleus from where it is removed by egg cytosol in vitro. In the egg, p56 is present in a subset of cytoplasmic membranes (MV2 beta) which contributes the bulk of the nuclear envelope during male pronuclear formation. p56-containing vesicles are required for nuclear envelope assembly and have a chromatin-binding capacity that is mediated by p56. Lamin B is not present in these vesicles and is imported into the nucleus from a soluble pool at a later stage of pronuclear formation. Lamin B incorporation and addition of new membranes are necessary for pronuclear swelling and nuclear envelope growth. We suggest that p56 is a sea urchin LBR homologue that targets membranes to chromatin and later anchors the membrane to the lamina.

  9. Reprogramming chromatin

    DEFF Research Database (Denmark)

    Ehrensberger, Andreas Hasso; Svejstrup, Jesper Qualmann

    2012-01-01

    attributed to high kinetic barriers that affect all cells equally and can only be overcome by rare stochastic events. The barriers to reprogramming are likely to involve transformations of chromatin state because (i) inhibitors of chromatin-modifying enzymes can enhance the efficiency of reprogramming...... and (ii) knockdown or knock-out of chromatin-modifying enzymes can lower the efficiency of reprogramming. Here, we review the relationship between chromatin state transformations (chromatin reprogramming) and cellular reprogramming, with an emphasis on transcription factors, chromatin remodeling factors...

  10. Genome-Wide Mapping Targets of the Metazoan Chromatin Remodeling Factor NURF Reveals Nucleosome Remodeling at Enhancers, Core Promoters and Gene Insulators.

    Directory of Open Access Journals (Sweden)

    So Yeon Kwon

    2016-04-01

    Full Text Available NURF is a conserved higher eukaryotic ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF is able to alter chromatin dynamics to control transcription and genome organization. Previous biochemical and genetic analysis of the specificity-subunit of Drosophila NURF (Nurf301/Enhancer of Bithorax (E(bx has defined NURF as a critical regulator of homeotic, heat-shock and steroid-responsive gene transcription. It has been speculated that NURF controls pathway specific transcription by co-operating with sequence-specific transcription factors to remodel chromatin at dedicated enhancers. However, conclusive in vivo demonstration of this is lacking and precise regulatory elements targeted by NURF are poorly defined. To address this, we have generated a comprehensive map of in vivo NURF activity, using MNase-sequencing to determine at base pair resolution NURF target nucleosomes, and ChIP-sequencing to define sites of NURF recruitment. Our data show that, besides anticipated roles at enhancers, NURF interacts physically and functionally with the TRF2/DREF basal transcription factor to organize nucleosomes downstream of active promoters. Moreover, we detect NURF remodeling and recruitment at distal insulator sites, where NURF functionally interacts with and co-localizes with DREF and insulator proteins including CP190 to establish nucleosome-depleted domains. This insulator function of NURF is most apparent at subclasses of insulators that mark the boundaries of chromatin domains, where multiple insulator proteins co-associate. By visualizing the complete repertoire of in vivo NURF chromatin targets, our data provide new insights into how chromatin remodeling can control genome organization and regulatory interactions.

  11. Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors.

    Science.gov (United States)

    Heisig, Julia; Weber, David; Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

    2012-01-01

    HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.

  12. miR-151-5p, targeting chromatin remodeler SMARCA5, as a marker for the BRCAness phenotype.

    Science.gov (United States)

    Tommasi, Stefania; Pinto, Rosamaria; Danza, Katia; Pilato, Brunella; Palumbo, Orazio; Micale, Lucia; De Summa, Simona

    2016-12-06

    In recent years, the assessment of biomarkers useful for "precision medicine" has been a hot topic in research. The involvement of microRNAs in the pathogenesis of breast cancer has been highly investigated with the aim of being able to molecularly stratify this highly heterogeneous disease. Our aim was to identify microRNAs targeting DNA repair machinery, through Affymetrix GeneChip miRNA Arrays, in a cohort of BRCA-related and sporadic breast cancers. Moreover, we analyzed microRNA expression taking into account our previous results on the expression of PARP1, because of its importance in targeted therapy. miR-361-5p and miR-151-5p were found to be overexpressed in PARP1-upregulating BRCA-germline mutated and sporadic breast tumors. Pathway enrichment analysis was performed to identify potential target genes to be analyzed in the validation step in an independent cohort. Our results confirmed the overexpression of miR-151-5p and, interestingly, its role in the targeting of SMARCA5, a chromatin remodeler. This result was also confirmed in vitro, both through luciferase assay and by analyzing endogenous levels of SMARCA5 in MCF-7 cell lines using miR-151-5p mimic and inhibitor. In conclusion, our data showed the possibility of considering the overexpression of PARP1 and miR-151-5p as biomarkers useful to correctly treat sporadic breast cancers, which eventually could be considered as BRCAness tumors, with PARP-inhibitors.

  13. The sequence-specific transcription factor c-Jun targets Cockayne syndrome protein B to regulate transcription and chromatin structure.

    Science.gov (United States)

    Lake, Robert J; Boetefuer, Erica L; Tsai, Pei-Fang; Jeong, Jieun; Choi, Inchan; Won, Kyoung-Jae; Fan, Hua-Ying

    2014-04-01

    Cockayne syndrome is an inherited premature aging disease associated with numerous developmental and neurological defects, and mutations in the gene encoding the CSB protein account for the majority of Cockayne syndrome cases. Accumulating evidence suggests that CSB functions in transcription regulation, in addition to its roles in DNA repair, and those defects in this transcriptional activity might contribute to the clinical features of Cockayne syndrome. Transcription profiling studies have so far uncovered CSB-dependent effects on gene expression; however, the direct targets of CSB's transcriptional activity remain largely unknown. In this paper, we report the first comprehensive analysis of CSB genomic occupancy during replicative cell growth. We found that CSB occupancy sites display a high correlation to regions with epigenetic features of promoters and enhancers. Furthermore, we found that CSB occupancy is enriched at sites containing the TPA-response element. Consistent with this binding site preference, we show that CSB and the transcription factor c-Jun can be found in the same protein-DNA complex, suggesting that c-Jun can target CSB to specific genomic regions. In support of this notion, we observed decreased CSB occupancy of TPA-response elements when c-Jun levels were diminished. By modulating CSB abundance, we found that CSB can influence the expression of nearby genes and impact nucleosome positioning in the vicinity of its binding site. These results indicate that CSB can be targeted to specific genomic loci by sequence-specific transcription factors to regulate transcription and local chromatin structure. Additionally, comparison of CSB occupancy sites with the MSigDB Pathways database suggests that CSB might function in peroxisome proliferation, EGF receptor transactivation, G protein signaling and NF-κB activation, shedding new light on the possible causes and mechanisms of Cockayne syndrome.

  14. The sequence-specific transcription factor c-Jun targets Cockayne syndrome protein B to regulate transcription and chromatin structure.

    Directory of Open Access Journals (Sweden)

    Robert J Lake

    2014-04-01

    Full Text Available Cockayne syndrome is an inherited premature aging disease associated with numerous developmental and neurological defects, and mutations in the gene encoding the CSB protein account for the majority of Cockayne syndrome cases. Accumulating evidence suggests that CSB functions in transcription regulation, in addition to its roles in DNA repair, and those defects in this transcriptional activity might contribute to the clinical features of Cockayne syndrome. Transcription profiling studies have so far uncovered CSB-dependent effects on gene expression; however, the direct targets of CSB's transcriptional activity remain largely unknown. In this paper, we report the first comprehensive analysis of CSB genomic occupancy during replicative cell growth. We found that CSB occupancy sites display a high correlation to regions with epigenetic features of promoters and enhancers. Furthermore, we found that CSB occupancy is enriched at sites containing the TPA-response element. Consistent with this binding site preference, we show that CSB and the transcription factor c-Jun can be found in the same protein-DNA complex, suggesting that c-Jun can target CSB to specific genomic regions. In support of this notion, we observed decreased CSB occupancy of TPA-response elements when c-Jun levels were diminished. By modulating CSB abundance, we found that CSB can influence the expression of nearby genes and impact nucleosome positioning in the vicinity of its binding site. These results indicate that CSB can be targeted to specific genomic loci by sequence-specific transcription factors to regulate transcription and local chromatin structure. Additionally, comparison of CSB occupancy sites with the MSigDB Pathways database suggests that CSB might function in peroxisome proliferation, EGF receptor transactivation, G protein signaling and NF-κB activation, shedding new light on the possible causes and mechanisms of Cockayne syndrome.

  15. Chromatin computation.

    Directory of Open Access Journals (Sweden)

    Barbara Bryant

    Full Text Available In living cells, DNA is packaged along with protein and RNA into chromatin. Chemical modifications to nucleotides and histone proteins are added, removed and recognized by multi-functional molecular complexes. Here I define a new computational model, in which chromatin modifications are information units that can be written onto a one-dimensional string of nucleosomes, analogous to the symbols written onto cells of a Turing machine tape, and chromatin-modifying complexes are modeled as read-write rules that operate on a finite set of adjacent nucleosomes. I illustrate the use of this "chromatin computer" to solve an instance of the Hamiltonian path problem. I prove that chromatin computers are computationally universal--and therefore more powerful than the logic circuits often used to model transcription factor control of gene expression. Features of biological chromatin provide a rich instruction set for efficient computation of nontrivial algorithms in biological time scales. Modeling chromatin as a computer shifts how we think about chromatin function, suggests new approaches to medical intervention, and lays the groundwork for the engineering of a new class of biological computing machines.

  16. Chromatin Computation

    Science.gov (United States)

    Bryant, Barbara

    2012-01-01

    In living cells, DNA is packaged along with protein and RNA into chromatin. Chemical modifications to nucleotides and histone proteins are added, removed and recognized by multi-functional molecular complexes. Here I define a new computational model, in which chromatin modifications are information units that can be written onto a one-dimensional string of nucleosomes, analogous to the symbols written onto cells of a Turing machine tape, and chromatin-modifying complexes are modeled as read-write rules that operate on a finite set of adjacent nucleosomes. I illustrate the use of this “chromatin computer” to solve an instance of the Hamiltonian path problem. I prove that chromatin computers are computationally universal – and therefore more powerful than the logic circuits often used to model transcription factor control of gene expression. Features of biological chromatin provide a rich instruction set for efficient computation of nontrivial algorithms in biological time scales. Modeling chromatin as a computer shifts how we think about chromatin function, suggests new approaches to medical intervention, and lays the groundwork for the engineering of a new class of biological computing machines. PMID:22567109

  17. A RECQ5–RNA polymerase II association identified by targeted proteomic analysis of human chromatin

    OpenAIRE

    Aygün, Ozan; Svejstrup, Jesper; Liu, Yilun

    2008-01-01

    Although the active forms of factors involved in DNA-related processes such as DNA replication, repair, and transcription are associated with chromatin, proteins are rarely purified from this source. Here, we describe a protocol for the isolation of chromatin-associated factors and use it to identify proteins interacting with human RNA polymerase II (RNAPII). Our data establish RECQ5 helicase as a bona fide RNAPII-associated protein. The RECQ5–RNAPII interaction is direct and is mediated by t...

  18. Chromatin deregulation in disease.

    Science.gov (United States)

    Mirabella, Anne C; Foster, Benjamin M; Bartke, Till

    2016-03-01

    The regulation of chromatin by epigenetic mechanisms plays a central role in gene expression and is essential for development and maintenance of cell identity and function. Aberrant chromatin regulation is observed in many diseases where it leads to defects in epigenetic gene regulation resulting in pathological gene expression programmes. These defects are caused by inherited or acquired mutations in genes encoding enzymes that deposit or remove DNA and histone modifications and that shape chromatin architecture. Chromatin deregulation often results in neurodevelopmental disorders and intellectual disabilities, frequently linked to physical and developmental abnormalities, but can also cause neurodegenerative diseases, immunodeficiency, or muscle wasting syndromes. Epigenetic diseases can either be of monogenic origin or manifest themselves as complex multifactorial diseases such as in congenital heart disease, autism spectrum disorders, or cancer in which mutations in chromatin regulators are contributing factors. The environment directly influences the epigenome and can induce changes that cause or predispose to diseases through risk factors such as stress, malnutrition or exposure to harmful chemicals. The plasticity of chromatin regulation makes targeting the enzymatic machinery an attractive strategy for therapeutic intervention and an increasing number of small molecule inhibitors against a variety of epigenetic regulators are in clinical use or under development. In this review, we will give an overview of the molecular lesions that underlie epigenetic diseases, and we will discuss the impact of the environment and prospects for epigenetic therapies.

  19. Expanding the druggable space of the LSD1/CoREST epigenetic target: new potential binding regions for drug-like molecules, peptides, protein partners, and chromatin.

    Directory of Open Access Journals (Sweden)

    James C Robertson

    Full Text Available Lysine specific demethylase-1 (LSD1/KDM1A in complex with its corepressor protein CoREST is a promising target for epigenetic drugs. No therapeutic that targets LSD1/CoREST, however, has been reported to date. Recently, extended molecular dynamics (MD simulations indicated that LSD1/CoREST nanoscale clamp dynamics is regulated by substrate binding and highlighted key hinge points of this large-scale motion as well as the relevance of local residue dynamics. Prompted by the urgent need for new molecular probes and inhibitors to understand LSD1/CoREST interactions with small-molecules, peptides, protein partners, and chromatin, we undertake here a configurational ensemble approach to expand LSD1/CoREST druggability. The independent algorithms FTMap and SiteMap and our newly developed Druggable Site Visualizer (DSV software tool were used to predict and inspect favorable binding sites. We find that the hinge points revealed by MD simulations at the SANT2/Tower interface, at the SWIRM/AOD interface, and at the AOD/Tower interface are new targets for the discovery of molecular probes to block association of LSD1/CoREST with chromatin or protein partners. A fourth region was also predicted from simulated configurational ensembles and was experimentally validated to have strong binding propensity. The observation that this prediction would be prevented when using only the X-ray structures available (including the X-ray structure bound to the same peptide underscores the relevance of protein dynamics in protein interactions. A fifth region was highlighted corresponding to a small pocket on the AOD domain. This study sets the basis for future virtual screening campaigns targeting the five novel regions reported herein and for the design of LSD1/CoREST mutants to probe LSD1/CoREST binding with chromatin and various protein partners.

  20. High-resolution chromatin immunoprecipitation (ChIP) sequencing reveals novel binding targets and prognostic role for SOX11 in mantle cell lymphoma.

    Science.gov (United States)

    Kuo, P-Y; Leshchenko, V V; Fazzari, M J; Perumal, D; Gellen, T; He, T; Iqbal, J; Baumgartner-Wennerholm, S; Nygren, L; Zhang, F; Zhang, W; Suh, K S; Goy, A; Yang, D T; Chan, W-C; Kahl, B S; Verma, A K; Gascoyne, R D; Kimby, E; Sander, B; Ye, B H; Melnick, A M; Parekh, S

    2015-03-05

    Sex determining region Y-box 11 (SOX11) expression is specific for mantle cell lymphoma (MCL) as compared with other non-Hodgkin's lymphomas. However, the function and direct-binding targets of SOX11 in MCL are largely unknown. We used high-resolution chromatin immunoprecipitation sequencing to identify the direct target genes of SOX11 in a genome-wide, unbiased manner and elucidate its functional significance. Pathway analysis identified WNT, PKA and TGF-beta signaling pathways as significantly enriched by SOX11-target genes. Quantitative chromatin immunoprecipitation sequencing and promoter reporter assays confirmed that SOX11 directly binds to individual genes and modulates their transcription activities in these pathways in MCL. Functional studies using RNA interference demonstrate that SOX11 directly regulates WNT in MCL. We analyzed SOX11 expression in three independent well-annotated tissue microarrays from the University of Wisconsin (UW), Karolinska Institute and British Columbia Cancer Agency. Our findings suggest that high SOX11 expression is associated with improved survival in a subset of MCL patients, particularly those treated with intensive chemotherapy. Transcriptional regulation of WNT and other biological pathways affected by SOX11-target genes may help explain the impact of SOX11 expression on patient outcomes.

  1. Use of chromatin remodeling ATPases as RNAi targets for parental control of western corn rootworm (Diabrotica virgifera virgifera) and Neotropical brown stink bug (Euschistus heros).

    Science.gov (United States)

    Fishilevich, Elane; Vélez, Ana M; Khajuria, Chitvan; Frey, Meghan L F; Hamm, Ronda L; Wang, Haichuan; Schulenberg, Greg A; Bowling, Andrew J; Pence, Heather E; Gandra, Premchand; Arora, Kanika; Storer, Nicholas P; Narva, Kenneth E; Siegfried, Blair D

    2016-04-01

    RNA interference (RNAi) is a gene silencing mechanism that is present in animals and plants and is triggered by double stranded RNA (dsRNA) or small interfering RNA (siRNA), depending on the organism. In the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), RNAi can be achieved by feeding rootworms dsRNA added to artificial diet or plant tissues transformed to express dsRNA. The effect of RNAi depends on the targeted gene function and can range from an absence of phenotypic response to readily apparent responses, including lethality. Furthermore, RNAi can directly affect individuals that consume dsRNA or the effect may be transferred to the next generation. Our previous work described the potential use of genes involved in embryonic development as a parental RNAi technology for the control of WCR. In this study, we describe the use of chromatin-remodeling ATPases as target genes to achieve parental gene silencing in two insect pests, a coleopteran, WCR, and a hemipteran, the Neotropical brown stink bug, Euschistus heros Fabricius (Hemiptera: Pentatomidae). Our results show that dsRNA targeting chromatin-remodeling ATPase transcripts, brahma, mi-2, and iswi strongly reduced the fecundity of the exposed females in both insect species. Additionally, knockdown of chd1 reduced the fecundity of E. heros. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Identification of Brassinosteroid Target Genes by Chromatin Immunoprecipitation Followed by High-Throughput Sequencing (ChIP-seq) and RNA-Sequencing.

    Science.gov (United States)

    Nolan, Trevor; Liu, Sanzhen; Guo, Hongqing; Li, Lei; Schnable, Patrick; Yin, Yanhai

    2017-01-01

    Brassinosteroids (BRs) play important roles in many growth and developmental processes. BRs signal to regulate BR-INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1) and BRASSINAZOLE-RESISTANT1 (BZR1) transcription factors (TFs), which, in turn, regulate several hundreds of transcription factors (termed BES1/BZR1-targeted TFs or BTFs) and thousands of genes to mediate various BR responses. Chromatin Immunoprecipitation followed by high-throughput sequencing (ChIP-seq) with BES1/BZR1 and BTFs is an important approach to identify BR target genes. In combination with RNA-sequencing experiments, these genomic methods have become powerful tools to detect BR target genes and reveal transcriptional networks underlying BR-regulated processes.

  3. Chromatin-bound IκBα regulates a subset of polycomb target genes in differentiation and cancer.

    Science.gov (United States)

    Mulero, María Carmen; Ferres-Marco, Dolors; Islam, Abul; Margalef, Pol; Pecoraro, Matteo; Toll, Agustí; Drechsel, Nils; Charneco, Cristina; Davis, Shelly; Bellora, Nicolás; Gallardo, Fernando; López-Arribillaga, Erika; Asensio-Juan, Elena; Rodilla, Verónica; González, Jessica; Iglesias, Mar; Shih, Vincent; Mar Albà, M; Di Croce, Luciano; Hoffmann, Alexander; Miyamoto, Shigeki; Villà-Freixa, Jordi; López-Bigas, Nuria; Keyes, William M; Domínguez, María; Bigas, Anna; Espinosa, Lluís

    2013-08-12

    IκB proteins are the primary inhibitors of NF-κB. Here, we demonstrate that sumoylated and phosphorylated IκBα accumulates in the nucleus of keratinocytes and interacts with histones H2A and H4 at the regulatory region of HOX and IRX genes. Chromatin-bound IκBα modulates Polycomb recruitment and imparts their competence to be activated by TNFα. Mutations in the Drosophila IκBα gene cactus enhance the homeotic phenotype of Polycomb mutants, which is not counteracted by mutations in dorsal/NF-κB. Oncogenic transformation of keratinocytes results in cytoplasmic IκBα translocation associated with a massive activation of Hox. Accumulation of cytoplasmic IκBα was found in squamous cell carcinoma (SCC) associated with IKK activation and HOX upregulation. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Five Friends of Methylated Chromatin Target of Protein-Arginine-Methyltransferase[Prmt]-1 (Chtop), a Complex Linking Arginine Methylation to Desumoylation*

    Science.gov (United States)

    Fanis, Pavlos; Gillemans, Nynke; Aghajanirefah, Ali; Pourfarzad, Farzin; Demmers, Jeroen; Esteghamat, Fatemehsadat; Vadlamudi, Ratna K.; Grosveld, Frank; Philipsen, Sjaak; van Dijk, Thamar B.

    2012-01-01

    Chromatin target of Prmt1 (Chtop) is a vertebrate-specific chromatin-bound protein that plays an important role in transcriptional regulation. As its mechanism of action remains unclear, we identified Chtop-interacting proteins using a biotinylation-proteomics approach. Here we describe the identification and initial characterization of Five Friends of Methylated Chtop (5FMC). 5FMC is a nuclear complex that can only be recruited by Chtop when the latter is arginine-methylated by Prmt1. It consists of the co-activator Pelp1, the Sumo-specific protease Senp3, Wdr18, Tex10, and Las1L. Pelp1 functions as the core of 5FMC, as the other components become unstable in the absence of Pelp1. We show that recruitment of 5FMC to Zbp-89, a zinc-finger transcription factor, affects its sumoylation status and transactivation potential. Collectively, our data provide a mechanistic link between arginine methylation and (de)sumoylation in the control of transcriptional activity. PMID:22872859

  5. Molecular Targets of Chromatin Repressive Mark H3K9me3 in Primate Progenitor Cells within Adult Neurogenic Niches

    Directory of Open Access Journals (Sweden)

    Michael R Foret

    2014-07-01

    Full Text Available Histone 3 Lysine 9 (H3K9 methylation is known to be associated with pericentric heterochromatin and important in genomic stability. In this study, we show that trimethylation at H3K9 (H3K9me3 is enriched in an adult neural stem cell niche- the subventricular zone (SVZ on the walls of the lateral ventricle in both rodent and non-human primate baboon brain. Previous studies have shown that there is significant correlation between baboon and human regarding genomic similarity and brain structure, suggesting that findings in baboon are relevant to human. To understand the function of H3K9me3 in this adult neurogenic niche, we performed genome-wide analyses using ChIP-Seq (chromatin immunoprecipitation and deep-sequencing and RNA-Seq for in vivo SVZ cells purified from baboon brain. Through integrated analyses of ChIP-Seq and RNA-Seq, we found that H3K9me3-enriched genes associated with cellular maintenance, post-transcriptional and translational modifications, signaling pathways, and DNA replication are expressed, while genes involved in axon/neuron, hepatic stellate cell, or immune-response activation are not expressed. As neurogenesis progresses in the adult SVZ, cell fate restriction is essential to direct proper lineage commitment. Our findings highlight that H3K9me3 repression in undifferentiated SVZ cells is engaged in the maintenance of cell type integrity, implicating a role for H3K9me3 as an epigenetic mechanism to control cell fate transition within this adult germinal niche.

  6. Chromatin and alternative splicing.

    Science.gov (United States)

    Alló, M; Schor, I E; Muñoz, M J; de la Mata, M; Agirre, E; Valcárcel, J; Eyras, E; Kornblihtt, A R

    2010-01-01

    Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNA-mediated transcriptional gene silencing.

  7. Chromatin Remodelers: From Function to Dysfunction

    Directory of Open Access Journals (Sweden)

    Gernot Längst

    2015-06-01

    Full Text Available Chromatin remodelers are key players in the regulation of chromatin accessibility and nucleosome positioning on the eukaryotic DNA, thereby essential for all DNA dependent biological processes. Thus, it is not surprising that upon of deregulation of those molecular machines healthy cells can turn into cancerous cells. Even though the remodeling enzymes are very abundant and a multitude of different enzymes and chromatin remodeling complexes exist in the cell, the particular remodeling complex with its specific nucleosome positioning features must be at the right place at the right time in order to ensure the proper regulation of the DNA dependent processes. To achieve this, chromatin remodeling complexes harbor protein domains that specifically read chromatin targeting signals, such as histone modifications, DNA sequence/structure, non-coding RNAs, histone variants or DNA bound interacting proteins. Recent studies reveal the interaction between non-coding RNAs and chromatin remodeling complexes showing importance of RNA in remodeling enzyme targeting, scaffolding and regulation. In this review, we summarize current understanding of chromatin remodeling enzyme targeting to chromatin and their role in cancer development.

  8. Defeating EpCAM(+) liver cancer stem cells by targeting chromatin remodeling enzyme CHD4 in human hepatocellular carcinoma.

    Science.gov (United States)

    Nio, Kouki; Yamashita, Taro; Okada, Hikari; Kondo, Mitsumasa; Hayashi, Takehiro; Hara, Yasumasa; Nomura, Yoshimoto; Zeng, Sha Sha; Yoshida, Mariko; Hayashi, Tomoyuki; Sunagozaka, Hajime; Oishi, Naoki; Honda, Masao; Kaneko, Shuichi

    2015-11-01

    Hepatocellular carcinoma is composed of a subset of cells with enhanced tumorigenicity and chemoresistance that are called cancer stem (or stem-like) cells. We explored the role of chromodomain-helicase-DNA-binding protein 4, which is encoded by the CHD4 gene and is known to epigenetically control gene regulation and DNA damage responses in EpCAM(+) liver cancer stem cells. Gene and protein expression profiles were determined by microarray and immunohistochemistry in 245 and 144 hepatocellular carcinoma patients, respectively. The relationship between gene/protein expression and prognosis was examined. The functional role of CHD4 was evaluated in primary hepatocellular carcinoma cells and in cell lines in vitro and in vivo. CHD4 was abundantly expressed in EpCAM(+) hepatocellular carcinoma with expression of hepatic stem cell markers and poor prognosis in two independent cohorts. In cell lines, CHD4 knockdown increased chemosensitivity and CHD4 overexpression induced epirubicin chemoresistance. To inhibit the functions of CHD4 that are mediated through histone deacetylase and poly (ADP-ribose) polymerase, we evaluated the effect of the histone deacetylase inhibitor suberohydroxamic acid and the poly (ADP-ribose) polymerase inhibitor AG-014699. Treatment with either suberohydroxamic acid or AG-014699 reduced the number of EpCAM(+) liver cancer stem cells in vitro, and suberohydroxamic acid and AG-014699 in combination successfully inhibited tumor growth in a mouse xenograft model. CHD4 plays a pivotal role in chemoresistance and the maintenance of stemness in liver cancer stem cells and is therefore a good target for the eradication of hepatocellular carcinoma. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  9. Chromatin is wonderful stuff.

    NARCIS (Netherlands)

    van Driel, R.

    2007-01-01

    Chromatin molecules have properties that set them aside from all other biomacromolecules in the cell. (i) Chromosomes, which are single chromatin molecules, are the largest macromolecules in eukaryotic cells. (ii) Chromatin molecules carry the cell's genetic and epigenetic information and all

  10. Chromatin Structure and Function

    CERN Document Server

    Wolffe, Alan P

    1999-01-01

    The Third Edition of Chromatin: Structure and Function brings the reader up-to-date with the remarkable progress in chromatin research over the past three years. It has been extensively rewritten to cover new material on chromatin remodeling, histone modification, nuclear compartmentalization, DNA methylation, and transcriptional co-activators and co-repressors. The book is written in a clear and concise fashion, with 60 new illustrations. Chromatin: Structure and Function provides the reader with a concise and coherent account of the nature, structure, and assembly of chromatin and its active

  11. Cell- and stage-specific chromatin structure across the Complement receptor 2 (CR2/CD21) promoter coincide with CBF1 and C/EBP-beta binding in B cells.

    Science.gov (United States)

    Cruickshank, Mark N; Fenwick, Emily; Karimi, Mahdad; Abraham, Lawrence J; Ulgiati, Daniela

    2009-08-01

    Stringent developmental transcription requires multiple transcription factor (TF) binding sites, cell-specific expression of signaling molecules, TFs and co-regulators and appropriate chromatin structure. During B-lymphopoiesis, human Complement receptor 2 (CR2/CD21) is detected on immature and mature B cells but not on B cell precursors and plasma cells. We examined cell- and stage-specific human CR2 gene regulation using cell lines modeling B-lymphopoiesis. Chromatin accessibility assays revealed a region between -409 and -262 with enhanced accessibility in mature B cells and pre-B cells, compared to either non-lymphoid or plasma cell-types, however, accessibility near the transcription start site (TSS) was elevated only in CR2-expressing B cells. A correlation between histone acetylation and CR2 expression was observed, while histone H3K4 dimethylation was enriched near the TSS in both CR2-expressing B cells and non-expressing pre-B cells. Candidate sites within the CR2 promoter were identified which could regulate chromatin, including a matrix attachment region associated with CDP, SATB1/BRIGHT and CEBP-beta sites as well as two CBF1 sites. ChIP assays verified that both CBF1 and C/EBP-beta bind the CR2 promoter in B cells raising the possibility that these factors facilitate or respond to alterations in chromatin structure to control the timing and/or level of CR2 transcription.

  12. The physics of chromatin

    OpenAIRE

    Schiessel, Helmut

    2003-01-01

    Recent progress has been made in the understanding of the physical properties of chromatin -- the dense complex of DNA and histone proteins that occupies the nuclei of plant and animal cells. Here I will focus on the two lowest levels of the hierarchy of DNA folding into the chromatin complex: (i) the nucleosome, the chromatin repeating unit consisting of a globular aggregate of eight histone proteins with the DNA wrapped around: its overcharging, the DNA unwrapping transition, the ''sliding'...

  13. SUMO and Chromatin Remodeling.

    Science.gov (United States)

    Wotton, David; Pemberton, Lucy F; Merrill-Schools, Jacqueline

    2017-01-01

    Many of the known SUMO substrates are nuclear proteins, which regulate gene expression and chromatin dynamics. Sumoylation, in general, appears to correlate with decreased transcriptional activity, and in many cases modulation of the chromatin template is implicated. Sumoylation of the core histones is associated with transcriptional silencing, and transcription factor sumoylation can decrease gene expression by promoting recruitment of chromatin modifying enzymes. Additionally, sumoylation of transcriptional corepressors and chromatin remodeling enzymes can influence interactions with other transcriptional regulators, and alter their enzymatic activity. In some cases, proteins that are components of transcriptional corepressor complexes have been shown to be SUMO E3 ligases, further emphasizing the integration of sumoylation with the regulation of chromatin remodeling. Despite the evidence suggesting that sumoylation is primarily repressive for access to chromatin, recent analyses suggest that protein sumoylation on the chromatin template may play important roles at highly expressed genes. Elucidating the dynamic interplay of sumoylation with other post-translational modifications of histones and chromatin associated proteins will be key to fully understanding the regulation of access to the chromatin template.

  14. Lamin B receptor plays a role in stimulating nuclear envelope production and targeting membrane vesicles to chromatin during nuclear envelope assembly through direct interaction with importin beta.

    Science.gov (United States)

    Ma, Yan; Cai, Shang; Lv, Quanlong; Jiang, Qing; Zhang, Quan; Sodmergen; Zhai, Zhonghe; Zhang, Chuanmao

    2007-02-01

    Lamin B receptor (LBR), a chromatin and lamin binding protein in the inner nuclear membrane, has been proposed to play a vital role in nuclear envelope (NE) assembly. But the specific role for LBR in NE assembly remains unknown. In the present study, we show that overexpression of LBR causes membrane overproduction, inducing NE invagination and membrane stack formation, and that these processes require the transmembrane domain of LBR. Biochemical analysis shows that the N-terminal domain of LBR directly interacts with importin beta in a Ran sensitive and importin alpha independent manner. Using an in vitro NE assembly assay, we also demonstrate that blocking full length LBR binding sites on importin beta, by the addition of the LBR N-terminal domain inhibits the recruitment of LBR-containing vesicles to importin beta- or Ran-coated beads to form NE structure. Our results suggest that LBR is recruited to chromatin through direct interaction with importin beta to contribute to the fusion of membrane vesicles and formation of the NE.

  15. Identification of Pou5f1, Sox2, and Nanog downstream target genes with statistical confidence by applying a novel algorithm to time course microarray and genome-wide chromatin immunoprecipitation data

    Directory of Open Access Journals (Sweden)

    Xin Li

    2008-06-01

    Full Text Available Abstract Background Target genes of a transcription factor (TF Pou5f1 (Oct3/4 or Oct4, which is essential for pluripotency maintenance and self-renewal of embryonic stem (ES cells, have previously been identified based on their response to Pou5f1 manipulation and occurrence of Chromatin-immunoprecipitation (ChIP-binding sites in promoters. However, many responding genes with binding sites may not be direct targets because response may be mediated by other genes and ChIP-binding site may not be functional in terms of transcription regulation. Results To reduce the number of false positives, we propose to separate responding genes into groups according to direction, magnitude, and time of response, and to apply the false discovery rate (FDR criterion to each group individually. Using this novel algorithm with stringent statistical criteria (FDR Pou5f1 suppression and published ChIP data, we identified 420 tentative target genes (TTGs for Pou5f1. The majority of TTGs (372 were down-regulated after Pou5f1 suppression, indicating that the Pou5f1 functions as an activator of gene expression when it binds to promoters. Interestingly, many activated genes are potent suppressors of transcription, which include polycomb genes, zinc finger TFs, chromatin remodeling factors, and suppressors of signaling. Similar analysis showed that Sox2 and Nanog also function mostly as transcription activators in cooperation with Pou5f1. Conclusion We have identified the most reliable sets of direct target genes for key pluripotency genes – Pou5f1, Sox2, and Nanog, and found that they predominantly function as activators of downstream gene expression. Thus, most genes related to cell differentiation are suppressed indirectly.

  16. An injected bacterial effector targets chromatin access for transcription factor NF-kappaB to alter transcription of host genes involved in immune responses.

    Science.gov (United States)

    Arbibe, Laurence; Kim, Dong Wook; Batsche, Eric; Pedron, Thierry; Mateescu, Bogdan; Muchardt, Christian; Parsot, Claude; Sansonetti, Philippe J

    2007-01-01

    Phosphorylation of histone H3 at Ser10 increases chromatin accessibility to transcription factor NF-kappaB on a subset of genes involved in immune responses. Here we report that a bacterial pathogen abrogated phosphorylation of histone H3 to 'shape' the transcriptional responses of infected host cells. We identify the Shigella flexneri protein effector OspF as a dually specific phosphatase that dephosphorylated mitogen-activated protein kinases in the nucleus, thus preventing histone H3 phosphorylation at Ser10 in a gene-specific way. That activity of OspF enabled shigella to block the activation of a subset of NF-kappaB-responsive genes, leading to compromised recruitment of polymorphonuclear leukocytes to infected tissues. S. flexneri has thus evolved the capacity to precisely modulate host cell epigenetic 'information' as a strategy for repressing innate immunity.

  17. Capitalizing on disaster: Establishing chromatin specificity behind the replication fork.

    Science.gov (United States)

    Ramachandran, Srinivas; Ahmad, Kami; Henikoff, Steven

    2017-04-01

    Eukaryotic genomes are packaged into nucleosomal chromatin, and genomic activity requires the precise localization of transcription factors, histone modifications and nucleosomes. Classic work described the progressive reassembly and maturation of bulk chromatin behind replication forks. More recent proteomics has detailed the molecular machines that accompany the replicative polymerase to promote rapid histone deposition onto the newly replicated DNA. However, localized chromatin features are transiently obliterated by DNA replication every S phase of the cell cycle. Genomic strategies now observe the rebuilding of locus-specific chromatin features, and reveal surprising delays in transcription factor binding behind replication forks. This implies that transient chromatin disorganization during replication is a central juncture for targeted transcription factor binding within genomes. We propose that transient occlusion of regulatory elements by disorganized nucleosomes during chromatin maturation enforces specificity of factor binding. © 2017 WILEY Periodicals, Inc.

  18. The Fun30 chromatin remodeler Fft3 controls nuclear organization and chromatin structure of insulators and subtelomeres in fission yeast.

    Directory of Open Access Journals (Sweden)

    Babett Steglich

    2015-03-01

    Full Text Available In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3∆ cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR elements and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and the nuclear envelope.

  19. Chromatin replication and epigenome maintenance

    DEFF Research Database (Denmark)

    Alabert, Constance; Groth, Anja

    2012-01-01

    initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA...

  20. Chromatin replication and histone dynamics

    DEFF Research Database (Denmark)

    Alabert, Constance; Jasencakova, Zuzana; Groth, Anja

    2017-01-01

    organization into chromatin. We reveal how specialized replication-coupled mechanisms rapidly assemble newly synthesized DNA into nucleosomes, while the complete restoration of chromatin organization including histone marks is a continuous process taking place throughout the cell cycle. Because failure...

  1. Chromatin dynamics in plants

    NARCIS (Netherlands)

    Fransz, P.F.; Jong, de J.H.

    2002-01-01

    Recent studies in yeast, animals and plants have provided major breakthroughs in unraveling the molecular mechanism of higher-order gene regulation. In conjunction with the DNA code, proteins that are involved in chromatin remodeling, histone modification and epigenetic imprinting form a large

  2. Reprogramming the chromatin landscape

    DEFF Research Database (Denmark)

    Miranda, Tina B; Voss, Ty C; Sung, Myong-Hee

    2013-01-01

    , mechanistic details defining the cellular interactions between ER and GR are poorly understood. We investigated genome-wide binding profiles for ER and GR upon coactivation and characterized the status of the chromatin landscape. We describe a novel mechanism dictating the molecular interplay between ER...

  3. Analysis of Chromatin Organisation

    Science.gov (United States)

    Szeberenyi, Jozsef

    2011-01-01

    Terms to be familiar with before you start to solve the test: chromatin, nucleases, sucrose density gradient centrifugation, melting point, gel electrophoresis, ethidium bromide, autoradiography, Southern blotting, Northern blotting, Sanger sequencing, restriction endonucleases, exonucleases, linker DNA, chloroform extraction, nucleosomes,…

  4. Proteomic interrogation of human chromatin.

    Directory of Open Access Journals (Sweden)

    Mariana P Torrente

    Full Text Available Chromatin proteins provide a scaffold for DNA packaging and a basis for epigenetic regulation and genomic maintenance. Despite understanding its functional roles, mapping the chromatin proteome (i.e. the "Chromatome" is still a continuing process. Here, we assess the biological specificity and proteomic extent of three distinct chromatin preparations by identifying proteins in selected chromatin-enriched fractions using mass spectrometry-based proteomics. These experiments allowed us to produce a chromatin catalog, including several proteins ranging from highly abundant histone proteins to less abundant members of different chromatin machinery complexes. Using a Normalized Spectral Abundance Factor approach, we quantified relative abundances of the proteins across the chromatin enriched fractions giving a glimpse into their chromosomal abundance. The large-scale data sets also allowed for the discovery of a variety of novel post-translational modifications on the identified chromatin proteins. With these comparisons, we find one of the probed methods to be qualitatively superior in specificity for chromatin proteins, but inferior in proteomic extent, evidencing a compromise that must be made between biological specificity and broadness of characterization. Additionally, we attempt to identify proteins in eu- and heterochromatin, verifying the enrichments by characterizing the post-translational modifications detected on histone proteins from these chromatin regions. In summary, our results provide insights into the value of different methods to extract chromatin-associated proteins and provide starting points to study the factors that may be involved in directing gene expression and other chromatin-related processes.

  5. HAMLET interacts with histones and chromatin in tumor cell nuclei.

    Science.gov (United States)

    Düringer, Caroline; Hamiche, Ali; Gustafsson, Lotta; Kimura, Hiroshi; Svanborg, Catharina

    2003-10-24

    HAMLET is a folding variant of human alpha-lactalbumin in an active complex with oleic acid. HAMLET selectively enters tumor cells, accumulates in their nuclei and induces apoptosis-like cell death. This study examined the interactions of HAMLET with nuclear constituents and identified histones as targets. HAMLET was found to bind histone H3 strongly and to lesser extent histones H4 and H2B. The specificity of these interactions was confirmed using BIAcore technology and chromatin assembly assays. In vivo in tumor cells, HAMLET co-localized with histones and perturbed the chromatin structure; HAMLET was found associated with chromatin in an insoluble nuclear fraction resistant to salt extraction. In vitro, HAMLET bound strongly to histones and impaired their deposition on DNA. We conclude that HAMLET interacts with histones and chromatin in tumor cell nuclei and propose that this interaction locks the cells into the death pathway by irreversibly disrupting chromatin organization.

  6. The AID-induced DNA damage response in chromatin

    DEFF Research Database (Denmark)

    Daniel, Jeremy A; Nussenzweig, André

    2013-01-01

    Chemical modifications to the DNA and histone protein components of chromatin can modulate gene expression and genome stability. Understanding the physiological impact of changes in chromatin structure remains an important question in biology. As one example, in order to generate antibody diversity...... with somatic hypermutation and class switch recombination, chromatin must be made accessible for activation-induced cytidine deaminase (AID)-mediated deamination of cytosines in DNA. These lesions are recognized and removed by various DNA repair pathways but, if not handled properly, can lead to formation...... of oncogenic chromosomal translocations. In this review, we focus the discussion on how chromatin-modifying activities and -binding proteins contribute to the native chromatin environment in which AID-induced DNA damage is targeted and repaired. Outstanding questions remain regarding the direct roles...

  7. High resolution Chromatin Immunoprecipitation (ChIP) sequencing reveals novel bindings targets and prognostic role for SOX11 in Mantle cell lymphoma

    Science.gov (United States)

    Kuo, Pei-Yu; Leshchenko, Violetta V.; Fazzari, Melissa J.; Perumal, Deepak; Gellen, Tobias; He, Tianfang; Iqbal, Javeed; Baumgartner-Wennerholm, Stefanie; Nygren, Lina; Zhang, Fan; Zhang, Weijia; Suh, K. Stephen; Goy, Andre; Yang, David T.; Chan, Wing-Chung; Kahl, Brad S.; Verma, Amit K.; Gascoyne, Randy D.; Kimby, Eva; Sander, Birgitta; Ye, B. Hilda; Melnick, Ari M.; Parekh, Samir

    2015-01-01

    SOX11 (Sex determining region Y-box 11) expression is specific for MCL as compared to other Non-Hodgkin's lymphomas. However, the function and direct binding targets of SOX11 in MCL are largely unknown. We used high-resolution ChIP-Seq to identify the direct target genes of SOX11 in a genome-wide, unbiased manner and elucidate its functional significance. Pathway analysis identified WNT, PKA and TGF-beta signaling pathways as significantly enriched by SOX11 target genes. qCHIP and promoter reporter assays confirmed that SOX11 directly binds to individual genes and modulates their transcription activities in these pathways in MCL. Functional studies using RNA interference demonstrate that SOX11 directly regulates WNT in MCL. We analyzed SOX11 expression in three independent well-annotated tissue microarrays from the University of Wisconsin (UW), Karolinska Institute and British Columbia Cancer Agency (BCCA). Our findings suggest that high SOX11 expression is associated with improved survival in a subset of MCL patients, particularly those treated with intensive chemotherapy. Transcriptional regulation of WNT and other biological pathways affected by SOX11 target genes may help explain the impact of SOX11 expression on patient outcomes. PMID:24681958

  8. Analysis of Chromatin Dynamics during Glucocorticoid Receptor Activation

    Science.gov (United States)

    Burd, Craig J.; Ward, James M.; Crusselle-Davis, Valerie J.; Kissling, Grace E.; Phadke, Dhiral; Shah, Ruchir R.

    2012-01-01

    Steroid hormone receptors initiate a genetic program tightly regulated by the chromatin environment of the responsive regions. Using the glucocorticoid receptor (GR) as a model factor for transcriptional initiation, we classified chromatin structure through formaldehyde-assisted isolation of regulatory elements (FAIRE). We looked at dynamic changes in FAIRE signals during GR activation specifically at regions of receptor interaction. We found a distribution of GR-responsive regions with diverse responses to activation and chromatin modulation. The majority of GR binding regions demonstrate increases in FAIRE signal in response to ligand. However, the majority GR-responsive regions shared a similar FAIRE signal in the basal chromatin state, suggesting a common chromatin structure for GR recruitment. Supporting this notion, global FAIRE sequencing (seq) data indicated an enrichment of signal surrounding the GR binding site prior to activation. Brg-1 knockdown showed response element-specific effects of ATPase-dependent chromatin remodeling. FAIRE induction was universally decreased by Brg-1 depletion, but to varying degrees in a target specific manner. Taken together, these data suggest classes of nuclear receptor response regions that react to activation through different chromatin regulatory events and identify a chromatin structure that classifies the majority of response elements tested. PMID:22451486

  9. The nucleosome: orchestrating DNA damage signaling and repair within chromatin.

    Science.gov (United States)

    Agarwal, Poonam; Miller, Kyle M

    2016-10-01

    DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling events that extensively modulates chromatin structure and organization to coordinate DDR factor recruitment to the break and repair, whilst also promoting the maintenance of normal chromatin functions within the damaged region. For example, DDR pathways must avoid conflicts between other DNA-based processes that function within the context of chromatin, including transcription and replication. The molecular mechanisms governing the recognition, target specificity, and recruitment of DDR factors and enzymes to the fundamental repeating unit of chromatin, i.e., the nucleosome, are poorly understood. Here we present our current view of how chromatin recognition by DDR factors is achieved at the level of the nucleosome. Emerging evidence suggests that the nucleosome surface, including the nucleosome acidic patch, promotes the binding and activity of several DNA damage factors on chromatin. Thus, in addition to interactions with damaged DNA and histone modifications, nucleosome recognition by DDR factors plays a key role in orchestrating the requisite chromatin response to maintain both genome and epigenome integrity.

  10. Probing Chromatin-modifying Enzymes with Chemical Tools

    KAUST Repository

    Fischle, Wolfgang

    2016-02-04

    Chromatin is the universal template of genetic information in all eukaryotic organisms. Chemical modifications of the DNA-packaging histone proteins and the DNA bases are crucial signaling events in directing the use and readout of eukaryotic genomes. The enzymes that install and remove these chromatin modifications as well as the proteins that bind these marks govern information that goes beyond the sequence of DNA. Therefore, these so-called epigenetic regulators are intensively studied and represent promising drug targets in modern medicine. We summarize and discuss recent advances in the field of chemical biology that have provided chromatin research with sophisticated tools for investigating the composition, activity, and target sites of chromatin modifying enzymes and reader proteins.

  11. Chromatin dynamics in genome stability

    DEFF Research Database (Denmark)

    Nair, Nidhi; Shoaib, Muhammad; Sørensen, Claus Storgaard

    2017-01-01

    Genomic DNA is compacted into chromatin through packaging with histone and non-histone proteins. Importantly, DNA accessibility is dynamically regulated to ensure genome stability. This is exemplified in the response to DNA damage where chromatin relaxation near genomic lesions serves to promote ...... of chromatin structure regulation in maintaining genome integrity by multiple mechanisms including facilitating DNA repair and directly suppressing endogenous DNA damage.......Genomic DNA is compacted into chromatin through packaging with histone and non-histone proteins. Importantly, DNA accessibility is dynamically regulated to ensure genome stability. This is exemplified in the response to DNA damage where chromatin relaxation near genomic lesions serves to promote...... access of relevant enzymes to specific DNA regions for signaling and repair. Furthermore, recent data highlight genome maintenance roles of chromatin through the regulation of endogenous DNA-templated processes including transcription and replication. Here, we review research that shows the importance...

  12. A SWI/SNF Chromatin Remodelling Protein Controls Cytokinin Production through the Regulation of Chromatin Architecture

    KAUST Repository

    Jégu, Teddy

    2015-10-12

    Chromatin architecture determines transcriptional accessibility to DNA and consequently gene expression levels in response to developmental and environmental stimuli. Recently, chromatin remodelers such as SWI/SNF complexes have been recognized as key regulators of chromatin architecture. To gain insight into the function of these complexes during root development, we have analyzed Arabidopsis knock-down lines for one sub-unit of SWI/SNF complexes: BAF60. Here, we show that BAF60 is a positive regulator of root development and cell cycle progression in the root meristem via its ability to down-regulate cytokinin production. By opposing both the deposition of active histone marks and the formation of a chromatin regulatory loop, BAF60 negatively regulates two crucial target genes for cytokinin biosynthesis (IPT3 and IPT7) and one cell cycle inhibitor (KRP7). Our results demonstrate that SWI/SNF complexes containing BAF60 are key factors governing the equilibrium between formation and dissociation of a chromatin loop controlling phytohormone production and cell cycle progression.

  13. Chromatinization of the KSHV Genome During the KSHV Life Cycle

    Energy Technology Data Exchange (ETDEWEB)

    Uppal, Timsy [Department of Microbiology and Immunology, School of Medicine, University of Nevada, 1664 N Virginia Street, MS 320, Reno, NV 89557 (United States); Jha, Hem C. [Department of Microbiology and the Tumor Virology Program of the Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, 201E Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104 (United States); Verma, Subhash C. [Department of Microbiology and Immunology, School of Medicine, University of Nevada, 1664 N Virginia Street, MS 320, Reno, NV 89557 (United States); Robertson, Erle S., E-mail: erle@mail.med.upenn.edu [Department of Microbiology and the Tumor Virology Program of the Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, 201E Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104 (United States)

    2015-01-14

    Kaposi’s sarcoma-associated herpesvirus (KSHV) belongs to the gamma herpesvirus family and is the causative agent of various lymphoproliferative diseases in humans. KSHV, like other herpesviruses, establishes life-long latent infection with the expression of a limited number of viral genes. Expression of these genes is tightly regulated by both the viral and cellular factors. Recent advancements in identifying the expression profiles of viral transcripts, using tilling arrays and next generation sequencing have identified additional coding and non-coding transcripts in the KSHV genome. Determining the functions of these transcripts will provide a better understanding of the mechanisms utilized by KSHV in altering cellular pathways involved in promoting cell growth and tumorigenesis. Replication of the viral genome is critical in maintaining the existing copies of the viral episomes during both latent and lytic phases of the viral life cycle. The replication of the viral episome is facilitated by viral components responsible for recruiting chromatin modifying enzymes and replication factors for altering the chromatin complexity and replication initiation functions, respectively. Importantly, chromatin modification of the viral genome plays a crucial role in determining whether the viral genome will persist as latent episome or undergo lytic reactivation. Additionally, chromatinization of the incoming virion DNA, which lacks chromatin structure, in the target cells during primary infection, helps in establishing latent infection. Here, we discuss the recent advancements on our understating of KSHV genome chromatinization and the consequences of chromatin modifications on viral life cycle.

  14. Chromatinization of the KSHV Genome During the KSHV Life Cycle

    Directory of Open Access Journals (Sweden)

    Timsy Uppal

    2015-01-01

    Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV belongs to the gamma herpesvirus family and is the causative agent of various lymphoproliferative diseases in humans. KSHV, like other herpesviruses, establishes life-long latent infection with the expression of a limited number of viral genes. Expression of these genes is tightly regulated by both the viral and cellular factors. Recent advancements in identifying the expression profiles of viral transcripts, using tilling arrays and next generation sequencing have identified additional coding and non-coding transcripts in the KSHV genome. Determining the functions of these transcripts will provide a better understanding of the mechanisms utilized by KSHV in altering cellular pathways involved in promoting cell growth and tumorigenesis. Replication of the viral genome is critical in maintaining the existing copies of the viral episomes during both latent and lytic phases of the viral life cycle. The replication of the viral episome is facilitated by viral components responsible for recruiting chromatin modifying enzymes and replication factors for altering the chromatin complexity and replication initiation functions, respectively. Importantly, chromatin modification of the viral genome plays a crucial role in determining whether the viral genome will persist as latent episome or undergo lytic reactivation. Additionally, chromatinization of the incoming virion DNA, which lacks chromatin structure, in the target cells during primary infection, helps in establishing latent infection. Here, we discuss the recent advancements on our understating of KSHV genome chromatinization and the consequences of chromatin modifications on viral life cycle.

  15. Rapid and reversible epigenome editing by endogenous chromatin regulators.

    Science.gov (United States)

    Braun, Simon M G; Kirkland, Jacob G; Chory, Emma J; Husmann, Dylan; Calarco, Joseph P; Crabtree, Gerald R

    2017-09-15

    Understanding the causal link between epigenetic marks and gene regulation remains a central question in chromatin biology. To edit the epigenome we developed the FIRE-Cas9 system for rapid and reversible recruitment of endogenous chromatin regulators to specific genomic loci. We enhanced the dCas9-MS2 anchor for genome targeting with Fkbp/Frb dimerizing fusion proteins to allow chemical-induced proximity of a desired chromatin regulator. We find that mSWI/SNF (BAF) complex recruitment is sufficient to oppose Polycomb within minutes, leading to activation of bivalent gene transcription in mouse embryonic stem cells. Furthermore, Hp1/Suv39h1 heterochromatin complex recruitment to active promoters deposits H3K9me3 domains, resulting in gene silencing that can be reversed upon washout of the chemical dimerizer. This inducible recruitment strategy provides precise kinetic information to model epigenetic memory and plasticity. It is broadly applicable to mechanistic studies of chromatin in mammalian cells and is particularly suited to the analysis of endogenous multi-subunit chromatin regulator complexes.Understanding the link between epigenetic marks and gene regulation requires the development of new tools to directly manipulate chromatin. Here the authors demonstrate a Cas9-based system to recruit chromatin remodelers to loci of interest, allowing rapid, reversible manipulation of epigenetic states.

  16. Optical tweezers stretching of chromatin

    NARCIS (Netherlands)

    Pope, L.H.; Bennink, Martin L.; Greve, Jan

    2003-01-01

    Recently significant success has emerged from exciting research involving chromatin stretching using optical tweezers. These experiments, in which a single chromatin fibre is attached by one end to a micron-sized bead held in an optical trap and to a solid surface or second bead via the other end,

  17. Epigenetics & chromatin: Interactions and processes

    NARCIS (Netherlands)

    S. Henikoff (Steven); F.G. Grosveld (Frank)

    2013-01-01

    textabstractOn 11 to 13 March 2013, BioMed Central will be hosting its inaugural conference, Epigenetics & Chromatin: Interactions and Processes, at Harvard Medical School, Cambridge, MA, USA. Epigenetics & Chromatin has now launched a special article series based on the general themes of the

  18. Chromatin condensation during terminal erythropoiesis.

    Science.gov (United States)

    Zhao, Baobing; Yang, Jing; Ji, Peng

    2016-09-02

    Mammalian terminal erythropoiesis involves gradual but dramatic chromatin condensation steps that are essential for cell differentiation. Chromatin and nuclear condensation is followed by a unique enucleation process, which is believed to liberate more spaces for hemoglobin enrichment and enable the generation of a physically flexible mature red blood cell. Although these processes have been known for decades, the mechanisms are still unclear. Our recent study reveals an unexpected nuclear opening formation during mouse terminal erythropoiesis that requires caspase-3 activity. Major histones, except H2AZ, are partially released from the opening, which is important for chromatin condensation. Block of the nuclear opening through caspase inhibitor or knockdown of caspase-3 inhibits chromatin condensation and enucleation. We also demonstrate that nuclear opening and histone release are cell cycle regulated. These studies reveal a novel mechanism for chromatin condensation in mammalia terminal erythropoiesis.

  19. HACking the centromere chromatin code: insights from human artificial chromosomes.

    Science.gov (United States)

    Bergmann, Jan H; Martins, Nuno M C; Larionov, Vladimir; Masumoto, Hiroshi; Earnshaw, William C

    2012-07-01

    The centromere is a specialized chromosomal region that serves as the assembly site of the kinetochore. At the centromere, CENP-A nucleosomes form part of a chromatin landscape termed centrochromatin. This chromatin environment conveys epigenetic marks regulating kinetochore formation. Recent work sheds light on the intricate relationship between centrochromatin state, the CENP-A assembly pathway and the maintenance of centromere function. Here, we review the emerging picture of how chromatin affects mammalian kinetochore formation. We place particular emphasis on data obtained from Human Artificial Chromosome (HAC) biology and the targeted engineering of centrochromatin using synthetic HACs. We discuss implications of these findings, which indicate that a delicate balance of histone modifications and chromatin state dictates both de novo centromere formation and the maintenance of centromere identity in dividing cell populations.

  20. Chromatin remodeling in plant development.

    Science.gov (United States)

    Jarillo, José A; Piñeiro, Manuel; Cubas, Pilar; Martínez-Zapater, José M

    2009-01-01

    Plant development results from specific patterns of gene expression that are tightly regulated in a spatio-temporal manner. Chromatin remodeling plays a central role in establishing these expression patterns and maintaining epigenetic transcriptional states through successive rounds of mitosis that take place within a cell lineage. Plant epigenetic switches occur not only at the embryo stage, but also during postembryonic developmental transitions, suggesting that chromatin remodeling activities in plants can provide a higher degree of regulatory flexibility which probably underlies their developmental plasticity. Here, we highlight recent progress in the understanding of plant chromatin dynamic organization, facilitating the activation or repression of specific sets of genes involved in different developmental programs and integrating them with the response to environmental signals. Chromatin conformation controls gene expression both in actively dividing undifferentiated cells and in those already fate-determined. In this context, we first describe chromatin reorganization activities required to maintain meristem function stable through DNA replication and cell division. Organ initiation at the apex, with emphasis on reproductive development, is next discussed to uncover the chromatin events involved in the establishment and maintenance of expression patterns associated with differentiating cells; this is illustrated with the complex epigenetic regulation of the Arabidopsis floral repressor FLOWERING LOCUS C (FLC). Finally, we discuss the involvement of chromatin remodeling in plant responses to environmental cues and to different types of stress conditions.

  1. Chromatin and Transcription in Yeast

    Science.gov (United States)

    Rando, Oliver J.; Winston, Fred

    2012-01-01

    Understanding the mechanisms by which chromatin structure controls eukaryotic transcription has been an intense area of investigation for the past 25 years. Many of the key discoveries that created the foundation for this field came from studies of Saccharomyces cerevisiae, including the discovery of the role of chromatin in transcriptional silencing, as well as the discovery of chromatin-remodeling factors and histone modification activities. Since that time, studies in yeast have continued to contribute in leading ways. This review article summarizes the large body of yeast studies in this field. PMID:22345607

  2. Cytoplasmic chromatin triggers inflammation in senescence and cancer.

    Science.gov (United States)

    Dou, Zhixun; Ghosh, Kanad; Vizioli, Maria Grazia; Zhu, Jiajun; Sen, Payel; Wangensteen, Kirk J; Simithy, Johayra; Lan, Yemin; Lin, Yanping; Zhou, Zhuo; Capell, Brian C; Xu, Caiyue; Xu, Mingang; Kieckhaefer, Julia E; Jiang, Tianying; Shoshkes-Carmel, Michal; Tanim, K M Ahasan Al; Barber, Glen N; Seykora, John T; Millar, Sarah E; Kaestner, Klaus H; Garcia, Benjamin A; Adams, Peter D; Berger, Shelley L

    2017-10-19

    Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

  3. Can chromatin conformation technologies bring light into human molecular pathology?

    Science.gov (United States)

    Kubiak, Marta; Lewandowska, Marzena Anna

    2015-01-01

    Regulation of gene expression in eukaryotes involves many complex processes, in which chromatin structure plays an important role. In addition to the epigenetic effects, such as DNA methylation and phosphorylation or histone modifications, gene expression is also controlled by the spatial organization of chromatin. For example, distant regulatory elements (enhancers, insulators) may come into direct physical interaction with target genes or other regulatory elements located in genomic regions of up to several hundred kilobases in size. Such long-range interactions result in the formation of chromatin loops. In the last several years, there has been a rapid increase in our knowledge of the spatial organization of chromatin in the nucleus through the chromosome conformation capture (3C) technology. Here we review and compare the original 3C and 3C-based methods including chromosome conformation capture-on-chip (4C), chromosome conformation capture carbon copy (5C), hi-resolution chromosome confomation capture (HiC). In this article, we discuss different aspects of how the nuclear organization of chromatin is associated with gene expression regulation and how this knowledge is useful in translational medicine and clinical applications. We demonstrate that the knowledge of the chromatin 3D organization may help understand the mechanisms of gene expression regulation of genes involved in the development of human diseases, such as CFTR (responsible for cystic fibrosis) or IGFBP3 (associated with breast cancer pathogenesis). Additionally, 3C-derivative methods have been also useful in the diagnosis of some leukemia subtypes.

  4. Brain Function and Chromatin Plasticity

    OpenAIRE

    Dulac, Catherine

    2010-01-01

    The characteristics of epigenetic control, including the potential for long-lasting, stable effects on gene expression that outlive an initial transient signal, could be of singular importance for post-mitotic neurons, which are subject to changes with short- to long-lasting influence on their activity and connectivity. Persistent changes in chromatin structure are thought to contribute to mechanisms of epigenetic inheritance. Recent advances in chromatin biology offer new avenues to investig...

  5. A chromatin insulator driving three-dimensional Polycomb response element (PRE) contacts and Polycomb association with the chromatin fiber

    Science.gov (United States)

    Comet, Itys; Schuettengruber, Bernd; Sexton, Tom; Cavalli, Giacomo

    2011-01-01

    Regulation of gene expression involves long-distance communication between regulatory elements and target promoters, but how this is achieved remains unknown. Insulator elements have been proposed to modulate the communication between regulatory elements and promoters due to their ability to insulate genes from regulatory elements or to take part in long-distance interactions. Using a high-resolution chromatin conformation capture (H3C) method, we show that the Drosophila gypsy insulator behaves as a conformational chromatin border that is able to prohibit contacts between a Polycomb response element (PRE) and a distal promoter. On the other hand, two spaced gypsy elements form a chromatin loop that is able to bring an upstream PRE in contact with a downstream gene to mediate its repression. Chromatin immunoprecipitation (ChIP) profiles of the Polycomb protein and its associated H3K27me3 histone mark reflect this insulator-dependent chromatin conformation, suggesting that Polycomb action at a distance can be organized by local chromatin topology. PMID:21262819

  6. Chromatin Architecture Defines the Glucocorticoid Response

    Science.gov (United States)

    Burd, Craig J.; Archer, Trevor K.

    2013-01-01

    The glucocorticoid receptor (GR) functions to regulate a wide group of physiological processes through hormone inducible interaction with genomic loci and subsequent manipulation of the transcriptional output of target genes. Despite expression in a wide variety of tissues, the GR has diverse roles that are regulated tightly in a cell type specific manner. With the advent of whole genome approaches, the details of that diversity and the mechanisms regulating them are beginning to be elucidated. This review aims describe the recent advances detailing the role chromatin structure plays in dictating GR specificity. PMID:23545159

  7. LINE-1 activation after fertilization regulates global chromatin accessibility in the early mouse embryo.

    Science.gov (United States)

    Jachowicz, Joanna W; Bing, Xinyang; Pontabry, Julien; Bošković, Ana; Rando, Oliver J; Torres-Padilla, Maria-Elena

    2017-10-01

    After fertilization, to initiate development, gametes are reprogramed to become totipotent. Approximately half of the mammalian genome consists of repetitive elements, including retrotransposons, some of which are transcribed after fertilization. Retrotransposon activation is generally assumed to be a side effect of the extensive chromatin remodeling underlying the epigenetic reprogramming of gametes. Here, we used a targeted epigenomic approach to address whether specific retrotransposon families play a direct role in chromatin organization and developmental progression. We demonstrate that premature silencing of LINE-1 elements decreases chromatin accessibility, whereas prolonged activation prevents the gradual chromatin compaction that occurs naturally in developmental progression. Preventing LINE-1 activation and interfering with its silencing decreases developmental rates independently of the coding nature of the LINE-1 transcript, thus suggesting that LINE-1 functions primarily at the chromatin level. Our data suggest that activation of LINE-1 regulates global chromatin accessibility at the beginning of development and indicate that retrotransposon activation is integral to the developmental program.

  8. Chromatin challenges during DNA replication and repair

    DEFF Research Database (Denmark)

    Groth, Anja; Rocha, Walter; Verreault, Alain

    2007-01-01

    Inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. To orchestrate DNA-replication and -repair processes in the context of chromatin is a challenge, both in terms of accessibility and maintenance of chromatin organization. To meet...... the challenge of maintenance, cells have evolved efficient nucleosome-assembly pathways and chromatin-maturation mechanisms that reproduce chromatin organization in the wake of DNA replication and repair. The aim of this Review is to describe how these pathways operate and to highlight how the epigenetic...... landscape may be stably maintained even in the face of dramatic changes in chromatin structure....

  9. High-resolution, genome-wide mapping of chromatin modifications by GMAT.

    Science.gov (United States)

    Roh, Tae-Young; Zhao, Keji

    2008-01-01

    One major postgenomic challenge is to characterize the epigenomes that control genome functions. The epigenomes are mainly defined by the specific association of nonhistone proteins with chromatin and the covalent modifications of chromatin, including DNA methylation and posttranslational histone modifications. The in vivo protein-binding and chromatin-modification patterns can be revealed by the chromatin immunoprecipitation assay (ChIP). By combining the ChIP assays and the serial analysis of gene expression (SAGE) protocols, we have developed an unbiased and high-resolution genome-wide mapping technique (GMAT) to determine the genome-wide protein-targeting and chromatin-modification patterns. GMAT has been successfully applied to mapping the target sites of the histone acetyltransferase, Gcn5p, in yeast and to the discovery of the histone acetylation islands as an epigenetic mark for functional regulatory elements in the human genome.

  10. Chromatin remodeling in mammalian embryos.

    Science.gov (United States)

    Cabot, Birgit; Cabot, Ryan A

    2018-03-01

    The mammalian embryo undergoes a dramatic amount of epigenetic remodeling during the first week of development. In this review, we discuss several epigenetic changes that happen over the course of cleavage development, focusing on covalent marks (e.g., histone methylation and acetylation) and non-covalent remodeling (chromatin remodeling via remodeling complexes; e.g., SWI/SNF-mediated chromatin remodeling). Comparisons are also drawn between remodeling events that occur in embryos from a variety of mammalian species. © 2018 Society for Reproduction and Fertility.

  11. Global quantitative modeling of chromatin factor interactions.

    Directory of Open Access Journals (Sweden)

    Jian Zhou

    2014-03-01

    Full Text Available Chromatin is the driver of gene regulation, yet understanding the molecular interactions underlying chromatin factor combinatorial patterns (or the "chromatin codes" remains a fundamental challenge in chromatin biology. Here we developed a global modeling framework that leverages chromatin profiling data to produce a systems-level view of the macromolecular complex of chromatin. Our model ultilizes maximum entropy modeling with regularization-based structure learning to statistically dissect dependencies between chromatin factors and produce an accurate probability distribution of chromatin code. Our unsupervised quantitative model, trained on genome-wide chromatin profiles of 73 histone marks and chromatin proteins from modENCODE, enabled making various data-driven inferences about chromatin profiles and interactions. We provided a highly accurate predictor of chromatin factor pairwise interactions validated by known experimental evidence, and for the first time enabled higher-order interaction prediction. Our predictions can thus help guide future experimental studies. The model can also serve as an inference engine for predicting unknown chromatin profiles--we demonstrated that with this approach we can leverage data from well-characterized cell types to help understand less-studied cell type or conditions.

  12. Global Quantitative Modeling of Chromatin Factor Interactions

    Science.gov (United States)

    Zhou, Jian; Troyanskaya, Olga G.

    2014-01-01

    Chromatin is the driver of gene regulation, yet understanding the molecular interactions underlying chromatin factor combinatorial patterns (or the “chromatin codes”) remains a fundamental challenge in chromatin biology. Here we developed a global modeling framework that leverages chromatin profiling data to produce a systems-level view of the macromolecular complex of chromatin. Our model ultilizes maximum entropy modeling with regularization-based structure learning to statistically dissect dependencies between chromatin factors and produce an accurate probability distribution of chromatin code. Our unsupervised quantitative model, trained on genome-wide chromatin profiles of 73 histone marks and chromatin proteins from modENCODE, enabled making various data-driven inferences about chromatin profiles and interactions. We provided a highly accurate predictor of chromatin factor pairwise interactions validated by known experimental evidence, and for the first time enabled higher-order interaction prediction. Our predictions can thus help guide future experimental studies. The model can also serve as an inference engine for predicting unknown chromatin profiles — we demonstrated that with this approach we can leverage data from well-characterized cell types to help understand less-studied cell type or conditions. PMID:24675896

  13. Chromatin states modify network motifs contributing to cell-specific functions

    Science.gov (United States)

    Zhao, Hongying; Liu, Tingting; Liu, Ling; Zhang, Guanxiong; Pang, Lin; Yu, Fulong; Fan, Huihui; Ping, Yanyan; Wang, Li; Xu, Chaohan; Xiao, Yun; Li, Xia

    2015-01-01

    Epigenetic modification can affect many important biological processes, such as cell proliferation and apoptosis. It can alter chromatin conformation and contribute to gene regulation. To investigate how chromatin states associated with network motifs, we assembled chromatin state-modified regulatory networks by combining 269 ChIP-seq data and chromatin states in four cell types. We found that many chromatin states were significantly associated with network motifs, especially for feedforward loops (FFLs). These distinct chromatin state compositions contribute to different expression levels and translational control of targets in FFLs. Strikingly, the chromatin state-modified FFLs were highly cell-specific and, to a large extent, determined cell-selective functions, such as the embryonic stem cell-specific bivalent modification-related FFL with an important role in poising developmentally important genes for expression. Besides, comparisons of chromatin state-modified FFLs between cancerous/stem and primary cell lines revealed specific type of chromatin state alterations that may act together with motif structural changes cooperatively contribute to cell-to-cell functional differences. Combination of these alterations could be helpful in prioritizing candidate genes. Together, this work highlights that a dynamic epigenetic dimension can help network motifs to control cell-specific functions. PMID:26169043

  14. Oxidative stress signaling to chromatin in health and disease

    KAUST Repository

    Kreuz, Sarah

    2016-06-20

    Oxidative stress has a significant impact on the development and progression of common human pathologies, including cancer, diabetes, hypertension and neurodegenerative diseases. Increasing evidence suggests that oxidative stress globally influences chromatin structure, DNA methylation, enzymatic and non-enzymatic post-translational modifications of histones and DNA-binding proteins. The effects of oxidative stress on these chromatin alterations mediate a number of cellular changes, including modulation of gene expression, cell death, cell survival and mutagenesis, which are disease-driving mechanisms in human pathologies. Targeting oxidative stress-dependent pathways is thus a promising strategy for the prevention and treatment of these diseases. We summarize recent research developments connecting oxidative stress and chromatin regulation.

  15. Oxidative stress signaling to chromatin in health and disease

    Science.gov (United States)

    Kreuz, Sarah; Fischle, Wolfgang

    2016-01-01

    Oxidative stress has a significant impact on the development and progression of common human pathologies, including cancer, diabetes, hypertension and neurodegenerative diseases. Increasing evidence suggests that oxidative stress globally influences chromatin structure, DNA methylation, enzymatic and non-enzymatic post-translational modifications of histones and DNA-binding proteins. The effects of oxidative stress on these chromatin alterations mediate a number of cellular changes, including modulation of gene expression, cell death, cell survival and mutagenesis, which are disease-driving mechanisms in human pathologies. Targeting oxidative stress-dependent pathways is thus a promising strategy for the prevention and treatment of these diseases. We summarize recent research developments connecting oxidative stress and chromatin regulation. PMID:27319358

  16. Chromatin Regulation and the Histone Code in HIV Latency
.

    Science.gov (United States)

    Turner, Anne-Marie W; Margolis, David M

    2017-06-01

    The formation of a latent reservoir of Human Immunodeficiency Virus (HIV) infection hidden from immune clearance remains a significant obstacle to approaches to eradicate HIV infection. Towards an understanding of the mechanisms of HIV persistence, there is a growing body of work implicating epigenetic regulation of chromatin in establishment and maintenance of this latent reservoir. Here we discuss recent advances in the field of chromatin regulation, specifically in our understanding of the histone code, and how these discoveries relate to our current knowledge of the chromatin mechanisms linked to HIV transcriptional repression and the reversal of latency. We also examine mechanisms unexplored in the context of HIV latency and briefly discuss current therapies aimed at the induction of proviral expression within latently infected cells. We aim to emphasize that a greater understanding of the epigenetic mechanisms which govern HIV latency could lead to new therapeutic targets for latency reversal and clearance cure strategies.

  17. Role of chromatin in water stress responses in plants.

    Science.gov (United States)

    Han, Soon-Ki; Wagner, Doris

    2014-06-01

    As sessile organisms, plants are exposed to environmental stresses throughout their life. They have developed survival strategies such as developmental and morphological adaptations, as well as physiological responses, to protect themselves from adverse environments. In addition, stress sensing triggers large-scale transcriptional reprogramming directed at minimizing the deleterious effect of water stress on plant cells. Here, we review recent findings that reveal a role of chromatin in water stress responses. In addition, we discuss data in support of the idea that chromatin remodelling and modifying enzymes may be direct targets of stress signalling pathways. Modulation of chromatin regulator activity by these signaling pathways may be critical in minimizing potential trade-offs between growth and stress responses. Alterations in the chromatin organization and/or in the activity of chromatin remodelling and modifying enzymes may furthermore contribute to stress memory. Mechanistic insight into these phenomena derived from studies in model plant systems should allow future engineering of broadly drought-tolerant crop plants that do not incur unnecessary losses in yield or growth. © The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  18. The size of the thyroid hormone receptor in chromatin.

    Science.gov (United States)

    Gruol, D J; Kempner, E S

    1982-01-25

    We have used radiation inactivation and target theory to determine the size of the functional unit for T3 binding in rat liver chromatin. The process involves exposure of frozen chromatin samples to a beam of high energy electrons produced in a linear accelerator and subsequent measurement of the residual capacity to bind hormone. Our experiments were carried out using three forms of solubilized chromatin: 1) sonicated, containing the receptor in fragments which sedimented faster than 30 S; 2) digested by nuclease, containing the receptor in a form which sedimented at 5-6 S; 3) digested by nuclease and made 0.5 M in KCl, containing the receptor in a form which sedimented at 3.8 S. We have shown that in each sample preparation the receptor retained the ability to bind T3 with the same capacity and affinity that had previously been measured with high molecular weight chromatin. Irradiation caused a reduction in the capacity to bind T3 but did not change the affinity of the remaining receptors for the hormone. In each preparation, the radiation resulted in a simple exponential loss of binding capacity with dose, indicating that a single target size was detected. Within the variation of the measurements, the target size for each form of the receptor was the same, 59,000 daltons.

  19. Transcriptional networks and chromatin remodeling controlling adipogenesis

    DEFF Research Database (Denmark)

    Siersbæk, Rasmus; Nielsen, Ronni; Mandrup, Susanne

    2012-01-01

    remodeling have revealed 'snapshots' of this cascade and the chromatin landscape at specific time-points of differentiation. These studies demonstrate that multiple adipogenic transcription factors co-occupy hotspots characterized by an open chromatin structure and specific epigenetic modifications...

  20. Guarding against Collateral Damage during Chromatin Transactions

    DEFF Research Database (Denmark)

    Altmeyer, Matthias; Lukas, Jiri

    2013-01-01

    Signal amplifications are vital for chromatin function, yet they also bear the risk of transforming into unrestrained, self-escalating, and potentially harmful responses. Examples of inbuilt limitations are emerging, revealing how chromatin transactions are confined within physiological boundaries....

  1. A Long-Distance Chromatin Affair

    NARCIS (Netherlands)

    Denker, Annette; de Laat, Wouter

    2015-01-01

    Changes in transcription factor binding sequences result in correlated changes in chromatin composition locally and at sites hundreds of kilobases away. New studies demonstrate that this concordance is mediated via spatial chromatin interactions that constitute regulatory modules of the human

  2. Impact of chromatin structure on PR signaling

    DEFF Research Database (Denmark)

    Grøntved, Lars; Hager, Gordon L

    2012-01-01

    The progesterone receptor (PR) interacts with chromatin in a highly dynamic manner that requires ongoing chromatin remodeling, interaction with chaparones and activity of the proteasome. Here we discuss dynamic interaction of steroid receptor with chromatin, with special attention not only to PR ...

  3. Spectroscopic study of laser irradiated chromatin

    Science.gov (United States)

    Radu, Liliana; Mihailescu, I.; Gazdaru, Doina; Preoteasa, V.

    2013-04-01

    The effects of three UV excimer laser radiations, with wavelengths of 193, 248 and 282 nm respectively, on the structure of chromatin (the complex of deoxyribonucleic acid with proteins that exists in eukaryotic cells nuclei) were investigated. The chromatin was extracted from livers of Winstar rats. The spectroscopic methods used are: fluorescence (Förster) resonance energy transfer (FRET), time resolved fluorescence and steady-state fluorescence. A chromatin deoxyribonucleic acid radiolysis, a chromatin proteins damage and a change of the global chromatin structure on lasers action were indicated by this study. It exists some small differences between the actions of these three laser radiations.

  4. Spectroscopic study of laser irradiated chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Radu, Liliana, E-mail: liliana1radu@gmail.com [V. Babes National Institute, Department of Molecular Genetics and Radiobiology (Romania); Mihailescu, I. [National Institute for Lasers, Plasma and Radiation Physics, Department of Lasers (Romania); Gazdaru, Doina [Faculty of Physics, Bucharest University, Department of Biophysics (Romania); Preoteasa, V. [V. Babes National Institute, Department of Molecular Genetics and Radiobiology (Romania)

    2013-04-15

    The effects of three UV excimer laser radiations, with wavelengths of 193, 248 and 282 nm respectively, on the structure of chromatin (the complex of deoxyribonucleic acid with proteins that exists in eukaryotic cells nuclei) were investigated. The chromatin was extracted from livers of Winstar rats. The spectroscopic methods used are: fluorescence (Foerster) resonance energy transfer (FRET), time resolved fluorescence and steady-state fluorescence. A chromatin deoxyribonucleic acid radiolysis, a chromatin proteins damage and a change of the global chromatin structure on lasers action were indicated by this study. It exists some small differences between the actions of these three laser radiations.

  5. An evolutionary consequence of dosage compensation on Drosophila melanogaster female X-chromatin structure?

    Directory of Open Access Journals (Sweden)

    Oliver Brian

    2010-01-01

    Full Text Available Abstract Background X chromosomes are subject to dosage compensation in Drosophila males. Dosage compensation requires cis sequence features of the X chromosome that are present in both sexes by definition and trans acting factors that target chromatin modifying machinery to the X specifically in males. The evolution of this system could result in neutral X chromatin changes that will be apparent in females. Results We find that the general chromatin structure of female X chromosomes is distinct from autosomes. Additionally, specific histone marks associated with dosage compensation and active chromatin marks on the male X chromosome are also enriched on the X chromosomes of females, albeit to a lesser degree. Conclusions Our data indicate that X chromatin structure is fundamentally different from autosome structure in both sexes. We suggest that the differences between the X chromosomes and autosomes in females are a consequence of mechanisms that have evolved to ensure sufficient X chromosome expression in the soma of males.

  6. High-Frequency Promoter Firing Links THO Complex Function to Heavy Chromatin Formation

    DEFF Research Database (Denmark)

    Mouaikel, John; Causse, Sébastien Z; Rougemaille, Mathieu

    2013-01-01

    The THO complex is involved in transcription, genome stability, and messenger ribonucleoprotein (mRNP) formation, but its precise molecular function remains enigmatic. Under heat shock conditions, THO mutants accumulate large protein-DNA complexes that alter the chromatin density of target genes...... (heavy chromatin), defining a specific biochemical facet of THO function and a powerful tool of analysis. Here, we show that heavy chromatin distribution is dictated by gene boundaries and that the gene promoter is necessary and sufficient to convey THO sensitivity in these conditions. Single......-molecule fluorescence insitu hybridization measurements show that heavy chromatin formation correlates with an unusually high firing pace of the promoter with more than 20 transcription events per minute. Heavy chromatin formation closely follows the modulation of promoter firing and strongly correlates with polymerase...

  7. Plant chromatin warms up in Madrid

    Science.gov (United States)

    Jarillo, José A; Gaudin, Valerie; Hennig, Lars; Köhler, Claudia; Piñeiro, Manuel

    2014-01-01

    The 3rd European Workshop on Plant Chromatin (EWPC) was held on August 2013 in Madrid, Spain. A number of different topics on plant chromatin were presented during the meeting, including new factors mediating Polycomb Group protein function in plants, chromatin-mediated reprogramming in plant developmental transitions, the role of histone variants, and newly identified chromatin remodeling factors. The function of interactions between chromatin and transcription factors in the modulation of gene expression, the role of chromatin dynamics in the control of nuclear processes and the influence of environmental factors on chromatin organization were also reported. In this report, we highlight some of the new insights emerging in this growing area of research, presented at the 3rd EWPC. PMID:24504145

  8. Open chromatin in plant genomes.

    Science.gov (United States)

    Zhang, Wenli; Zhang, Tao; Wu, Yufeng; Jiang, Jiming

    2014-01-01

    Sensitivity to DNase I digestion is an indicator of the accessibility and configuration of chromatin in eukaryotic genomes. Open chromatin exhibits high sensitivity to DNase I cleavage. DNase I hypersensitive sites (DHSs) in eukaryotic genomes can be identified through DNase I treatment followed by sequencing (DNase-seq). DHSs are most frequently associated with various cis-regulatory DNA elements, including promoters, enhancers, and silencers in both animal and plant genomes. Genome-wide identification of DHSs provides an efficient method to interpret previously un-annotated regulatory DNA sequences. In this review, we provide an overview of the historical perspective of DHS research in eukaryotes. We summarize the main achievements of DHS research in model animal species and review the recent progress of DHS research in plants. We finally discuss possible future directions of using DHS as a tool in plant genomics research. © 2014 S. Karger AG, Basel.

  9. Chromatin structure in telomere dynamics

    Directory of Open Access Journals (Sweden)

    Alessandra eGalati

    2013-03-01

    Full Text Available The establishment of a specific nucleoprotein structure, the telomere, is required to ensure the protection of chromosome ends from being recognized as DNA damage sites. Telomere shortening below a critical length triggers a DNA damage response that leads to replicative senescence. In normal human somatic cells, characterized by telomere shortening with each cell division, telomere uncapping is a regulated process associated with cell turnover. Nevertheless, telomere dysfunction has also been associated with genomic instability, cell transformation and cancer. Despite the essential role telomeres play in chromosome protection and in tumorigenesis, our knowledge of the chromatin structure involved in telomere maintenance is still limited. Here we review the recent findings on chromatin modifications associated with the dynamic changes of telomeres from protected to de-protected state and their role in telomere functions.

  10. Nuclear phosphoinositide regulation of chromatin.

    Science.gov (United States)

    Hamann, Bree L; Blind, Raymond D

    2018-01-01

    Phospholipid signaling has clear connections to a wide array of cellular processes, particularly in gene expression and in controlling the chromatin biology of cells. However, most of the work elucidating how phospholipid signaling pathways contribute to cellular physiology have studied cytoplasmic membranes, while relatively little attention has been paid to the role of phospholipid signaling in the nucleus. Recent work from several labs has shown that nuclear phospholipid signaling can have important roles that are specific to this cellular compartment. This review focuses on the nuclear phospholipid functions and the activities of phospholipid signaling enzymes that regulate metazoan chromatin and gene expression. In particular, we highlight the roles that nuclear phosphoinositides play in several nuclear-driven physiological processes, such as differentiation, proliferation, and gene expression. Taken together, the recent discovery of several specifically nuclear phospholipid functions could have dramatic impact on our understanding of the fundamental mechanisms that enable tight control of cellular physiology. © 2017 Wiley Periodicals, Inc.

  11. A Testis-Specific Chaperone and the Chromatin Remodeler ISWI Mediate Repackaging of the Paternal Genome

    Directory of Open Access Journals (Sweden)

    Cécile M. Doyen

    2015-11-01

    Full Text Available During spermatogenesis, the paternal genome is repackaged into a non-nucleosomal, highly compacted chromatin structure. Bioinformatic analysis revealed that Drosophila sperm chromatin proteins are characterized by a motif related to the high-mobility group (HMG box, which we termed male-specific transcript (MST-HMG box. MST77F is a MST-HMG-box protein that forms an essential component of sperm chromatin. The deposition of MST77F onto the paternal genome requires the chaperone function of tNAP, a testis-specific NAP protein. MST77F, in turn, enables the stable incorporation of MST35Ba and MST35Bb into sperm chromatin. Following MST-HMG-box protein deposition, the ATP-dependent chromatin remodeler ISWI mediates the appropriate organization of sperm chromatin. Conversely, at fertilization, maternal ISWI targets the paternal genome and drives its repackaging into de-condensed nucleosomal chromatin. Failure of this transition in ISWI mutant embryos is followed by mitotic defects, aneuploidy, and haploid embryonic divisions. Thus, ISWI enables bi-directional transitions between two fundamentally different forms of chromatin.

  12. Glucocorticoid receptor binding to chromatin is selectively controlled by the coregulator Hic-5 and chromatin remodeling enzymes.

    Science.gov (United States)

    Lee, Brian H; Stallcup, Michael R

    2017-06-02

    The steroid hormone-activated glucocorticoid receptor (GR) regulates cellular stress pathways by binding to genomic regulatory elements of target genes and recruiting coregulator proteins to remodel chromatin and regulate transcription complex assembly. The coregulator hydrogen peroxide-inducible clone 5 (Hic-5) is required for glucocorticoid (GC) regulation of some genes but not others and blocks the regulation of a third gene set by inhibiting GR binding. How Hic-5 exerts these gene-specific effects and specifically how it blocks GR binding to some genes but not others is unclear. Here we show that site-specific blocking of GR binding is due to gene-specific requirements for ATP-dependent chromatin remodeling enzymes. By depletion of 11 different chromatin remodelers, we found that ATPases chromodomain helicase DNA-binding protein 9 (CHD9) and Brahma homologue (BRM, a product of the SMARCA2 gene) are required for GC-regulated expression of the blocked genes but not for other GC-regulated genes. Furthermore, CHD9 and BRM were required for GR occupancy and chromatin remodeling at GR-binding regions associated with blocked genes but not at GR-binding regions associated with other GC-regulated genes. Hic-5 selectively inhibits GR interaction with CHD9 and BRM, thereby blocking chromatin remodeling and robust GR binding at GR-binding sites associated with blocked genes. Thus, Hic-5 regulates GR binding site selection by a novel mechanism, exploiting gene-specific requirements for chromatin remodeling enzymes to selectively influence DNA occupancy and gene regulation by a transcription factor. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. High-resolution mapping reveals links of HP1 with active and inactive chromatin components.

    Directory of Open Access Journals (Sweden)

    Elzo de Wit

    2007-03-01

    Full Text Available Heterochromatin protein 1 (HP1 is commonly seen as a key factor of repressive heterochromatin, even though a few genes are known to require HP1-chromatin for their expression. To obtain insight into the targeting of HP1 and its interplay with other chromatin components, we have mapped HP1-binding sites on Chromosomes 2 and 4 in Drosophila Kc cells using high-density oligonucleotide arrays and the DNA adenine methyltransferase identification (DamID technique. The resulting high-resolution maps show that HP1 forms large domains in pericentric regions, but is targeted to single genes on chromosome arms. Intriguingly, HP1 shows a striking preference for exon-dense genes on chromosome arms. Furthermore, HP1 binds along entire transcription units, except for 5' regions. Comparison with expression data shows that most of these genes are actively transcribed. HP1 target genes are also marked by the histone variant H3.3 and dimethylated histone 3 lysine 4 (H3K4me2, which are both typical of active chromatin. Interestingly, H3.3 deposition, which is usually observed along entire transcription units, is limited to the 5' ends of HP1-bound genes. Thus, H3.3 and HP1 are mutually exclusive marks on active chromatin. Additionally, we observed that HP1-chromatin and Polycomb-chromatin are nonoverlapping, but often closely juxtaposed, suggesting an interplay between both types of chromatin. These results demonstrate that HP1-chromatin is transcriptionally active and has extensive links with several other chromatin components.

  14. Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration.

    Science.gov (United States)

    Matysiak, Julien; Lesbats, Paul; Mauro, Eric; Lapaillerie, Delphine; Dupuy, Jean-William; Lopez, Angelica P; Benleulmi, Mohamed Salah; Calmels, Christina; Andreola, Marie-Line; Ruff, Marc; Llano, Manuel; Delelis, Olivier; Lavigne, Marc; Parissi, Vincent

    2017-07-28

    Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.

  15. Proteomics of a fuzzy organelle: interphase chromatin

    Science.gov (United States)

    Kustatscher, Georg; Hégarat, Nadia; Wills, Karen L H; Furlan, Cristina; Bukowski-Wills, Jimi-Carlo; Hochegger, Helfrid; Rappsilber, Juri

    2014-01-01

    Chromatin proteins mediate replication, regulate expression, and ensure integrity of the genome. So far, a comprehensive inventory of interphase chromatin has not been determined. This is largely due to its heterogeneous and dynamic composition, which makes conclusive biochemical purification difficult, if not impossible. As a fuzzy organelle, it defies classical organellar proteomics and cannot be described by a single and ultimate list of protein components. Instead, we propose a new approach that provides a quantitative assessment of a protein's probability to function in chromatin. We integrate chromatin composition over a range of different biochemical and biological conditions. This resulted in interphase chromatin probabilities for 7635 human proteins, including 1840 previously uncharacterized proteins. We demonstrate the power of our large-scale data-driven annotation during the analysis of cyclin-dependent kinase (CDK) regulation in chromatin. Quantitative protein ontologies may provide a general alternative to list-based investigations of organelles and complement Gene Ontology. PMID:24534090

  16. Large-Scale Analysis of mRNA Translation States during Sucrose Starvation in Arabidopsis Cells Identifies Cell Proliferation and Chromatin Structure as Targets of Translational Control1[W

    Science.gov (United States)

    Nicolaï, M.; Roncato, M.A.; Canoy, A.S.; Rouquié, D.; Sarda, X.; Freyssinet, G.; Robaglia, C.

    2006-01-01

    Sucrose starvation of Arabidopsis (Arabidopsis thaliana) cell culture was used to identify translationally regulated genes by DNA microarray analysis. Cells were starved by subculture without sucrose, and total and polysomal RNA was extracted between 6 and 48 h. Probes were derived from both RNA populations and used to screen oligonucleotide microarrays. Out of 25,607 screened genes, 224 were found to be differentially accumulated in polysomal RNA following starvation and 21 were found to be invariant in polysomal RNA while their total RNA abundance was modified. Most of the mRNA appears to be translationally repressed (183/245 genes), which is consistent with a general decrease in metabolic activities during starvation. The parallel transcriptional analysis identifies 268 regulated genes. Comparison of transcriptional and translational gene lists highlights the importance of translational regulation (mostly repression) affecting genes involved in cell cycle and cell growth, these being overrepresented in translationally regulated genes, providing a molecular framework for the arrest of cell proliferation following starvation. Starvation-induced translational control also affects chromatin regulation genes, such as the HD1 histone deacetylase, and the level of histone H4 acetylation was found to increase during starvation. This suggests that regulation of the global nuclear transcriptional activity might be linked to cytoplasmic translational regulations. PMID:16632591

  17. The architects of crenarchaeal chromatin : A biophysical characterization of chromatin proteins from Sulfolobus solfataricus

    NARCIS (Netherlands)

    Driessen, Rosalie Paula Catharina

    2014-01-01

    Understanding of chromatin organization and compaction in Archaea is currently limited. The genome of several megabasepairs long is folded by a set of small chromatin proteins to fit into the micron-sized cell. A first step in understanding archaeal chromatin organization is to study the action of

  18. Single-molecule kinetic analysis of HP1-chromatin binding reveals a dynamic network of histone modification and DNA interactions.

    Science.gov (United States)

    Bryan, Louise C; Weilandt, Daniel R; Bachmann, Andreas L; Kilic, Sinan; Lechner, Carolin C; Odermatt, Pascal D; Fantner, Georg E; Georgeon, Sandrine; Hantschel, Oliver; Hatzimanikatis, Vassily; Fierz, Beat

    2017-10-13

    Chromatin recruitment of effector proteins involved in gene regulation depends on multivalent interaction with histone post-translational modifications (PTMs) and structural features of the chromatin fiber. Due to the complex interactions involved, it is currently not understood how effectors dynamically sample the chromatin landscape. Here, we dissect the dynamic chromatin interactions of a family of multivalent effectors, heterochromatin protein 1 (HP1) proteins, using single-molecule fluorescence imaging and computational modeling. We show that the three human HP1 isoforms are recruited and retained on chromatin by a dynamic exchange between histone PTM and DNA bound states. These interactions depend on local chromatin structure, the HP1 isoforms as well as on PTMs on HP1 itself. Of the HP1 isoforms, HP1α exhibits the longest residence times and fastest binding rates due to DNA interactions in addition to PTM binding. HP1α phosphorylation further increases chromatin retention through strengthening of multivalency while reducing DNA binding. As DNA binding in combination with specific PTM recognition is found in many chromatin effectors, we propose a general dynamic capture mechanism for effector recruitment. Multiple weak protein and DNA interactions result in a multivalent interaction network that targets effectors to a specific chromatin modification state, where their activity is required. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components

    DEFF Research Database (Denmark)

    Alabert, Constance; Bukowski-Wills, Jimi-Carlo; Lee, Sung-Po

    2014-01-01

    replication in human cells. NCC relies on biotin-dUTP labelling of replicating DNA, affinity purification and quantitative proteomics. Comparing nascent chromatin with mature post-replicative chromatin, we provide association dynamics for 3,995 proteins. The replication machinery and 485 chromatin factors...... with experimentally derived chromatin probabilities to predict a function in nascent chromatin for 93 uncharacterized proteins, and identify FAM111A as a replication factor required for PCNA loading. Together, this provides an extensive resource to understand genome and epigenome maintenance....

  20. A microscopic analysis of Arabidopsis chromatin

    NARCIS (Netherlands)

    Willemse, J.J.

    2007-01-01

    Genetic information of eukaryotic organisms is stored as DNA in the nuclei of their cells. Nuclear DNA is associated with several proteins, which together form chromatin. The most abundant chromatin proteins arehistones,they arrange the initial packaging step of the DNA. DNA

  1. Chromatin-modifying proteins in cancer

    DEFF Research Database (Denmark)

    Fog, Cathrine K; Jensen, Klaus T; Lund, Anders Henrik

    2007-01-01

    -despite the fact that all cells in the organism contain the same genetic information. A large amount of data gathered over the last decades has demonstrated that deregulation of chromatin-modifying proteins is etiologically involved in the development and progression of cancer. Here we discuss how epigenetic...... alterations influence cancer development and review known cancer-associated alterations in chromatin-modifying proteins....

  2. Open chromatin reveals the functional maize genome

    Science.gov (United States)

    Every cellular process mediated through nuclear DNA must contend with chromatin. As results from ENCODE show, open chromatin assays can efficiently integrate across diverse regulatory elements, revealing functional non-coding genome. In this study, we use a MNase hypersensitivity assay to discover o...

  3. Molecular structures guide the engineering of chromatin.

    Science.gov (United States)

    Tekel, Stefan J; Haynes, Karmella A

    2017-07-27

    Chromatin is a system of proteins, RNA, and DNA that interact with each other to organize and regulate genetic information within eukaryotic nuclei. Chromatin proteins carry out essential functions: packing DNA during cell division, partitioning DNA into sub-regions within the nucleus, and controlling levels of gene expression. There is a growing interest in manipulating chromatin dynamics for applications in medicine and agriculture. Progress in this area requires the identification of design rules for the chromatin system. Here, we focus on the relationship between the physical structure and function of chromatin proteins. We discuss key research that has elucidated the intrinsic properties of chromatin proteins and how this information informs design rules for synthetic systems. Recent work demonstrates that chromatin-derived peptide motifs are portable and in some cases can be customized to alter their function. Finally, we present a workflow for fusion protein design and discuss best practices for engineering chromatin to assist scientists in advancing the field of synthetic epigenetics. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Chromatin dynamics resolved with force spectroscopy

    NARCIS (Netherlands)

    Chien, Fan-Tso

    2011-01-01

    In eukaryotic cells, genomic DNA is organized in chromatin fibers composed of nucleosomes as structural units. A nucleosome contains 1.7 turns of DNA wrapped around a histone octamer and is connected to the adjacent nucleosomes with linker DNA. The folding of chromatin fibers effectively increases

  5. TOPICAL REVIEW: The physics of chromatin

    Science.gov (United States)

    Schiessel, Helmut

    2003-05-01

    Recent progress has been made in the understanding of the physical properties of chromatin - the dense complex of DNA and histone proteins that occupies the nuclei of plant and animal cells. Here I will focus on the two lowest levels of the hierarchy of DNA folding into the chromatin complex. (i) The nucleosome, the chromatin repeating unit consisting of a globular aggregate of eight histone proteins with the DNA wrapped around it: its overcharging, the DNA unwrapping transition, the 'sliding' of the octamer along the DNA. (ii) The 30 nm chromatin fibre, the necklace-like structure of nucleosomes connected via linker DNA: its geometry, its mechanical properties under stretching and its response to changing ionic conditions. I will stress that chromatin combines two seemingly contradictory features: (1) high compaction of DNA within the nuclear envelope and, at the same time, (2) accessibility to genes, promoter regions and gene regulatory sequences.

  6. Genome-Wide Analysis of Chromatin States Reveals Distinct Mechanisms of Sex-Dependent Gene Regulation in Male and Female Mouse Liver

    Science.gov (United States)

    Sugathan, Aarathi

    2013-01-01

    Chromatin state maps were developed to elucidate sex differences in chromatin structure and their impact on sex-differential chromatin accessibility and sex-biased gene expression in mouse liver. Genes in active, inactive, and poised chromatin states exhibited differential responsiveness to ligand-activated nuclear receptors and distinct enrichments for functional gene categories. Sex-biased genes were clustered by chromatin environments and mapped to DNase-hypersensitive sites (DHS) classified by sex bias in chromatin accessibility and enhancer modifications. Results were integrated with genome-wide binding data for five transcription factors implicated in growth hormone-regulated, sex-biased liver gene expression, leading to the following findings. (i) Sex-biased DHS, but not sex-biased genes, are frequently characterized by sex-differential chromatin states, indicating distal regulation. (ii) Trimethylation of histone H3 at K27 (H3K27me3) is a major sex-biased repressive mark at highly female-biased but not at highly male-biased genes. (iii) FOXA factors are associated with sex-dependent chromatin opening at male-biased but not female-biased regulatory sites. (iv) Sex-biased STAT5 binding is enriched at sex-biased DHS marked as active enhancers and preferentially targets sex-biased genes with sex-differences in local chromatin marks. (v) The male-biased repressor BCL6 preferentially targets female-biased genes and regulatory sites in a sex-independent chromatin state. (vi) CUX2, a female-specific repressor of male-biased genes, also activates strongly female-biased genes, in association with loss of H3K27me3 marks. Chromatin states are thus a major determinant of sex-biased chromatin accessibility and gene expression, with FOXA pioneer factors proposed to confer sex-dependent chromatin opening and STAT5, but not BCL6, regulating sex-biased genes by binding to sites in a sex-biased chromatin state. PMID:23836885

  7. Organization of DNA in chromatin.

    Science.gov (United States)

    Sobell, H M; Tsai, C C; Gilbert, S G; Jain, S C; Sakore, T D

    1976-01-01

    Conformational changes in DNA that accompany drug intercalation have led us to ask if DNA first bends or "kinks" to accept an intercalative drug or dye. Kinking is made possible by altering the normal C2' endo deoxyribose sugar ring puckering in B DNA to a mixed sugar puckering pattern of the type C3' and partially unstacking base-pairs. A kinking scheme such as this would require minimal stereochemical rearrangement and would also involve small energies. This has prompted us to ask more generally if a conformational change such as this could be used by proteins in their interactions with nucleic acids. In this papter we describe an interesting superhelical DNA structure formed by kinking DNA every 10 base-pairs. The structure may be used in the organization of DNA in chromatin. Images PMID:1067602

  8. Organization of DNA in chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Sobell, H.M.; Tsai, C.C.; Gilbert, S.G.; Jain, S.C.; Sakore, T.D.

    1976-09-01

    Conformational changes in DNA that accompany drug intercalation have led us to ask if DNA first bends or ''kinks'' to accept an intercalative drug or dye. Kinking is made possible by altering the normal C2' endo deoxyribose sugar ring puckering in B DNA to a mixed sugar puckering pattern of the type C3' endo (3'--5')C2' endo and partially unstacking base-paris. A kinking scheme such as this would require minimal stereochemical rearrangement and would also involve small energies. This has prompted us to ask more generally if a conformational change such as this could be used by proteins in their interactions with nucleic acids. In this paper we describe an interesting superhelical DNA structure formed by kinking DNA every 10 base-pairs. The structure may be used in the organization of DNA in chromatin.

  9. Inheritance of epigenetic chromatin silencing.

    Science.gov (United States)

    David-Rus, Diana; Mukhopadhyay, Swagatam; Lebowitz, Joel L; Sengupta, Anirvan M

    2009-05-07

    Maintenance of alternative chromatin states through cell divisions pose some fundamental constraints on the dynamics of histone modifications. In this paper, we study the systems biology of epigenetic inheritance by defining and analyzing general classes of mathematical models. We discuss how the number of modification states involved plays an essential role in the stability of epigenetic states. In addition, DNA duplication and the consequent dilution of marked histones act as a large perturbation for a stable state of histone modifications. The requirement that this large perturbation falls into the basin of attraction of the original state sometimes leads to additional constraints on effective models. Two such models, inspired by two different biological systems, are compared in their fulfilling the requirements of multistability and of recovery after DNA duplication. We conclude that in the presence of multiple histone modifications that characterize alternative epigenetic stable states, these requirements are more easily fulfilled.

  10. Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis

    Directory of Open Access Journals (Sweden)

    Alexandre A.S.F. Raposo

    2015-03-01

    Full Text Available The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program.

  11. RNAi-mediated gene silencing reveals involvement of Arabidopsis chromatin-related genes in Agrobacterium-mediated root transformation

    OpenAIRE

    Crane, Yan Ma; Gelvin, Stanton B.

    2007-01-01

    We investigated the effect of RNAi-mediated gene silencing of 109 Arabidopsis thaliana chromatin-related genes (termed “chromatin genes” hereafter) on Agrobacterium-mediated root transformation. Each of the RNAi lines contains a single- or low-copy-number insertion of a hairpin construction that silences the endogenous copy of the target gene. We used three standard transient and stable transformation assays to screen 340 independent RNAi lines, representing 109 target genes, for the rat (res...

  12. Undifferentiated embryonic cell transcription factor 1 regulates ESC chromatin organization and gene expression

    DEFF Research Database (Denmark)

    Kooistra, Susanne M; van den Boom, Vincent; Thummer, Rajkumar P

    2010-01-01

    Previous reports showed that embryonic stem (ES) cells contain hyperdynamic and globally transcribed chromatin-properties that are important for ES cell pluripotency and differentiation. Here, we demonstrate a role for undifferentiated embryonic cell transcription factor 1 (UTF1) in regulating ES...... cell chromatin structure. Using chromatin immunoprecipitation-on-chip analysis, we identified >1,700 UTF1 target genes that significantly overlap with previously identified Nanog, Oct4, Klf-4, c-Myc, and Rex1 targets. Gene expression profiling showed that UTF1 knock down results in increased expression...... of a large set of genes, including a significant number of UTF1 targets. UTF1 knock down (KD) ES cells are, irrespective of the increased expression of several self-renewal genes, Leukemia inhibitory factor (LIF) dependent. However, UTF1 KD ES cells are perturbed in their differentiation in response...

  13. Cryo-electron microscopy of chromatin biology.

    Science.gov (United States)

    Wilson, Marcus D; Costa, Alessandro

    2017-06-01

    The basic unit of chromatin, the nucleosome core particle (NCP), controls how DNA in eukaryotic cells is compacted, replicated and read. Since its discovery, biochemists have sought to understand how this protein-DNA complex can help to control so many diverse tasks. Recent electron-microscopy (EM) studies on NCP-containing assemblies have helped to describe important chromatin transactions at a molecular level. With the implementation of recent technical advances in single-particle EM, our understanding of how nucleosomes are recognized and read looks to take a leap forward. In this review, the authors highlight recent advances in the architectural understanding of chromatin biology elucidated by EM.

  14. Extensive Variation in Chromatin States Across Humans

    KAUST Repository

    Kasowski, M.

    2013-10-17

    The majority of disease-associated variants lie outside protein-coding regions, suggesting a link between variation in regulatory regions and disease predisposition. We studied differences in chromatin states using five histone modifications, cohesin, and CTCF in lymphoblastoid lines from 19 individuals of diverse ancestry. We found extensive signal variation in regulatory regions, which often switch between active and repressed states across individuals. Enhancer activity is particularly diverse among individuals, whereas gene expression remains relatively stable. Chromatin variability shows genetic inheritance in trios, correlates with genetic variation and population divergence, and is associated with disruptions of transcription factor binding motifs. Overall, our results provide insights into chromatin variation among humans.

  15. Modeling gene regulation from paired expression and chromatin accessibility data.

    Science.gov (United States)

    Duren, Zhana; Chen, Xi; Jiang, Rui; Wang, Yong; Wong, Wing Hung

    2017-06-20

    The rapid increase of genome-wide datasets on gene expression, chromatin states, and transcription factor (TF) binding locations offers an exciting opportunity to interpret the information encoded in genomes and epigenomes. This task can be challenging as it requires joint modeling of context-specific activation of cis-regulatory elements (REs) and the effects on transcription of associated regulatory factors. To meet this challenge, we propose a statistical approach based on paired expression and chromatin accessibility (PECA) data across diverse cellular contexts. In our approach, we model (i) the localization to REs of chromatin regulators (CRs) based on their interaction with sequence-specific TFs, (ii) the activation of REs due to CRs that are localized to them, and (iii) the effect of TFs bound to activated REs on the transcription of target genes (TGs). The transcriptional regulatory network inferred by PECA provides a detailed view of how trans- and cis-regulatory elements work together to affect gene expression in a context-specific manner. We illustrate the feasibility of this approach by analyzing paired expression and accessibility data from the mouse Encyclopedia of DNA Elements (ENCODE) and explore various applications of the resulting model.

  16. Combinatorial epigenetic patterns as quantitative predictors of chromatin biology.

    Science.gov (United States)

    Cieślik, Marcin; Bekiranov, Stefan

    2014-01-28

    Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is the most widely used method for characterizing the epigenetic states of chromatin on a genomic scale. With the recent availability of large genome-wide data sets, often comprising several epigenetic marks, novel approaches are required to explore functionally relevant interactions between histone modifications. Computational discovery of "chromatin states" defined by such combinatorial interactions enabled descriptive annotations of genomes, but more quantitative approaches are needed to progress towards predictive models. We propose non-negative matrix factorization (NMF) as a new unsupervised method to discover combinatorial patterns of epigenetic marks that frequently co-occur in subsets of genomic regions. We show that this small set of combinatorial "codes" can be effectively displayed and interpreted. NMF codes enable dimensionality reduction and have desirable statistical properties for regression and classification tasks. We demonstrate the utility of codes in the quantitative prediction of Pol2-binding and the discrimination between Pol2-bound promoters and enhancers. Finally, we show that specific codes can be linked to molecular pathways and targets of pluripotency genes during differentiation. We have introduced and evaluated a new computational approach to represent combinatorial patterns of epigenetic marks as quantitative variables suitable for predictive modeling and supervised machine learning. To foster widespread adoption of this method we make it available as an open-source software-package - epicode at https://github.com/mcieslik-mctp/epicode.

  17. Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

    KAUST Repository

    Bigeard, Jean

    2014-07-10

    In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

  18. Roles of chromatin insulator proteins in higher-order chromatin organization and transcription regulation

    Science.gov (United States)

    Vogelmann, Jutta; Valeri, Alessandro; Guillou, Emmanuelle; Cuvier, Olivier; Nollmann, Marcelo

    2013-01-01

    Eukaryotic chromosomes are condensed into several hierarchical levels of complexity: DNA is wrapped around core histones to form nucleosomes, nucleosomes form a higher-order structure called chromatin, and chromatin is subsequently compartmentalized in part by the combination of multiple specific or unspecific long-range contacts. The conformation of chromatin at these three levels greatly influences DNA metabolism and transcription. One class of chromatin regulatory proteins called insulator factors may organize chromatin both locally, by setting up barriers between heterochromatin and euchromatin, and globally by establishing platforms for long-range interactions. Here, we review recent data revealing a global role of insulator proteins in the regulation of transcription through the formation of clusters of long-range interactions that impact different levels of chromatin organization. PMID:21983085

  19. DNA methylation-independent removable insulator controls chromatin remodeling at the HOXA locus via retinoic acid signaling.

    Science.gov (United States)

    Ishihara, Ko; Nakamoto, Masafumi; Nakao, Mitsuyoshi

    2016-12-15

    Chromatin insulators partition the genome into functional units to control gene expression, particularly in complex chromosomal regions. The CCCTC-binding factor (CTCF) is an insulator-binding protein that functions in transcriptional regulation and higher-order chromatin formation. Variable CTCF-binding sites have been identified to be cell type-specific partly due to differential DNA methylation. Here, we show that DNA methylation-independent removable CTCF insulator is responsible for retinoic acid (RA)-mediated higher-order chromatin remodeling in the human HOXA gene locus. Detailed chromatin analysis characterized multiple CTCF-enriched sites and RA-responsive enhancers at this locus. These regulatory elements and transcriptionally silent HOXA genes are closely positioned under basal conditions. Notably, upon RA signaling, the RAR/RXR transcription factor induced loss of adjacent CTCF binding and changed the higher-order chromatin conformation of the overall locus. Targeted disruption of a CTCF site by genome editing with zinc finger nucleases and CRISPR/Cas9 system showed that the site is required for chromatin conformations that maintain the initial associations among insulators, enhancers and promoters. The results indicate that the initial chromatin conformation affects subsequent RA-induced HOXA gene activation. Our study uncovers that a removable insulator spatiotemporally switches higher-order chromatin and multiple gene activities via cooperation of CTCF and key transcription factors. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques

    Science.gov (United States)

    Bystricky, Kerstin; Heun, Patrick; Gehlen, Lutz; Langowski, Jörg; Gasser, Susan M.

    2004-11-01

    Little is known about how chromatin folds in its native state. Using optimized in situ hybridization and live imaging techniques have determined compaction ratios and fiber flexibility for interphase chromatin in budding yeast. Unlike previous studies, ours examines nonrepetitive chromatin at intervals short enough to be meaningful for yeast chromosomes and functional domains in higher eukaryotes. We reconcile high-resolution fluorescence in situ hybridization data from intervals of 14-100 kb along single chromatids with measurements of whole chromosome arms (122-623 kb in length), monitored in intact cells through the targeted binding of bacterial repressors fused to GFP derivatives. The results are interpreted with a flexible polymer model and suggest that interphase chromatin exists in a compact higher-order conformation with a persistence length of 170-220 nm and a mass density of 110-150 bp/nm. These values are equivalent to 7-10 nucleosomes per 11-nm turn within a 30-nm-like fiber structure. Comparison of long and short chromatid arm measurements demonstrates that chromatin fiber extension is also influenced by nuclear geometry. The observation of this surprisingly compact chromatin structure for transcriptionally competent chromatin in living yeast cells suggests that the passage of RNA polymerase II requires a very transient unfolding of higher-order chromatin structure. higher-order structure | 30-nm fiber | nucleosomes

  1. Histone deacetylase 2 is required for chromatin condensation and subsequent enucleation of cultured mouse fetal erythroblasts.

    Science.gov (United States)

    Ji, Peng; Yeh, Victor; Ramirez, Tzutzuy; Murata-Hori, Maki; Lodish, Harvey F

    2010-12-01

    During the final stages of differentiation of mammalian erythroid cells, the chromatin is condensed and enucleated. We previously reported that Rac GTPases and their downstream target, mammalian homolog of Drosophila diaphanous 2 (mDia2), are required for enucleation of in vitro cultured mouse fetal liver erythroblasts. However, it is not clear how chromatin condensation is achieved and whether it is required for enucleation. Mouse fetal liver erythroblasts were purified from embryonic day 14.5 pregnant mice and cultured in erythropoietin-containing medium. Enucleation was determined by flow-cytometry based analysis after treatment with histone deacetylase inhibitors or infection with lentiviral short hairpin RNA. We showed that histone deacetylases play critical roles in chromatin condensation and enucleation in cultured mouse fetal liver erythroblasts. Enzymatic inhibition of histone deacetylases by trichostatin A or valproic acid prior to the start of enucleation blocked chromatin condensation, contractile actin ring formation and enucleation. We further demonstrated that histone deacetylases 1, 2, 3 and 5 are highly expressed in mouse fetal erythroblasts. Short hairpin RNA down-regulation of histone deacetylase 2, but not of the other histone deacetylases, phenotypically mimicked the effect of trichostatin A or valproic acid treatment, causing significant inhibition of chromatin condensation and enucleation. Importantly, knock-down of histone deacetylase 2 did not affect erythroblast proliferation, differentiation, or apoptosis. These results identify histone deacetylase 2 as an important regulator, mediating chromatin condensation and enucleation in the final stages of mammalian erythropoiesis.

  2. The Multifaceted Contributions of Chromatin to HIV-1 Integration, Transcription, and Latency.

    Science.gov (United States)

    De Crignis, E; Mahmoudi, T

    2017-01-01

    The capacity of the human immunodeficiency virus (HIV-1) to establish latent infections constitutes a major barrier to the development of a cure for HIV-1. In latent infection, replication competent HIV-1 provirus is integrated within the host genome but remains silent, masking the infected cells from the activity of the host immune response. Despite the progress in elucidating the molecular players that regulate HIV-1 gene expression, the mechanisms driving the establishment and maintenance of latency are still not fully understood. Transcription from the HIV-1 genome occurs in the context of chromatin and is subjected to the same regulatory mechanisms that drive cellular gene expression. Much like in eukaryotic genes, the nucleosomal landscape of the HIV-1 promoter and its position within genomic chromatin are determinants of its transcriptional activity. Understanding the multilayered chromatin-mediated mechanisms that underpin HIV-1 integration and expression is of utmost importance for the development of therapeutic strategies aimed at reducing the pool of latently infected cells. In this review, we discuss the impact of chromatin structure on viral integration, transcriptional regulation and latency, and the host factors that influence HIV-1 replication by regulating chromatin organization. Finally, we describe therapeutic strategies under development to target the chromatin-HIV-1 interplay. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. High-throughput assessment of context-dependent effects of chromatin proteins.

    Science.gov (United States)

    Brueckner, Laura; van Arensbergen, Joris; Akhtar, Waseem; Pagie, Ludo; van Steensel, Bas

    2016-01-01

    Chromatin proteins control gene activity in a concerted manner. We developed a high-throughput assay to study the effects of the local chromatin environment on the regulatory activity of a protein of interest. The assay combines a previously reported multiplexing strategy based on barcoded randomly integrated reporters with Gal4-mediated tethering. We applied the assay to Drosophila heterochromatin protein 1a (HP1a), which is mostly known as a repressive protein but has also been linked to transcriptional activation. Recruitment to over 1000 genomic locations revealed that HP1a is a potent repressor able to silence even highly expressing reporter genes. However, the local chromatin context can modulate HP1a function. In pericentromeric regions, HP1a-induced repression was enhanced by twofold. In regions marked by a H3K36me3-rich chromatin signature, HP1a-dependent silencing was significantly decreased. We found no evidence for an activating function of HP1a in our experimental system. Furthermore, we did not observe stable transmission of repression over mitotic divisions after loss of targeted HP1a. The multiplexed tethered reporter assay should be applicable to a large number of chromatin proteins and will be a useful tool to dissect combinatorial regulatory interactions in chromatin.

  4. Chromatin Pioneers | Center for Cancer Research

    Science.gov (United States)

    Taking advantage of their ability to explore provocative ideas, NCI investigators pioneered the study of chromatin to demonstrate its functional importance and lay the groundwork for understanding its role in cancer and other diseases.

  5. Chromatin epigenomic domain folding: size matters

    Directory of Open Access Journals (Sweden)

    Bertrand R. Caré

    2015-09-01

    Full Text Available In eukaryotes, chromatin is coated with epigenetic marks which induce differential gene expression profiles and eventually lead to different cellular phenotypes. One of the challenges of contemporary cell biology is to relate the wealth of epigenomic data with the observed physical properties of chromatin. In this study, we present a polymer physics framework that takes into account the sizes of epigenomic domains. We build a model of chromatin as a block copolymer made of domains with various sizes. This model produces a rich set of conformations which is well explained by finite-size scaling analysis of the coil-globule transition of epigenomic domains. Our results suggest that size-dependent folding of epigenomic domains may be a crucial physical mechanism able to provide chromatin with tissue-specific folding states, these being associated with differential gene expression.

  6. 3C-digital PCR for quantification of chromatin interactions.

    Science.gov (United States)

    Du, Meijun; Wang, Liang

    2016-12-06

    Chromosome conformation capture (3C) is a powerful and widely used technique for detecting the physical interactions between chromatin regions in vivo. The principle of 3C is to convert physical chromatin interactions into specific DNA ligation products, which are then detected by quantitative polymerase chain reaction (qPCR). However, 3C-qPCR assays are often complicated by the necessity of normalization controls to correct for amplification biases. In addition, qPCR is often limited to a certain cycle number, making it difficult to detect fragment ligations with low frequency. Recently, digital PCR (dPCR) technology has become available, which allows for highly sensitive nucleic acid quantification. Main advantage of dPCR is its high precision of absolute nucleic acid quantification without requirement of normalization controls. To demonstrate the utility of dPCR in quantifying chromatin interactions, we examined two prostate cancer risk loci at 8q24 and 2p11.2 for their interaction target genes MYC and CAPG in LNCaP cell line. We designed anchor and testing primers at known regulatory element fragments and target gene regions, respectively. dPCR results showed that interaction frequency between the regulatory element and MYC gene promoter was 0.7 (95% CI 0.40-1.10) copies per 1000 genome copies while other regions showed relatively low ligation frequencies. The dPCR results also showed that the ligation frequencies between the regulatory element and two EcoRI fragments containing CAPG gene promoter were 1.9 copies (95% CI 1.41-2.47) and 1.3 copies per 1000 genome copies (95% CI 0.76-1.92), respectively, while the interaction signals were reduced on either side of the promoter region of CAPG gene. Additionally, we observed comparable results from 3C-dPCR and 3C-qPCR at 2p11.2 in another cell line (DU145). Compared to traditional 3C-qPCR, our results show that 3C-dPCR is much simpler and more sensitive to detect weak chromatin interactions. It may eliminate

  7. Probabilistic modelling of chromatin code landscape reveals functional diversity of enhancer-like chromatin states

    Science.gov (United States)

    Zhou, Jian; Troyanskaya, Olga G.

    2016-01-01

    Interpreting the functional state of chromatin from the combinatorial binding patterns of chromatin factors, that is, the chromatin codes, is crucial for decoding the epigenetic state of the cell. Here we present a systematic map of Drosophila chromatin states derived from data-driven probabilistic modelling of dependencies between chromatin factors. Our model not only recapitulates enhancer-like chromatin states as indicated by widely used enhancer marks but also divides these states into three functionally distinct groups, of which only one specific group possesses active enhancer activity. Moreover, we discover a strong association between one specific enhancer state and RNA Polymerase II pausing, linking transcription regulatory potential and chromatin organization. We also observe that with the exception of long-intron genes, chromatin state transition positions in transcriptionally active genes align with an absolute distance to their corresponding transcription start site, regardless of gene length. Using our method, we provide a resource that helps elucidate the functional and spatial organization of the chromatin code landscape. PMID:26841971

  8. Etiology and Evaluation of Sperm Chromatin Anomalies

    Directory of Open Access Journals (Sweden)

    Marziyeh Tavalaee

    2008-01-01

    Full Text Available Evidence suggests that human sperm chromatin anomalies adversely affect reproductive outcomesand infertile men possess substantially amount of sperm with chromatin anomalies than fertilemen.Routine semen analysis evaluates parameters such as sperm motility and morphology, but doesnot examine the nuclear DNA integrity of spermatozoa. It has been suggested that altered nuclearchromatin structure or damaged DNA in spermatozoa could modify the special cellular functionsof human spermatozoa, and thereby affect the fertility potential. Intra-cytoplasmic sperm injection(ICSI bypass the barriers to fertilization for such a sperm, then the effect of chromatin anomalies onthe development remains a concern. Therefore, it is essential to develop and use accurate diagnostictests, which may provide better prognostic capabilities than the standard sperm assessments. Thisreview discusses our current understanding of the structure and organization of sperm DNA,the different procedures for assessment of sperm chromatin anomalies including comet assay,Chromomycin A3 (CMA3, sperm chromatin structure assay (SCSA, acridine orange test (AOT,terminal TdT-mediated dUTP-nick-end labelling (TUNEL assay, aniline blue and sperm chromatindispersion (SCD test and the impact of chromatin anomalies on reproductive outcome.

  9. Ku80 facilitates chromatin binding of the telomere binding protein, TRF2.

    Science.gov (United States)

    Fink, Lauren S; Lerner, Chad A; Torres, Paulina F; Sell, Christian

    2010-09-15

    The Ku70/80 heterodimer is central to non-homologous end joining repair of DNA double-strand breaks and the Ku80 gene appears to be essential for human but not rodent cell survival. The Ku70/80 heterodimer is located at telomeres but its precise function in telomere maintenance is not known. In order to examine the role of Ku80 beyond DNA repair in more detail, we have taken a knockdown approach using a human fibroblast strain. Following targeted Ku80 knockdown, telomere defects are observed and the steady state levels of the TRF2 protein are reduced. Inhibitor studies indicate that this loss of TRF2 is mediated by the proteasome and degradation of TRF2 following Ku depletion appears to involve a decrease in chromatin binding of TRF2, suggesting that the Ku heterodimer enhances TRF2 chromatin association and that non-chromatin bound TRF2 is targeted to the proteasome.

  10. Chromatin Dynamics during Lytic Infection with Herpes Simplex Virus 1

    Science.gov (United States)

    Conn, Kristen L.; Schang, Luis M.

    2013-01-01

    Latent HSV-1 genomes are chromatinized with silencing marks. Since 2004, however, there has been an apparent inconsistency in the studies of the chromatinization of the HSV-1 genomes in lytically infected cells. Nuclease protection and chromatin immunoprecipitation assays suggested that the genomes were not regularly chromatinized, having only low histone occupancy. However, the chromatin modifications associated with transcribed and non-transcribed HSV-1 genes were those associated with active or repressed transcription, respectively. Moreover, the three critical HSV-1 transcriptional activators all had the capability to induce chromatin remodelling, and interacted with critical chromatin modifying enzymes. Depletion or overexpression of some, but not all, chromatin modifying proteins affected HSV-1 transcription, but often in unexpected manners. Since 2010, it has become clear that both cellular and HSV-1 chromatins are highly dynamic in infected cells. These dynamics reconcile the weak interactions between HSV-1 genomes and chromatin proteins, detected by nuclease protection and chromatin immunoprecipitation, with the proposed regulation of HSV-1 gene expression by chromatin, supported by the marks in the chromatin in the viral genomes and the abilities of the HSV-1 transcription activators to modulate chromatin. It also explains the sometimes unexpected results of interventions to modulate chromatin remodelling activities in infected cells. PMID:23863878

  11. The physical size of transcription factors is key to transcriptional regulation in chromatin domains

    Science.gov (United States)

    Maeshima, Kazuhiro; Kaizu, Kazunari; Tamura, Sachiko; Nozaki, Tadasu; Kokubo, Tetsuro; Takahashi, Koichi

    2015-02-01

    Genetic information, which is stored in the long strand of genomic DNA as chromatin, must be scanned and read out by various transcription factors. First, gene-specific transcription factors, which are relatively small (˜50 kDa), scan the genome and bind regulatory elements. Such factors then recruit general transcription factors, Mediators, RNA polymerases, nucleosome remodellers, and histone modifiers, most of which are large protein complexes of 1-3 MDa in size. Here, we propose a new model for the functional significance of the size of transcription factors (or complexes) for gene regulation of chromatin domains. Recent findings suggest that chromatin consists of irregularly folded nucleosome fibres (10 nm fibres) and forms numerous condensed domains (e.g., topologically associating domains). Although the flexibility and dynamics of chromatin allow repositioning of genes within the condensed domains, the size exclusion effect of the domain may limit accessibility of DNA sequences by transcription factors. We used Monte Carlo computer simulations to determine the physical size limit of transcription factors that can enter condensed chromatin domains. Small gene-specific transcription factors can penetrate into the chromatin domains and search their target sequences, whereas large transcription complexes cannot enter the domain. Due to this property, once a large complex binds its target site via gene-specific factors it can act as a ‘buoy’ to keep the target region on the surface of the condensed domain and maintain transcriptional competency. This size-dependent specialization of target-scanning and surface-tethering functions could provide novel insight into the mechanisms of various DNA transactions, such as DNA replication and repair/recombination.

  12. A Computer Lab Exploring Evolutionary Aspects of Chromatin Structure and Dynamics for an Undergraduate Chromatin Course

    Science.gov (United States)

    Eirin-Lopez, Jose M.

    2013-01-01

    The study of chromatin constitutes one of the most active research fields in life sciences, being subject to constant revisions that continuously redefine the state of the art in its knowledge. As every other rapidly changing field, chromatin biology requires clear and straightforward educational strategies able to efficiently translate such a…

  13. Ligation-mediated PCR for chromatin-structure analysis of interphase and metaphase chromatin.

    Science.gov (United States)

    Hershkovitz, M; Riggs, A D

    1997-02-01

    Chromatin structure is becoming increasingly recognized as important for a full understanding of gene function and cell memory. With regard to cell memory, which involves the transfer of chromatin-encoded epigenetic information from one cell generation to another, the detailed structure of metaphase chromatin is of crucial importance. In this paper we describe methods for the use of dimethyl sulfate, DNase I, and potassium permanganate for in vivo footprinting and chromatin analysis, with special emphasis on studies of metaphase cells. We review the use of ligation-mediated PCR for the analysis of chromatin, including the human phosphoglycerate kinase promoter, and also report initial studies of a matrix attachment region near the human beta-interferon gene.

  14. Quantitative analysis of chromatin accessibility in mouse embryonic fibroblasts.

    Science.gov (United States)

    Zhuo, Baowen; Yu, Juan; Chang, Luyuan; Lei, Jiafan; Wen, Zengqi; Liu, Cuifang; Mao, Guankun; Wang, Kehui; Shen, Jie; Xu, Xueqing

    2017-11-04

    Genomic DNA of eukaryotic cells is hierarchically packaged into chromatin by histones. The dynamic organization of chromatin fibers plays a critical role in the regulation of gene transcription and other DNA-associated biological processes. Recently, numerous approaches have been developed to map the chromatin organization by characterizing chromatin accessibilities in genome-wide. However, reliable methods to quantitatively map chromatin accessibility are not well-established, especially not on a genome-wide scale. Here, we developed a modified MNase-seq for mouse embryonic fibroblasts, wherein chromatin was partially digested at multiple digestion times using micrococcal nuclease (MNase), allowing quantitative analysis of local yet genome-wide chromatin compaction. Our results provide strong evidence that the chromatin accessibility at promoter regions are positively correlated with gene activity. In conclusion, our assay is an ideal tool for the quantitative study of gene regulation in the perspective of chromatin accessibility. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. New mitotic regulators released from chromatin.

    Science.gov (United States)

    Yokoyama, Hideki; Gruss, Oliver J

    2013-12-16

    Faithful action of the mitotic spindle segregates duplicated chromosomes into daughter cells. Perturbations of this process result in chromosome mis-segregation, leading to chromosomal instability and cancer development. Chromosomes are not simply passengers segregated by spindle microtubules but rather play a major active role in spindle assembly. The GTP bound form of the Ran GTPase (RanGTP), produced around chromosomes, locally activates spindle assembly factors. Recent studies have uncovered that chromosomes organize mitosis beyond spindle formation. They distinctly regulate other mitotic events, such as spindle maintenance in anaphase, which is essential for chromosome segregation. Furthermore, the direct function of chromosomes is not only to produce RanGTP but, in addition, to release key mitotic regulators from chromatin. Chromatin-remodeling factors and nuclear pore complex proteins, which have established functions on chromatin in interphase, dissociate from mitotic chromatin and function in spindle assembly or maintenance. Thus, chromosomes actively organize their own segregation using chromatin-releasing mitotic regulators as well as RanGTP.

  16. Chromatin proteins: key responders to stress.

    Directory of Open Access Journals (Sweden)

    Karen T Smith

    Full Text Available Environments can be ever-changing and stresses are commonplace. In order for organisms to survive, they need to be able to respond to change and adapt to new conditions. Fortunately, many organisms have systems in place that enable dynamic adaptation to immediate stresses and changes within the environment. Much of this cellular response is coordinated by modulating the structure and accessibility of the genome. In eukaryotic cells, the genome is packaged and rolled up by histone proteins to create a series of DNA/histone core structures known as nucleosomes; these are further condensed into chromatin. The degree and nature of the condensation can in turn determine which genes are transcribed. Histones can be modified chemically by a large number of proteins that are thereby responsible for dynamic changes in gene expression. In this Primer we discuss findings from a study published in this issue of PLoS Biology by Weiner et al. that highlight how chromatin structure and chromatin binding proteins alter transcription in response to environmental changes and stresses. Their study reveals the importance of chromatin in mediating the speed and amplitude of stress responses in cells and suggests that chromatin is a critically important component of the cellular response to stress.

  17. New mitotic regulators released from chromatin

    Directory of Open Access Journals (Sweden)

    Hideki eYokoyama

    2013-12-01

    Full Text Available Faithful action of the mitotic spindle segregates duplicated chromosomes into daughter cells. Perturbations of this process result in chromosome mis-segregation, leading to chromosomal instability and cancer development. Chromosomes are not simply passengers segregated by spindle microtubules but rather play a major active role in spindle assembly. The GTP bound form of the Ran GTPase (RanGTP, produced around chromosomes, locally activates spindle assembly factors. Recent studies have uncovered that chromosomes organize mitosis beyond spindle formation. They distinctly regulate other mitotic events, such as spindle maintenance in anaphase, which is essential for chromosome segregation. Furthermore, the direct function of chromosomes is not only to produce RanGTP but, in addition, to release key mitotic regulators from chromatin. Chromatin-remodeling factors and nuclear pore complex proteins, which have established functions on chromatin in interphase, dissociate from mitotic chromatin and function in spindle assembly or maintenance. Thus, chromosomes actively organize their own segregation using chromatin-releasing mitotic regulators as well as RanGTP.

  18. Dot1 binding induces chromatin rearrangements by histone methylation-dependent and -independent mechanisms

    Directory of Open Access Journals (Sweden)

    Stulemeijer Iris JE

    2011-02-01

    Full Text Available Abstract Background Methylation of histone H3 lysine 79 (H3K79 by Dot1 is highly conserved among species and has been associated with both gene repression and activation. To eliminate indirect effects and examine the direct consequences of Dot1 binding and H3K79 methylation, we investigated the effects of targeting Dot1 to different positions in the yeast genome. Results Targeting Dot1 did not activate transcription at a euchromatic locus. However, chromatin-bound Dot1 derepressed heterochromatin-mediated gene silencing over a considerable distance. Unexpectedly, Dot1-mediated derepression was established by both a H3K79 methylation-dependent and a methylation-independent mechanism; the latter required the histone acetyltransferase Gcn5. By monitoring the localization of a fluorescently tagged telomere in living cells, we found that the targeting of Dot1, but not its methylation activity, led to the release of a telomere from the repressive environment at the nuclear periphery. This probably contributes to the activity-independent derepression effect of Dot1. Conclusions Targeting of Dot1 promoted gene expression by antagonizing gene repression through both histone methylation and chromatin relocalization. Our findings show that binding of Dot1 to chromatin can positively affect local gene expression by chromatin rearrangements over a considerable distance.

  19. [Structural studies of chromatin remodeling factors].

    Science.gov (United States)

    Volokh, O I; Derkacheva, N I; Studitsky, V M; Sokolova, O S

    2016-01-01

    Changes of chromatin structure require participation of chromatin remodeling factors (CRFs), which are ATP-dependent multisubunit complexes that change the structure of the nucleosome without covalently modifying its components. CRFs act together with other protein factors to regulate the extent of chromatin condensation. Four CRF families are currently distinguished based on their structural and biochemical characteristics: SWI/SNF, ISWI, Mi-2/CHD, and SWR/INO80. X-ray diffraction analysis and electron microscopy are the main methods to obtain structural information about macromolecules. CRFs are difficult to obtain in crystal because of their large sizes and structural heterogeneity, and transmission electron microscopy (TEM) is mostly employed in their structural studies. The review considers all structures obtained for CRFs by TEM and discusses several models of CRF-nucleosome interactions.

  20. The Chromatin Fiber: Multiscale Problems and Approaches

    Science.gov (United States)

    Ozer, Gungor; Luque, Antoni; Schlick, Tamar

    2015-01-01

    The structure of chromatin, affected by many factors from DNA linker lengths to posttranslational modifications, is crucial to the regulation of eukaryotic cells. Combined experimental and computational methods have led to new insights into its structural and dynamical features, from interactions due to the flexible core histone tails of the nucleosomes to the physical mechanism driving the formation of chromosomal domains. Here we present a perspective of recent advances in chromatin modeling techniques at the atomic, mesoscopic, and chromosomal scales with a view toward developing multiscale computational strategies to integrate such findings. Innovative modeling methods that connect molecular to chromosomal scales are crucial for interpreting experiments and eventually deciphering the complex dynamic organization and function of chromatin in the cell. PMID:26057099

  1. Protocol: methodology for chromatin immunoprecipitation (ChIP in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Strenkert Daniela

    2011-11-01

    Full Text Available Abstract We report on a detailed chromatin immunoprecipitation (ChIP protocol for the unicellular green alga Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example for the normalization of ChIP results as determined by real-time PCR.

  2. EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex.

    Science.gov (United States)

    Shapira, Suzanne N; Lim, Hee-Woong; Rajakumari, Sona; Sakers, Alexander P; Ishibashi, Jeff; Harms, Matthew J; Won, Kyoung-Jae; Seale, Patrick

    2017-04-01

    The transcription factor early B-cell factor 2 (EBF2) is an essential mediator of brown adipocyte commitment and terminal differentiation. However, the mechanisms by which EBF2 regulates chromatin to activate brown fat-specific genes in adipocytes were unknown. ChIP-seq (chromatin immunoprecipitation [ChIP] followed by deep sequencing) analyses in brown adipose tissue showed that EBF2 binds and regulates the activity of lineage-specific enhancers. Mechanistically, EBF2 physically interacts with the chromatin remodeler BRG1 and the BAF chromatin remodeling complex in brown adipocytes. We identified the histone reader protein DPF3 as a brown fat-selective component of the BAF complex that was required for brown fat gene programming and mitochondrial function. Loss of DPF3 in brown adipocytes reduced chromatin accessibility at EBF2-bound enhancers and led to a decrease in basal and catecholamine-stimulated expression of brown fat-selective genes. Notably, Dpf3 is a direct transcriptional target of EBF2 in brown adipocytes, thereby establishing a regulatory module through which EBF2 activates and also recruits DPF3-anchored BAF complexes to chromatin. Together, these results reveal a novel mechanism by which EBF2 cooperates with a tissue-specific chromatin remodeling complex to activate brown fat identity genes. © 2017 Shapira et al.; Published by Cold Spring Harbor Laboratory Press.

  3. Replicating chromatin: a tale of histones

    DEFF Research Database (Denmark)

    Groth, Anja

    2009-01-01

    framework of chromatin and carry information to specify higher-order organization and gene expression. When replication forks traverse the chromosomes, nucleosomes are transiently disrupted, allowing the replication machinery to gain access to DNA. Histone recycling, together with new deposition, ensures...... reassembly on nascent DNA strands. The aim of this review is to discuss how histones - new and old - are handled at the replication fork, highlighting new mechanistic insights and revisiting old paradigms.......Chromatin serves structural and functional roles crucial for genome stability and correct gene expression. This organization must be reproduced on daughter strands during replication to maintain proper overlay of epigenetic fabric onto genetic sequence. Nucleosomes constitute the structural...

  4. Targeted Sister Chromatid Cohesion by Sir2

    Science.gov (United States)

    Wu, Ching-Shyi; Chen, Yu-Fan; Gartenberg, Marc R.

    2011-01-01

    The protein complex known as cohesin binds pericentric regions and other sites of eukaryotic genomes to mediate cohesion of sister chromatids. In budding yeast Saccharomyces cerevisiae, cohesin also binds silent chromatin, a repressive chromatin structure that functionally resembles heterochromatin of higher eukaryotes. We developed a protein-targeting assay to investigate the mechanistic basis for cohesion of silent chromatin domains. Individual silencing factors were tethered to sites where pairing of sister chromatids could be evaluated by fluorescence microscopy. We report that the evolutionarily conserved Sir2 histone deacetylase, an essential silent chromatin component, was both necessary and sufficient for cohesion. The cohesin genes were required, but the Sir2 deacetylase activity and other silencing factors were not. Binding of cohesin to silent chromatin was achieved with a small carboxyl terminal fragment of Sir2. Taken together, these data define a unique role for Sir2 in cohesion of silent chromatin that is distinct from the enzyme's role as a histone deacetylase. PMID:21304892

  5. The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells.

    Science.gov (United States)

    Daer, René M; Cutts, Josh P; Brafman, David A; Haynes, Karmella A

    2017-03-17

    In order to efficiently edit eukaryotic genomes, it is critical to test the impact of chromatin dynamics on CRISPR/Cas9 function and develop strategies to adapt the system to eukaryotic contexts. So far, research has extensively characterized the relationship between the CRISPR endonuclease Cas9 and the composition of the RNA-DNA duplex that mediates the system's precision. Evidence suggests that chromatin modifications and DNA packaging can block eukaryotic genome editing by custom-built DNA endonucleases like Cas9; however, the underlying mechanism of Cas9 inhibition is unclear. Here, we demonstrate that closed, gene-silencing-associated chromatin is a mechanism for the interference of Cas9-mediated DNA editing. Our assays use a transgenic cell line with a drug-inducible switch to control chromatin states (open and closed) at a single genomic locus. We show that closed chromatin inhibits binding and editing at specific target sites and that artificial reversal of the silenced state restores editing efficiency. These results provide new insights to improve Cas9-mediated editing in human and other mammalian cells.

  6. Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape

    Science.gov (United States)

    Camp, J. Gray; Frank, Christopher L.; Lickwar, Colin R.; Guturu, Harendra; Rube, Tomas; Wenger, Aaron M.; Chen, Jenny; Bejerano, Gill; Crawford, Gregory E.

    2014-01-01

    Microbiota regulate intestinal physiology by modifying host gene expression along the length of the intestine, but the underlying regulatory mechanisms remain unresolved. Transcriptional specificity occurs through interactions between transcription factors (TFs) and cis-regulatory regions (CRRs) characterized by nucleosome-depleted accessible chromatin. We profiled transcriptome and accessible chromatin landscapes in intestinal epithelial cells (IECs) from mice reared in the presence or absence of microbiota. We show that regional differences in gene transcription along the intestinal tract were accompanied by major alterations in chromatin accessibility. Surprisingly, we discovered that microbiota modify host gene transcription in IECs without significantly impacting the accessible chromatin landscape. Instead, microbiota regulation of host gene transcription might be achieved by differential expression of specific TFs and enrichment of their binding sites in nucleosome-depleted CRRs near target genes. Our results suggest that the chromatin landscape in IECs is preprogrammed by the host in a region-specific manner to permit responses to microbiota through binding of open CRRs by specific TFs. PMID:24963153

  7. Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation.

    Science.gov (United States)

    Kieffer-Kwon, Kyong-Rim; Nimura, Keisuke; Rao, Suhas S P; Xu, Jianliang; Jung, Seolkyoung; Pekowska, Aleksandra; Dose, Marei; Stevens, Evan; Mathe, Ewy; Dong, Peng; Huang, Su-Chen; Ricci, Maria Aurelia; Baranello, Laura; Zheng, Ying; Tomassoni Ardori, Francesco; Resch, Wolfgang; Stavreva, Diana; Nelson, Steevenson; McAndrew, Michael; Casellas, Adriel; Finn, Elizabeth; Gregory, Charles; St Hilaire, Brian Glenn; Johnson, Steven M; Dubois, Wendy; Cosma, Maria Pia; Batchelor, Eric; Levens, David; Phair, Robert D; Misteli, Tom; Tessarollo, Lino; Hager, Gordon; Lakadamyali, Melike; Liu, Zhe; Floer, Monique; Shroff, Hari; Aiden, Erez Lieberman; Casellas, Rafael

    2017-08-17

    50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells. Published by Elsevier Inc.

  8. Chromatin association of UHRF1 during the cell cycle

    KAUST Repository

    Al-Gashgari, Bothayna

    2017-05-01

    Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is a nuclear protein that associates with chromatin. Regardless of the various functions of UHRF1 in the cell, one of its more important functions is its role in the maintenance of DNA methylation patterns by the recruitment of DNMT1. Studies on UHRF1 based on this function have revealed the importance of UHRF1 during the cell cycle. Moreover, based on different studies various factors were described to be involved in the regulation of UHRF1 with different functionalities that can control its binding affinity to different targets on chromatin. These factors are regulated differently in a cell cycle specific manner. In light of this, we propose that UHRF1 has different binding behaviors during the cell cycle in regard to its association with chromatin. In this project, we first analyzed the binding behavior of endogenous UHRF1 from different unsynchronized cell systems in pull-down assays with peptides and oligonucleotides. Moreover, to analyze UHRF1 binding behavior during the cell cycle, we used two different approaches. First we sorted Jurkat and HT1080 cells based on their cell cycle stage using FACS analysis. Additionally, we synchronized HeLa cells to different stages of the cell cycle by chemical treatments, and used extracts from cellsorting and cell synchronization experiments for pull-down assays. We observed that UHRF1 in different cell systems has different preferences in regard to its binding to H3 unmodified and H3K9me3. Moreover, we detected that UHRF1, in general, displays different patterns between different stages of cell cycle; however, we cannot draw a final model for UHRF1 binding pattern during cell cycle.

  9. Chromatin Repressive Complexes in Stem Cells, Development, and Cancer

    DEFF Research Database (Denmark)

    Laugesen, Anne; Helin, Kristian

    2014-01-01

    The chromatin environment is essential for the correct specification and preservation of cell identity through modulation and maintenance of transcription patterns. Many chromatin regulators are required for development, stem cell maintenance, and differentiation. Here, we review the roles...

  10. Nuclear c-Abl-mediated tyrosine phosphorylation induces chromatin structural changes through histone modifications that include H4K16 hypoacetylation

    Energy Technology Data Exchange (ETDEWEB)

    Aoyama, Kazumasa; Fukumoto, Yasunori; Ishibashi, Kenichi; Kubota, Sho; Morinaga, Takao; Horiike, Yasuyoshi; Yuki, Ryuzaburo; Takahashi, Akinori; Nakayama, Yuji; Yamaguchi, Naoto, E-mail: nyama@p.chiba-u.ac.jp

    2011-12-10

    c-Abl tyrosine kinase, which is ubiquitously expressed, has three nuclear localization signals and one nuclear export signal and can shuttle between the nucleus and the cytoplasm. c-Abl plays important roles in cell proliferation, adhesion, migration, and apoptosis. Recently, we developed a pixel imaging method for quantitating the level of chromatin structural changes and showed that nuclear Src-family tyrosine kinases are involved in chromatin structural changes upon growth factor stimulation. Using this method, we show here that nuclear c-Abl induces chromatin structural changes in a manner dependent on the tyrosine kinase activity. Expression of nuclear-targeted c-Abl drastically increases the levels of chromatin structural changes, compared with that of c-Abl. Intriguingly, nuclear-targeted c-Abl induces heterochromatic profiles of histone methylation and acetylation, including hypoacetylation of histone H4 acetylated on lysine 16 (H4K16Ac). The level of heterochromatic histone modifications correlates with that of chromatin structural changes. Adriamycin-induced DNA damage stimulates translocation of c-Abl into the nucleus and induces chromatin structural changes together with H4K16 hypoacetylation. Treatment with trichostatin A, a histone deacetylase inhibitor, blocks chromatin structural changes but not nuclear tyrosine phosphorylation by c-Abl. These results suggest that nuclear c-Abl plays an important role in chromatin dynamics through nuclear tyrosine phosphorylation-induced heterochromatic histone modifications.

  11. Chromatin Modifications Associated With Diabetes and Obesity.

    Science.gov (United States)

    Schones, Dustin E; Leung, Amy; Natarajan, Rama

    2015-07-01

    The incidence of obesity across the globe has doubled over the past several decades, leading to escalating rates of diabetes mellitus, cardiovascular disease, and other complications. Given this dramatic rise in disease incidence, understanding the cause of these diseases is therefore of paramount importance. Metabolic diseases, such as obesity and diabetes mellitus, result from a multitude of genetic and environmental factors. Although the genetic basis of these diseases has been extensively studied, the molecular pathways whereby environmental factors influence disease progression are only beginning to be understood. One manner by which environmental factors can contribute to disease progression is through modifications to chromatin. The highly structured packaging of the genome into the nucleus through chromatin has been shown to be fundamental to tissue-specific gene regulation. Modifications to chromatin can regulate gene expression and are involved in a myriad of biological functions, and hence, disruption of these modifications is central to many human diseases. These modifications can furthermore be epigenetic in nature, thereby contributing to prolonged disease risk. Recent work has demonstrated that modifications to chromatin are associated with the progression of both diabetes mellitus and obesity, which is the subject of this review. © 2015 American Heart Association, Inc.

  12. Keystone Symposia on Epigenomics and Chromatin Dynamics

    DEFF Research Database (Denmark)

    Ravnskjær, Kim

    2012-01-01

    Keystone Symposia kicked off the start of 2012 with two joint meetings on Epigenomics and Chromatin Dynamics and a star-studded list of speakers. Held in Keystone, CO, January 17-22, and organized by Steven Jacobsen and Steven Henikoff and by Bradley Cairns and Geneviève Almouzni, respectively...

  13. Fibronectin: a chromatin-associated protein?

    Science.gov (United States)

    Zardi, L; Siri, A; Carnemolla, B; Santi, L; Gardner, W D; Hoch, S O

    1979-11-01

    We have previously reported that chromatin preparations from human cultured fibroblasts contain a single homologous serum protein. In this paper we present evidence, based on immunological identity and physicochemical properties, that this serum protein is fibronectin. Furthermore, using a radioimmunoassay system, we have estimated that fibronectin represents about 0.7% of the total protein in both chromatin preparations and whole fibroblasts. Using a nitrocellulose filter assay system, we also show that fibronectin is a DNA-binding protein having an equilibrium constant of 4.6 x 10(-6) M. Equilibrium competition experiments have demonstrated that fibronectin has the ability to differentiate among nucleotides, indicating that fibronectin-DNA interaction is at least partially specific, and that a minimum polymer length of 12-18 nucleotides is required for effective binding to occur. Fibronectin has been isolated readily from plasma using DNA-affinity chromatography. We do not have direct evidence that fibronectin is an actual nonhistone chromosomal protein, but fibronectin is a DNA-binding protein (at least under in vitro assay conditions) and appears to be a normal constituent of chromatin as chromatin is currently isolated from cell nuclei.

  14. Chromatin and epigenetics in all their states

    NARCIS (Netherlands)

    Bey, Till; Jamge, Suraj; Klemme, Sonja; Komar, Dorota Natalia; Gall, Le Sabine; Mikulski, Pawel; Schmidt, Martin; Zicola, Johan; Berr, Alexandre

    2016-01-01

    In January 2016, the first Epigenetic and Chromatin Regulation of Plant Traits conference was held in Strasbourg, France. An all-star lineup of speakers, a packed audience of 130 participants from over 20 countries, and a friendly scientific atmosphere contributed to make this conference a

  15. Chromatin Dynamics of the mouse β-globin locus

    NARCIS (Netherlands)

    M.P.C. van de Corput (Mariëtte); E. de Boer (Ernie); T.A. Knoch (Tobias); W.A. van Cappellen (Gert); M. Lesnussa (Michael); H.J.F.M.M. Eussen (Bert)

    2010-01-01

    textabstractLately it has become more clear that (subtle) changes in 3D organization of chromatin can either trigger transcription or silence genes or gene clusters. It has also been postulated that due to changes in chromatin structure, a change in chromatin accessibility of transcription factors

  16. Spatially confined folding of chromatin in the interphase nucleus

    NARCIS (Netherlands)

    Mateos-Langerak, Julio; Bohn, Manfred; de Leeuw, Wim; Giromus, Osdilly; Manders, Erik M. M.; Verschure, Pernette J.; Indemans, Mireille H. G.; Gierman, Hinco J.; Heermann, Dieter W.; van Driel, Roel; Goetze, Sandra

    2009-01-01

    Genome function in higher eukaryotes involves major changes in the spatial organization of the chromatin fiber. Nevertheless, our understanding of chromatin folding is remarkably limited. Polymer models have been used to describe chromatin folding. However, none of the proposed models gives a

  17. Direct Chromatin PCR (DC-PCR): Hypotonic Conditions Allow Differentiation of Chromatin States during Thermal Cycling

    Science.gov (United States)

    Vatolin, Sergei; Khan, Shahper N.; Reu, Frederic J.

    2012-01-01

    Current methods to study chromatin configuration are not well suited for high throughput drug screening since they require large cell numbers and multiple experimental steps that include centrifugation for isolation of nuclei or DNA. Here we show that site specific chromatin analysis can be achieved in one step by simply performing direct chromatin PCR (DC-PCR) on cells. The basic underlying observation was that standard hypotonic PCR buffers prevent global cellular chromatin solubilization during thermal cycling while more loosely organized chromatin can be amplified. Despite repeated heating to >90°C, 41 of 61 tested 5′ sequences of silenced genes (CDKN2A, PU.1, IRF4, FOSB, CD34) were not amplifiable while 47 could be amplified from expressing cells. Two gene regions (IRF4, FOSB) even required pre-heating of cells in isotonic media to allow this differentiation; otherwise none of 19 assayed sequences yielded PCR products. Cells with baseline expression or epigenetic reactivation gave similar DC-PCR results. Silencing during differentiation of CD34 positive cord blood cells closed respective chromatin while treatment of myeloma cells with an IRF4 transcriptional inhibitor opened a site to DC-PCR that was occupied by RNA polymerase II and NFκB as determined by ChIP. Translation into real-time PCR can not be achieved with commercial real-time PCR buffers which potently open chromatin, but even with simple ethidium bromide addition to standard PCR mastermix we were able to identify hits in small molecules screens that suppressed IRF4 expression or reactivated CDKN2A in myeloma cells using densitometry or visual inspection of PCR plates under UV light. While need in drug development inspired this work, application to genome-wide analysis appears feasible using phi29 for selective amplification of open cellular chromatin followed by library construction from supernatants since such supernatants yielded similar results as gene specific DC-PCR. PMID:22984542

  18. Reading the Epigenetic State of Chromatin Alters its Accessibility

    Science.gov (United States)

    Gibson, Matthew D.

    The eukaryotic genome is organized into a structural polymer called chromatin. Ultimately, all access to genetic information is regulated by chromatin including access required for DNA replication, transcription, and repair. The basic repeating unit of chromatin is the nucleosome which is comprised of ˜147 bp of DNA tightly wrapped around a protein histone octamer core. The histone octamer is made up of eight proteins: two each of histones H2A, H2B, H3, and H4. Many mechanisms exist to regulate access to DNA but one of pivotal importance is the creation of unique nucleosomes through i) integration of histone variants and ii) deposition of post translational modifications (PTMs). These modifications help comprise the epigenome of a cell. Classically, the two mechanisms by which they function have been through a direct regulation of nucleosome dynamics, or through third party proteins which are able to recognize the variants or PTMs and facilitate work. The library of potential PTMs therefore forms a sort of histone code which regulates access to DNA. This thesis investigates the intersection of these mechanisms to determine whether the act of recognizing epigenetic information alters DNA accessibility. The primary method used to determine changes in DNA accessibility is though observing the effective binding affinity of a transcription factor to its target site buried within a recombinantly prepared nucleosome which has been modified to carry a PTM and to report on its wrapping state. We find different regulation depending both on the PTM we investigate and the specific PTM-binding protein. We first investigate the H3K36me3-binding protein PHF1 and find that while the PTM it recognizes, H3K36me3, does not alter DNA accessibility, the binding of its recognition domain and N-terminal domain can illicit a change of DNA accessibility of 8 +/- 2-fold. This means that 8 times less DNA binding protein is required to occupy its target site if the nucleosome is bound by PHF

  19. Lamins and lamin-binding proteins in functional chromatin organization.

    Science.gov (United States)

    Gotzmann, J; Foisner, R

    1999-01-01

    Lamins are the major components of the nuclear lamina, a two-dimensional filamentous network at the periphery of the nucleus in higher eukaryotes, directly underlying the inner nuclear membrane. Several integral proteins of the inner nuclear membrane bind to lamins and may link the nuclear membrane to the core lamina network. The lamins and the lamin-binding proteins lamina-associated polypeptide (LAP)2beta and lamin B receptor (LBR) have been described to bind to DNA or to interact with chromatin via histones, BAF-1, and HP1 chromodomain proteins, respectively, and may provide anchorage sites for chromatin fibers at the nuclear periphery. In addition, lamin A structures on intranuclear filaments, or lamin B in replication foci have been described in the nuclear interior, but their specific roles remain unclear. An isoform of the LAP2 protein family, LAP2alpha, has been found to colocalize with A-type lamins in the nucleoplasm and might be involved in intranuclear structure organization. In the course of cell-cycle-dependent dynamics of the nucleus in higher eukaryotes, lamins as well as lamin-binding proteins seem to possess important functions during various steps of post-mitotic nuclear reassembly, including cross-linking of chromatides, nuclear membrane targeting, nuclear lamina assembly, and the formation of a replication-competent nucleus.

  20. Capture-C reveals preformed chromatin interactions between HIF-binding sites and distant promoters.

    Science.gov (United States)

    Platt, James L; Salama, Rafik; Smythies, James; Choudhry, Hani; Davies, James Oj; Hughes, Jim R; Ratcliffe, Peter J; Mole, David R

    2016-10-01

    Hypoxia-inducible factor (HIF) directs an extensive transcriptional cascade that transduces numerous adaptive responses to hypoxia. Pan-genomic analyses, using chromatin immunoprecipitation and transcript profiling, have revealed large numbers of HIF-binding sites that are generally associated with hypoxia-inducible transcripts, even over long chromosomal distances. However, these studies do not define the specific targets of HIF-binding sites and do not reveal how induction of HIF affects chromatin conformation over distantly connected functional elements. To address these questions, we deployed a recently developed chromosome conformation assay that enables simultaneous high-resolution analyses from multiple viewpoints. These assays defined specific long-range interactions between intergenic HIF-binding regions and one or more promoters of hypoxia-inducible genes, revealing the existence of multiple enhancer-promoter, promoter-enhancer, and enhancer-enhancer interactions. However, neither short-term activation of HIF by hypoxia, nor long-term stabilization of HIF in von Hippel-Lindau (VHL)-defective cells greatly alters these interactions, indicating that at least under these conditions, HIF can operate on preexisting patterns of chromatin-chromatin interactions that define potential transcriptional targets and permit rapid gene activation by hypoxic stress. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  1. ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

    DEFF Research Database (Denmark)

    Dann, Geoffrey P; Liszczak, Glen P; Bagert, John D

    2017-01-01

    ATP-dependent chromatin remodellers regulate access to genetic information by controlling nucleosome positions in vivo. However, the mechanism by which remodellers discriminate between different nucleosome substrates is poorly understood. Many chromatin remodelling proteins possess conserved...... activity of all ISWI remodellers evaluated. This dependence also extends to CHD and SWI/SNF family remodellers, suggesting that the acidic patch may be generally required for chromatin remodelling. Critically, remodelling activity can be regulated by modifications neighbouring the acidic patch, signifying...... that it may act as a tunable interaction hotspot for ATP-dependent chromatin remodellers and, by extension, many other chromatin effectors that engage this region of the nucleosome surface....

  2. Quantification of chromatin condensation level by image processing.

    Science.gov (United States)

    Irianto, Jerome; Lee, David A; Knight, Martin M

    2014-03-01

    The level of chromatin condensation is related to the silencing/activation of chromosomal territories and therefore impacts on gene expression. Chromatin condensation changes during cell cycle, progression and differentiation, and is influenced by various physicochemical and epigenetic factors. This study describes a validated experimental technique to quantify chromatin condensation. A novel image processing procedure is developed using Sobel edge detection to quantify the level of chromatin condensation from nuclei images taken by confocal microscopy. The algorithm was developed in MATLAB and used to quantify different levels of chromatin condensation in chondrocyte nuclei achieved through alteration in osmotic pressure. The resulting chromatin condensation parameter (CCP) is in good agreement with independent multi-observer qualitative visual assessment. This image processing technique thereby provides a validated unbiased parameter for rapid and highly reproducible quantification of the level of chromatin condensation. Copyright © 2013 IPEM. Published by Elsevier Ltd. All rights reserved.

  3. Histone phosphorylation: a chromatin modification involved in diverse nuclear events.

    Science.gov (United States)

    Rossetto, Dorine; Avvakumov, Nikita; Côté, Jacques

    2012-10-01

    Histone posttranslational modifications are key components of diverse processes that modulate chromatin structure. These marks function as signals during various chromatin-based events, and act as platforms for recruitment, assembly or retention of chromatin-associated factors. The best-known function of histone phosphorylation takes place during cellular response to DNA damage, when phosphorylated histone H2A(X) demarcates large chromatin domains around the site of DNA breakage. However, multiple studies have also shown that histone phosphorylation plays crucial roles in chromatin remodeling linked to other nuclear processes. In this review, we summarize the current knowledge of histone phosphorylation and describe the many kinases and phosphatases that regulate it. We discuss the key roles played by this histone mark in DNA repair, transcription and chromatin compaction during cell division and apoptosis. Additionally, we describe the intricate crosstalk that occurs between phosphorylation and other histone modifications and allows for sophisticated control over the chromatin remodeling processes.

  4. SUMO-2 Orchestrates Chromatin Modifiers in Response to DNA Damage

    DEFF Research Database (Denmark)

    Hendriks, Ivo A; Treffers, Louise W; Verlaan-de Vries, Matty

    2015-01-01

    Small ubiquitin-like modifiers play critical roles in the DNA damage response (DDR). To increase our understanding of SUMOylation in the mammalian DDR, we employed a quantitative proteomics approach in order to identify dynamically regulated SUMO-2 conjugates and modification sites upon treatment...... with the DNA damaging agent methyl methanesulfonate (MMS). We have uncovered a dynamic set of 20 upregulated and 33 downregulated SUMO-2 conjugates, and 755 SUMO-2 sites, of which 362 were dynamic in response to MMS. In contrast to yeast, where a response is centered on homologous recombination, we identified......-targeted ubiquitin ligase RNF4 and degraded by the proteasome in response to DNA damage, JARID1C was SUMOylated and recruited to the chromatin to demethylate histone H3K4....

  5. Epigenetic effects of chromatin remodeling agents on organotypic cultures.

    Science.gov (United States)

    Sirchia, Silvia M; Faversani, Alice; Rovina, Davide; Russo, Maria V; Paganini, Leda; Savi, Federica; Augello, Claudia; Rosso, Lorenzo; Del Gobbo, Alessandro; Tabano, Silvia; Bosari, Silvano; Miozzo, Monica

    2016-03-01

    Tumor epigenetic defects are of increasing relevance to clinical practice, because they are 'druggable' targets for cancer therapy using chromatin-remodeling agents (CRAs). New evidences highlight the importance of the microenvironment on the epigenome regulation and the need to use culture models able to preserve tissue morphology, to better understand the action of CRAs. Methods & methods: We studied the epigenetic response induced by culturing and CRAs in a preclinical model, preserving ex vivo the original tissue microenvironment and morphology, assessing different epigenetic signatures. Our overall findings suggest that culturing and CRAs cause heterogeneous effects on the genes methylation; CRAs affect the global DNA methylation and can trigger an active DNA demethylation; the culture induces alterations in the histone deacetylase expression. Despite the limited number of cases, these findings can be considered a proof of concept of the possibility to test CRAs epigenetic effects on ex vivo tissues maintained in their native tissue architecture.

  6. Histone chaperone networks shaping chromatin function

    DEFF Research Database (Denmark)

    Hammond, Colin; Strømme, Caroline Bianchi; Huang, Hongda

    2017-01-01

    The association of histones with specific chaperone complexes is important for their folding, oligomerization, post-translational modification, nuclear import, stability, assembly and genomic localization. In this way, the chaperoning of soluble histones is a key determinant of histone availabili...... chaperone network and via co-chaperone complexes to match histone supply with demand, thereby promoting proper nucleosome assembly and maintaining epigenetic information by recycling modified histones evicted from chromatin....

  7. Bromodomain Protein Brd4 Associated with Acetylated Chromatin Is Important for Maintenance of Higher-order Chromatin Structure*

    Science.gov (United States)

    Wang, Ranran; Li, Qing; Helfer, Christine M.; Jiao, Jing; You, Jianxin

    2012-01-01

    Chromatin structure organization is crucial for regulating many fundamental cellular processes. However, the molecular mechanism that regulates the assembly of higher-order chromatin structure remains poorly understood. In this study, we demonstrate that Brd4 (bromodomain-containing protein 4) protein participates in the maintenance of the higher-order chromatin structure. Brd4, a member of the BET family of proteins, has been shown to play important roles in cellular growth control, cell cycle progression, and cancer development. We apply in situ single cell chromatin imaging and micrococcal nuclease (MNase) assay to show that Brd4 depletion leads to a large scale chromatin unfolding. A dominant-negative inhibitor encoding the double bromodomains (BDI/II) of Brd4 can competitively dissociate endogenous Brd4 from chromatin to trigger severely fragmented chromatin morphology. Mechanistic studies using Brd4 truncation mutants reveal that the Brd4 C-terminal domain is crucial for maintaining normal chromatin structure. Using bimolecular fluorescence complementation technology, we demonstrate that Brd4 molecules interact intermolecularly on chromatin and that replacing Brd4 molecules by BDI/II causes abnormal nucleosome aggregation and chromatin fragmentation. These studies establish a novel structural role of Brd4 in supporting the higher chromatin architecture. PMID:22334664

  8. Chromatin fiber allostery and the epigenetic code

    Science.gov (United States)

    Lesne, Annick; Foray, Nicolas; Cathala, Guy; Forné, Thierry; Wong, Hua; Victor, Jean-Marc

    2015-02-01

    The notion of allostery introduced for proteins about fifty years ago has been extended since then to DNA allostery, where a locally triggered DNA structural transition remotely controls other DNA-binding events. We further extend this notion and propose that chromatin fiber allosteric transitions, induced by histone-tail covalent modifications, may play a key role in transcriptional regulation. We present an integrated scenario articulating allosteric mechanisms at different scales: allosteric transitions of the condensed chromatin fiber induced by histone-tail acetylation modify the mechanical constraints experienced by the embedded DNA, thus possibly controlling DNA-binding of allosteric transcription factors or further allosteric mechanisms at the linker DNA level. At a higher scale, different epigenetic constraints delineate different statistically dominant subsets of accessible chromatin fiber conformations, which each favors the assembly of dedicated regulatory complexes, as detailed on the emblematic example of the mouse Igf2-H19 gene locus and its parental imprinting. This physical view offers a mechanistic and spatially structured explanation of the observed correlation between transcriptional activity and histone modifications. The evolutionary origin of allosteric control supports to speak of an ‘epigenetic code’, by which events involved in transcriptional regulation are encoded in histone modifications in a context-dependent way.

  9. Titration and hysteresis in epigenetic chromatin silencing

    Science.gov (United States)

    Dayarian, Adel; Sengupta, Anirvan M.

    2013-06-01

    Epigenetic mechanisms of silencing via heritable chromatin modifications play a major role in gene regulation and cell fate specification. We consider a model of epigenetic chromatin silencing in budding yeast and study the bifurcation diagram and characterize the bistable and the monostable regimes. The main focus of this paper is to examine how the perturbations altering the activity of histone modifying enzymes affect the epigenetic states. We analyze the implications of having the total number of silencing proteins, given by the sum of proteins bound to the nucleosomes and the ones available in the ambient, to be constant. This constraint couples different regions of chromatin through the shared reservoir of ambient silencing proteins. We show that the response of the system to perturbations depends dramatically on the titration effect caused by the above constraint. In particular, for a certain range of overall abundance of silencing proteins, the hysteresis loop changes qualitatively with certain jump replaced by continuous merger of different states. In addition, we find a nonmonotonic dependence of gene expression on the rate of histone deacetylation activity of Sir2. We discuss how these qualitative predictions of our model could be compared with experimental studies of the yeast system under anti-silencing drugs.

  10. Capturing Structural Heterogeneity in Chromatin Fibers.

    Science.gov (United States)

    Ekundayo, Babatunde; Richmond, Timothy J; Schalch, Thomas

    2017-10-13

    Chromatin fiber organization is implicated in processes such as transcription, DNA repair and chromosome segregation, but how nucleosomes interact to form higher-order structure remains poorly understood. We solved two crystal structures of tetranucleosomes with approximately 11-bp DNA linker length at 5.8 and 6.7 Å resolution. Minimal intramolecular nucleosome-nucleosome interactions result in a fiber model resembling a flat ribbon that is compatible with a two-start helical architecture, and that exposes histone and DNA surfaces to the environment. The differences in the two structures combined with electron microscopy reveal heterogeneous structural states, and we used site-specific chemical crosslinking to assess the diversity of nucleosome-nucleosome interactions through identification of structure-sensitive crosslink sites that provide a means to characterize fibers in solution. The chromatin fiber architectures observed here provide a basis for understanding heterogeneous chromatin higher-order structures as they occur in a genomic context. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Plant chromatin warms up in Madrid: meeting summary of the 3rd European Workshop on Plant Chromatin 2013, Madrid, Spain.

    Science.gov (United States)

    Jarillo, José A; Gaudin, Valérie; Hennig, Lars; Köhler, Claudia; Piñeiro, Manuel

    2014-04-01

    The 3rd European Workshop on Plant Chromatin (EWPC) was held on August 2013 in Madrid, Spain. A number of different topics on plant chromatin were presented during the meeting, including new factors mediating Polycomb Group protein function in plants, chromatin-mediated reprogramming in plant developmental transitions, the role of histone variants, and newly identified chromatin remodeling factors. The function of interactions between chromatin and transcription factors in the modulation of gene expression, the role of chromatin dynamics in the control of nuclear processes and the influence of environmental factors on chromatin organization were also reported. In this report, we highlight some of the new insights emerging in this growing area of research, presented at the 3rd EWPC.

  12. PTEN Interacts with Histone H1 and Controls Chromatin Condensation

    Science.gov (United States)

    Chen, Zhu Hong; Zhu, Minglu; Yang, Jingyi; Liang, Hui; He, Jinxue; He, Shiming; Wang, Pan; Kang, Xi; McNutt, Michael A.; Yin, Yuxin; Shen, Wen H.

    2014-01-01

    SUMMARY Chromatin organization and dynamics are integral to global gene transcription. Histone modification influences chromatin status and gene expression. PTEN plays multiple roles in tumor suppression, development and metabolism. Here we report on the interplay of PTEN, histone H1 and chromatin. We show that loss of PTEN leads to dissociation of histone H1 from chromatin and decondensation of chromatin. PTEN deletion also results in elevation of histone H4 acetylation at lysine 16, an epigenetic marker for chromatin activation. We found that PTEN and histone H1 physically interact through their C-terminal domains. Disruption of the PTEN C-terminus promotes the chromatin association of MOF acetyltransferase and induces H4K16 acetylation. Hyperacetylation of H4K16 impairs the association of PTEN with histone H1, which constitutes regulatory feedback that may deteriorate chromatin stability. Our results demonstrate that PTEN controls chromatin condensation, thus influencing gene expression. We propose that PTEN regulates global gene transcription profiling through histones and chromatin remodeling. PMID:25199838

  13. Recognition of chromatin by the plant alkaloid, ellipticine as a dual binder

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Amrita; Sanyal, Sulagna; Majumder, Parijat [Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics, Block-AF, Sector-1, Bidhan Nagar, Kolkata 700064, West Bengal (India); Chakraborty, Payal [Bionivid Technology Pvt Ltd, Kasturi Nagar, Bangalore 560043 (India); Jana, Kuladip [Division of Molecular Medicine, Centre for Translational Animal Research, Bose Institute, P-1/12 C.I.T. Scheme VIIM, Kolkata 700054, West Bengal (India); Das, Chandrima, E-mail: chandrima.das@saha.ac.in [Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics, Block-AF, Sector-1, Bidhan Nagar, Kolkata 700064, West Bengal (India); Dasgupta, Dipak, E-mail: dipak.dasgupta@saha.ac.in [Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics, Block-AF, Sector-1, Bidhan Nagar, Kolkata 700064, West Bengal (India)

    2015-07-10

    Recognition of core histone components of chromatin along with chromosomal DNA by a class of small molecule modulators is worth examining to evaluate their intracellular mode of action. A plant alkaloid ellipticine (ELP) which is a putative anticancer agent has so far been reported to function via DNA intercalation, association with topoisomerase II and binding to telomere region. However, its effect upon the potential intracellular target, chromatin is hitherto unreported. Here we have characterized the biomolecular recognition between ELP and different hierarchical levels of chromatin. The significant result is that in addition to DNA, it binds to core histone(s) and can be categorized as a ‘dual binder’. As a sequel to binding with histone(s) and core octamer, it alters post-translational histone acetylation marks. We have further demonstrated that it has the potential to modulate gene expression thereby regulating several key biological processes such as nuclear organization, transcription, translation and histone modifications. - Highlights: • Ellipticine acts a dual binder binding to both DNA and core histone(s). • It induces structural perturbations in chromatin, chromatosome and histone octamer. • It alters histones acetylation and affects global gene expression.

  14. Cotranscriptional Chromatin Remodeling by Small RNA Species: An HTLV-1 Perspective

    Directory of Open Access Journals (Sweden)

    Nishat Aliya

    2012-01-01

    Full Text Available Cell type specificity of human T cell leukemia virus 1 has been proposed as a possible reason for differential viral outcome in primary target cells versus secondary. Through chromatin remodeling, the HTLV-1 transactivator protein Tax interacts with cellular factors at the chromosomally integrated viral promoter to activate downstream genes and control viral transcription. RNA interference is the host innate defense mechanism mediated by short RNA species (siRNA or miRNA that regulate gene expression. There exists a close collaborative functioning of cellular transcription factors with miRNA in order to regulate the expression of a number of eukaryotic genes including those involved in suppression of cell growth, induction of apoptosis, as well as repressing viral replication and propagation. In addition, it has been suggested that retroviral latency is influenced by chromatin alterations brought about by miRNA. Since Tax requires the assembly of transcriptional cofactors to carry out viral gene expression, there might be a close association between miRNA influencing chromatin alterations and Tax-mediated LTR activation. Herein we explore the possible interplay between HTLV-1 infection and miRNA pathways resulting in chromatin reorganization as one of the mechanisms determining HTLV-1 cell specificity and viral fate in different cell types.

  15. A Polycomb Group Protein Is Retained at Specific Sites on Chromatin in Mitosis

    Science.gov (United States)

    Follmer, Nicole E.; Wani, Ajazul H.; Francis, Nicole J.

    2012-01-01

    Epigenetic regulation of gene expression, including by Polycomb Group (PcG) proteins, may depend on heritable chromatin states, but how these states can be propagated through mitosis is unclear. Using immunofluorescence and biochemical fractionation, we find PcG proteins associated with mitotic chromosomes in Drosophila S2 cells. Genome-wide sequencing of chromatin immunoprecipitations (ChIP–SEQ) from mitotic cells indicates that Posterior Sex Combs (PSC) is not present at well-characterized PcG targets including Hox genes in mitosis, but does remain at a subset of interphase sites. Many of these persistent sites overlap with chromatin domain borders described by Sexton et al. (2012), which are genomic regions characterized by low levels of long range contacts. Persistent PSC binding sites flank both Hox gene clusters. We hypothesize that disruption of long-range chromatin contacts in mitosis contributes to PcG protein release from most sites, while persistent binding at sites with minimal long-range contacts may nucleate re-establishment of PcG binding and chromosome organization after mitosis. PMID:23284300

  16. PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes.

    Science.gov (United States)

    Soto-Feliciano, Yadira M; Bartlebaugh, Jordan M E; Liu, Yunpeng; Sánchez-Rivera, Francisco J; Bhutkar, Arjun; Weintraub, Abraham S; Buenrostro, Jason D; Cheng, Christine S; Regev, Aviv; Jacks, Tyler E; Young, Richard A; Hemann, Michael T

    2017-05-15

    Developmental and lineage plasticity have been observed in numerous malignancies and have been correlated with tumor progression and drug resistance. However, little is known about the molecular mechanisms that enable such plasticity to occur. Here, we describe the function of the plant homeodomain finger protein 6 (PHF6) in leukemia and define its role in regulating chromatin accessibility to lineage-specific transcription factors. We show that loss of Phf6 in B-cell leukemia results in systematic changes in gene expression via alteration of the chromatin landscape at the transcriptional start sites of B-cell- and T-cell-specific factors. Additionally, Phf6KO cells show significant down-regulation of genes involved in the development and function of normal B cells, show up-regulation of genes involved in T-cell signaling, and give rise to mixed-lineage lymphoma in vivo. Engagement of divergent transcriptional programs results in phenotypic plasticity that leads to altered disease presentation in vivo, tolerance of aberrant oncogenic signaling, and differential sensitivity to frontline and targeted therapies. These findings suggest that active maintenance of a precise chromatin landscape is essential for sustaining proper leukemia cell identity and that loss of a single factor (PHF6) can cause focal changes in chromatin accessibility and nucleosome positioning that render cells susceptible to lineage transition. © 2017 Soto-Feliciano et al.; Published by Cold Spring Harbor Laboratory Press.

  17. Gene expression profiling of epigenetic chromatin modification enzymes and histone marks by cigarette smoke: implications for COPD and lung cancer.

    Science.gov (United States)

    Sundar, Isaac K; Rahman, Irfan

    2016-12-01

    Chromatin-modifying enzymes mediate DNA methylation and histone modifications on recruitment to specific target gene loci in response to various stimuli. The key enzymes that regulate chromatin accessibility for maintenance of modifications in DNA and histones, and for modulation of gene expression patterns in response to cigarette smoke (CS), are not known. We hypothesize that CS exposure alters the gene expression patterns of chromatin-modifying enzymes, which then affects multiple downstream pathways involved in the response to CS. We have, therefore, analyzed chromatin-modifying enzyme profiles and validated by quantitative real-time PCR (qPCR). We also performed immunoblot analysis of targeted histone marks in C57BL/6J mice exposed to acute and subchronic CS, and of lungs from nonsmokers, smokers, and patients with chronic obstructive pulmonary disease (COPD). We found a significant increase in expression of several chromatin modification enzymes, including DNA methyltransferases, histone acetyltransferases, histone methyltransferases, and SET domain proteins, histone kinases, and ubiquitinases. Our qPCR validation data revealed a significant downregulation of Dnmt1, Dnmt3a, Dnmt3b, Hdac2, Hdac4, Hat1, Prmt1, and Aurkb We identified targeted chromatin histone marks (H3K56ac and H4K12ac), which are induced by CS. Thus CS-induced genotoxic stress differentially affects the expression of epigenetic modulators that regulate transcription of target genes via DNA methylation and site-specific histone modifications. This may have implications in devising epigenetic-based therapies for COPD and lung cancer. Copyright © 2016 the American Physiological Society.

  18. Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development.

    Science.gov (United States)

    Pajoro, Alice; Madrigal, Pedro; Muiño, Jose M; Matus, José Tomás; Jin, Jian; Mecchia, Martin A; Debernardi, Juan M; Palatnik, Javier F; Balazadeh, Salma; Arif, Muhammad; Ó'Maoiléidigh, Diarmuid S; Wellmer, Frank; Krajewski, Pawel; Riechmann, José-Luis; Angenent, Gerco C; Kaufmann, Kerstin

    2014-03-03

    Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood. We characterized the relationship of chromatin accessibility, gene expression, and DNA binding of two MADS-domain proteins at different stages of Arabidopsis flower development. Dynamic changes in APETALA1 and SEPALLATA3 DNA binding correlated with changes in gene expression, and many of the target genes could be associated with the developmental stage in which they are transcriptionally controlled. We also observe dynamic changes in chromatin accessibility during flower development. Remarkably, DNA binding of APETALA1 and SEPALLATA3 is largely independent of the accessibility status of their binding regions and it can precede increases in DNA accessibility. These results suggest that APETALA1 and SEPALLATA3 may modulate chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes. Our findings indicate that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions, thereby addressing dynamics of regulatory networks throughout flower development. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility.

  19. A role for chromatin topology in imprinted domain regulation.

    Science.gov (United States)

    MacDonald, William A; Sachani, Saqib S; White, Carlee R; Mann, Mellissa R W

    2016-02-01

    Recently, many advancements in genome-wide chromatin topology and nuclear architecture have unveiled the complex and hidden world of the nucleus, where chromatin is organized into discrete neighbourhoods with coordinated gene expression. This includes the active and inactive X chromosomes. Using X chromosome inactivation as a working model, we utilized publicly available datasets together with a literature review to gain insight into topologically associated domains, lamin-associated domains, nucleolar-associating domains, scaffold/matrix attachment regions, and nucleoporin-associated chromatin and their role in regulating monoallelic expression. Furthermore, we comprehensively review for the first time the role of chromatin topology and nuclear architecture in the regulation of genomic imprinting. We propose that chromatin topology and nuclear architecture are important regulatory mechanisms for directing gene expression within imprinted domains. Furthermore, we predict that dynamic changes in chromatin topology and nuclear architecture play roles in tissue-specific imprint domain regulation during early development and differentiation.

  20. The impact of cellular metabolism on chromatin dynamics and epigenetics.

    Science.gov (United States)

    Reid, Michael A; Dai, Ziwei; Locasale, Jason W

    2017-11-01

    The substrates used to modify nucleic acids and chromatin are affected by nutrient availability and the activity of metabolic pathways. Thus, cellular metabolism constitutes a fundamental component of chromatin status and thereby of genome regulation. Here we describe the biochemical and genetic principles of how metabolism can influence chromatin biology and epigenetics, discuss the functional roles of this interplay in developmental and cancer biology, and present future directions in this rapidly emerging area.

  1. NET23/STING Promotes Chromatin Compaction from the Nuclear Envelope

    Science.gov (United States)

    de las Heras, Jose I.; Saiz-Ros, Natalia; Makarov, Alexandr A.; Lazou, Vassiliki; Meinke, Peter; Waterfall, Martin; Kelly, David A.; Schirmer, Eric C.

    2014-01-01

    Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture. PMID:25386906

  2. AIRE-PHD fingers are structural hubs to maintain the integrity of chromatin-associated interactome.

    Science.gov (United States)

    Gaetani, Massimiliano; Matafora, Vittoria; Saare, Mario; Spiliotopoulos, Dimitrios; Mollica, Luca; Quilici, Giacomo; Chignola, Francesca; Mannella, Valeria; Zucchelli, Chiara; Peterson, Pärt; Bachi, Angela; Musco, Giovanna

    2012-12-01

    Mutations in autoimmune regulator (AIRE) gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy. AIRE is expressed in thymic medullary epithelial cells, where it promotes the expression of peripheral-tissue antigens to mediate deletional tolerance, thereby preventing self-reactivity. AIRE contains two plant homeodomains (PHDs) which are sites of pathological mutations. AIRE-PHD fingers are important for AIRE transcriptional activity and presumably play a crucial role in the formation of multimeric protein complexes at chromatin level which ultimately control immunological tolerance. As a step forward the understanding of AIRE-PHD fingers in normal and pathological conditions, we investigated their structure and used a proteomic SILAC approach to assess the impact of patient mutations targeting AIRE-PHD fingers. Importantly, both AIRE-PHD fingers are structurally independent and mutually non-interacting domains. In contrast to D297A and V301M on AIRE-PHD1, the C446G mutation on AIRE-PHD2 destroys the structural fold, thus causing aberrant AIRE localization and reduction of AIRE target genes activation. Moreover, mutations targeting AIRE-PHD1 affect the formation of a multimeric protein complex at chromatin level. Overall our results reveal the importance of AIRE-PHD domains in the interaction with chromatin-associated nuclear partners and gene regulation confirming the role of PHD fingers as versatile protein interaction hubs for multiple binding events.

  3. Chromatin insulators: regulatory mechanisms and epigenetic inheritance

    Science.gov (United States)

    Bushey, Ashley M.; Dorman, Elizabeth R.; Corces, Victor G.

    2008-01-01

    Enhancer-blocking insulators are DNA elements that disrupt the communication between a regulatory sequence, such as an enhancer or a silencer, and a promoter. Insulators participate in both transcriptional regulation and global nuclear organization, two features of chromatin that are thought to be maintained from one generation to the next through epigenetic mechanisms. Furthermore, there are many regulatory mechanisms in place that enhance or hinder insulator activity. These modes of regulation could be used to establish cell-type specific insulator activity that is epigenetically inherited along a cell and/or organismal lineage. This review will discuss the evidence for epigenetic inheritance and regulation of insulator function. PMID:18851828

  4. Regulation of chromatin structure in the cardiovascular system.

    Science.gov (United States)

    Rosa-Garrido, Manuel; Karbassi, Elaheh; Monte, Emma; Vondriska, Thomas M

    2013-01-01

    It has been appreciated for some time that cardiovascular disease involves large-scale transcriptional changes in various cell types. What has become increasingly clear only in the past few years, however, is the role of chromatin remodeling in cardiovascular phenotypes in normal physiology, as well as in development and disease. This review summarizes the state of the chromatin field in terms of distinct mechanisms to regulate chromatin structure in vivo, identifying when these modes of regulation have been demonstrated in cardiovascular tissues. We describe areas in which a better understanding of chromatin structure is leading to new insights into the fundamental biology of cardiovascular disease. 

  5. Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes*

    Science.gov (United States)

    Hoffman, Elizabeth A.; Frey, Brian L.; Smith, Lloyd M.; Auble, David T.

    2015-01-01

    Formaldehyde has been used for decades to probe macromolecular structure and function and to trap complexes, cells, and tissues for further analysis. Formaldehyde crosslinking is routinely employed for detection and quantification of protein-DNA interactions, interactions between chromatin proteins, and interactions between distal segments of the chromatin fiber. Despite widespread use and a rich biochemical literature, important aspects of formaldehyde behavior in cells have not been well described. Here, we highlight features of formaldehyde chemistry relevant to its use in analyses of chromatin complexes, focusing on how its properties may influence studies of chromatin structure and function. PMID:26354429

  6. Formaldehyde crosslinking: a tool for the study of chromatin complexes.

    Science.gov (United States)

    Hoffman, Elizabeth A; Frey, Brian L; Smith, Lloyd M; Auble, David T

    2015-10-30

    Formaldehyde has been used for decades to probe macromolecular structure and function and to trap complexes, cells, and tissues for further analysis. Formaldehyde crosslinking is routinely employed for detection and quantification of protein-DNA interactions, interactions between chromatin proteins, and interactions between distal segments of the chromatin fiber. Despite widespread use and a rich biochemical literature, important aspects of formaldehyde behavior in cells have not been well described. Here, we highlight features of formaldehyde chemistry relevant to its use in analyses of chromatin complexes, focusing on how its properties may influence studies of chromatin structure and function. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Myogenic Differential Methylation: Diverse Associations with Chromatin Structure

    Directory of Open Access Journals (Sweden)

    Sruti Chandra

    2014-06-01

    Full Text Available Employing a new algorithm for identifying differentially methylated regions (DMRs from reduced representation bisulfite sequencing profiles, we identified 1972 hypermethylated and 3250 hypomethylated myogenic DMRs in a comparison of myoblasts (Mb and myotubes (Mt with 16 types of nonmuscle cell cultures. DMRs co-localized with a variety of chromatin structures, as deduced from ENCODE whole-genome profiles. Myogenic hypomethylation was highly associated with both weak and strong enhancer-type chromatin, while hypermethylation was infrequently associated with enhancer-type chromatin. Both myogenic hypermethylation and hypomethylation often overlapped weak transcription-type chromatin and Polycomb-repressed-type chromatin. For representative genes, we illustrate relationships between DNA methylation, the local chromatin state, DNaseI hypersensitivity, and gene expression. For example, MARVELD2 exhibited myogenic hypermethylation in transcription-type chromatin that overlapped a silenced promoter in Mb and Mt while TEAD4 had myogenic hypomethylation in intronic subregions displaying enhancer-type or transcription-type chromatin in these cells. For LSP1, alternative promoter usage and active promoter-type chromatin were linked to highly specific myogenic or lymphogenic hypomethylated DMRs. Lastly, despite its myogenesis-associated expression, TBX15 had multiple hypermethylated myogenic DMRs framing its promoter region. This could help explain why TBX15 was previously reported to be underexpressed and, unexpectedly, its promoter undermethylated in placentas exhibiting vascular intrauterine growth restriction.

  8. Probing Chromatin Modifications in Response to ERK Signaling.

    Science.gov (United States)

    Oksuz, Ozgur; Tee, Wee-Wei

    2017-01-01

    Chromatin immunoprecipitation (ChIP) is a technique used to determine the association of proteins or histone modifications with chromatin regions in living cells or tissues, and is used extensively in the chromatin biology field to study transcriptional and epigenetic mechanisms. Increasing evidence points to an epigenetic coordination of signaling cascades, such as ERK, that regulate key processes in development and disease, revealing novel principles of gene regulation. Here we describe a detailed protocol for performing chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) for probing histone modifications regulated by ERK signaling in mouse ESCs.

  9. Effect of chromatin structure on quantitative ultrasound parameters.

    Science.gov (United States)

    Pasternak, Maurice; Doss, Lilian; Farhat, Golnaz; Al-Mahrouki, Azza; Kim, Christina Hyunjung; Kolios, Michael; Tran, William Tyler; Czarnota, Gregory J

    2017-03-21

    High-frequency ultrasound (~20 MHz) techniques were investigated using in vitro and ex vivo models to determine whether alterations in chromatin structure are responsible for ultrasound backscatter changes in biological samples. Acute myeloid leukemia (AML) cells and their isolated nuclei were exposed to various chromatin altering treatments. These included 10 different ionic environments, DNA cleaving and unfolding agents, as well as DNA condensing agents. Raw radiofrequency (RF) data was used to generate quantitative ultrasound parameters from spectral and form factor analyses. Chromatin structure was evaluated using electron microscopy. Results indicated that trends in quantitative ultrasound parameters mirrored trends in biophysical chromatin structure parameters. In general, higher ordered states of chromatin compaction resulted in increases to ultrasound paramaters of midband fit, spectral intercept, and estimated scatterer concentration, while samples with decondensed forms of chromatin followed an opposite trend. Experiments with isolated nuclei demonstrated that chromatin changes alone were sufficient to account for these observations. Experiments with ex vivo samples indicated similar effects of chromatin structure changes. The results obtained in this research provide a mechanistic explanation for ultrasound investigations studying scattering from cells and tissues undergoing biological processes affecting chromatin.

  10. The Chromatin Scaffold Protein SAFB1 Renders Chromatin Permissive for DNA Damage Signaling

    DEFF Research Database (Denmark)

    Altmeyer, Matthias; Toledo Lazaro, Luis Ignacio; Gudjonsson, Thorkell

    2013-01-01

    Although the general relevance of chromatin modifications for genotoxic stress signaling, cell-cycle checkpoint activation, and DNA repair is well established, how these modifications reach initial thresholds in order to trigger robust responses remains largely unexplored. Here, we identify the c...

  11. MDM2 Associates with Polycomb Repressor Complex 2 and Enhances Stemness-Promoting Chromatin Modifications Independent of p53

    DEFF Research Database (Denmark)

    Wienken, Magdalena; Dickmanns, Antje; Nemajerova, Alice

    2016-01-01

    associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell...

  12. p53 binding sites in normal and cancer cells are characterized by distinct chromatin context.

    Science.gov (United States)

    Bao, Feifei; LoVerso, Peter R; Fisk, Jeffrey N; Zhurkin, Victor B; Cui, Feng

    2017-08-18

    The tumor suppressor protein p53 interacts with DNA in a sequence-dependent manner. Thousands of p53 binding sites have been mapped genome-wide in normal and cancer cells. However, the way p53 selectively binds its cognate sites in different types of cells is not fully understood. Here, we performed a comprehensive analysis of 25 published p53 cistromes and identified 3,551 and 6,039 'high-confidence' binding sites in normal and cancer cells, respectively. Our analysis revealed 2 distinct epigenetic features underlying p53-DNA interactions in vivo. First, p53 binding sites are associated with transcriptionally active histone marks (H3K4me3 and H3K36me3) in normal-cell chromatin, but with repressive histone marks (H3K27me3) in cancer-cell chromatin. Second, p53 binding sites in cancer cells are characterized by a lower level of DNA methylation than their counterparts in normal cells, probably related to global hypomethylation in cancers. Intriguingly, regardless of the cell type, p53 sites are highly enriched in the endogenous retroviral elements of the ERV1 family, highlighting the importance of this repeat family in shaping the transcriptional network of p53. Moreover, the p53 sites exhibit an unusual combination of chromatin patterns: high nucleosome occupancy and, at the same time, high sensitivity to DNase I. Our results suggest that p53 can access its target sites in a chromatin environment that is non-permissive to most DNA-binding transcription factors, which may allow p53 to act as a pioneer transcription factor in the context of chromatin.

  13. Mapping chromatin interactions with 5C technology

    Science.gov (United States)

    Ferraiuolo, Maria A.; Sanyal, Amartya; Naumova, Natalia; Dekker, Job; Dostie, Josée

    2013-01-01

    In eukaryotes, genome organization can be observed on many levels and at different scales. This organization is important not only to reduce chromosome length but also for the proper execution of various biological processes. High-resolution mapping of spatial chromatin structure was made possible by the development of the chromosome conformation capture (3C) technique. 3C uses chemical cross-linking followed by proximity-based ligation of fragmented DNA to capture frequently interacting chromatin segments in cell populations. Several 3C-related methods capable of higher chromosome conformation mapping throughput were reported afterwards. These techniques include the 3C-carbon copy (5C) approach, which offers the advantage of being highly quantitative and reproducible. We provide here a reference protocol for the production of 5C libraries analyzed by next-generation sequencing or onto microarrays. A procedure used to verify that 3C library templates bear the high quality required to produce superior 5C libraries is also described. We believe that this comprehensive detailed protocol will help guide researchers in probing spatial genome organization and its role in various biological processes. PMID:23137922

  14. HDAC up-regulation in early colon field carcinogenesis is involved in cell tumorigenicity through regulation of chromatin structure.

    Science.gov (United States)

    Stypula-Cyrus, Yolanda; Damania, Dhwanil; Kunte, Dhananjay P; Cruz, Mart Dela; Subramanian, Hariharan; Roy, Hemant K; Backman, Vadim

    2013-01-01

    Normal cell function is dependent on the proper maintenance of chromatin structure. Regulation of chromatin structure is controlled by histone modifications that directly influence chromatin architecture and genome function. Specifically, the histone deacetylase (HDAC) family of proteins modulate chromatin compaction and are commonly dysregulated in many tumors, including colorectal cancer (CRC). However, the role of HDAC proteins in early colorectal carcinogenesis has not been previously reported. We found HDAC1, HDAC2, HDAC3, HDAC5, and HDAC7 all to be up-regulated in the field of human CRC. Furthermore, we observed that HDAC2 up-regulation is one of the earliest events in CRC carcinogenesis and observed this in human field carcinogenesis, the azoxymethane-treated rat model, and in more aggressive colon cancer cell lines. The universality of HDAC2 up-regulation suggests that HDAC2 up-regulation is a novel and important early event in CRC, which may serve as a biomarker. HDAC inhibitors (HDACIs) interfere with tumorigenic HDAC activity; however, the precise mechanisms involved in this process remain to be elucidated. We confirmed that HDAC inhibition by valproic acid (VPA) targeted the more aggressive cell line. Using nuclease digestion assays and transmission electron microscopy imaging, we observed that VPA treatment induced greater changes in chromatin structure in the more aggressive cell line. Furthermore, we used the novel imaging technique partial wave spectroscopy (PWS) to quantify nanoscale alterations in chromatin. We noted that the PWS results are consistent with the biological assays, indicating a greater effect of VPA treatment in the more aggressive cell type. Together, these results demonstrate the importance of HDAC activity in early carcinogenic events and the unique role of higher-order chromatin structure in determining cell tumorigenicity.

  15. HDAC up-regulation in early colon field carcinogenesis is involved in cell tumorigenicity through regulation of chromatin structure.

    Directory of Open Access Journals (Sweden)

    Yolanda Stypula-Cyrus

    Full Text Available Normal cell function is dependent on the proper maintenance of chromatin structure. Regulation of chromatin structure is controlled by histone modifications that directly influence chromatin architecture and genome function. Specifically, the histone deacetylase (HDAC family of proteins modulate chromatin compaction and are commonly dysregulated in many tumors, including colorectal cancer (CRC. However, the role of HDAC proteins in early colorectal carcinogenesis has not been previously reported. We found HDAC1, HDAC2, HDAC3, HDAC5, and HDAC7 all to be up-regulated in the field of human CRC. Furthermore, we observed that HDAC2 up-regulation is one of the earliest events in CRC carcinogenesis and observed this in human field carcinogenesis, the azoxymethane-treated rat model, and in more aggressive colon cancer cell lines. The universality of HDAC2 up-regulation suggests that HDAC2 up-regulation is a novel and important early event in CRC, which may serve as a biomarker. HDAC inhibitors (HDACIs interfere with tumorigenic HDAC activity; however, the precise mechanisms involved in this process remain to be elucidated. We confirmed that HDAC inhibition by valproic acid (VPA targeted the more aggressive cell line. Using nuclease digestion assays and transmission electron microscopy imaging, we observed that VPA treatment induced greater changes in chromatin structure in the more aggressive cell line. Furthermore, we used the novel imaging technique partial wave spectroscopy (PWS to quantify nanoscale alterations in chromatin. We noted that the PWS results are consistent with the biological assays, indicating a greater effect of VPA treatment in the more aggressive cell type. Together, these results demonstrate the importance of HDAC activity in early carcinogenic events and the unique role of higher-order chromatin structure in determining cell tumorigenicity.

  16. c-Myb Binding Sites in Haematopoietic Chromatin Landscapes

    Science.gov (United States)

    Bengtsen, Mads; Klepper, Kjetil; Gundersen, Sveinung; Cuervo, Ignacio; Drabløs, Finn; Hovig, Eivind; Sandve, Geir Kjetil; Gabrielsen, Odd Stokke; Eskeland, Ragnhild

    2015-01-01

    Strict control of tissue-specific gene expression plays a pivotal role during lineage commitment. The transcription factor c-Myb has an essential role in adult haematopoiesis and functions as an oncogene when rearranged in human cancers. Here we have exploited digital genomic footprinting analysis to obtain a global picture of c-Myb occupancy in the genome of six different haematopoietic cell-types. We have biologically validated several c-Myb footprints using c-Myb knockdown data, reporter assays and DamID analysis. We show that our predicted conserved c-Myb footprints are highly dependent on the haematopoietic cell type, but that there is a group of gene targets common to all cell-types analysed. Furthermore, we find that c-Myb footprints co-localise with active histone mark H3K4me3 and are significantly enriched at exons. We analysed co-localisation of c-Myb footprints with 104 chromatin regulatory factors in K562 cells, and identified nine proteins that are enriched together with c-Myb footprints on genes positively regulated by c-Myb and one protein enriched on negatively regulated genes. Our data suggest that c-Myb is a transcription factor with multifaceted target regulation depending on cell type. PMID:26208222

  17. Multiple modes of chromatin remodeling by Forkhead box proteins.

    Science.gov (United States)

    Lalmansingh, Avin S; Karmakar, Sudipan; Jin, Yetao; Nagaich, Akhilesh K

    2012-07-01

    Forkhead box (FOX) proteins represent a large family of transcriptional regulators unified by their DNA binding domain (DBD) known as a 'forkhead' or 'winged helix' domain. Over 40 FOX genes have been identified in the mammalian genome. FOX proteins share significant sequence similarities in the DBD which allow them to bind to a consensus DNA response element. However, their modes of action are quite diverse as they regulate gene expression by acting as pioneer factors, transcription factors, or both. This review focuses on the mechanisms of chromatin remodeling with an emphasis on three sub-classes-FOXA, FOXO, and FOXP members. FOXA proteins serve as pioneer factors to open up local chromatin structure and thereby increase accessibility of chromatin to factors regulating transcription. FOXP proteins, in contrast, function as classic transcription factors to recruit a variety of chromatin modifying enzymes to regulate gene expression. FOXO proteins represent a hybrid subclass having dual roles as pioneering factors and transcription factors. A subset of FOX proteins interacts with condensed mitotic chromatin and may function as 'bookmarking' agents to maintain transcriptional competence at specific genomic sites. The overall diversity in chromatin remodeling function by FOX proteins is related to unique structural motifs present within the DBD flanking regions that govern selective interactions with core histones and/or chromatin coregulatory proteins. This article is part of a Special Issue entitled: Chromatin in time and space. Published by Elsevier B.V.

  18. (epigenetic) rule: Chromatin domains as functional and structural ...

    Indian Academy of Sciences (India)

    The nuclear sub- structure which provides a skeletal framework for periodic attachment of the 30nm chromatin fiber to form loop structures has been termed as the .... Various features of the human genome that transcend its primary. DNA sequence, such as chromatin packaging, his- tone modifications, and DNA methylation ...

  19. Human sperm chromatin epigenetic potential: genomics, proteomics, and male infertility

    Directory of Open Access Journals (Sweden)

    Judit Castillo

    2015-01-01

    Full Text Available The classical idea about the function of the mammalian sperm chromatin is that it serves to transmit a highly protected and transcriptionally inactive paternal genome, largely condensed by protamines, to the next generation. In addition, recent sperm chromatin genome-wide dissection studies indicate the presence of a differential distribution of the genes and repetitive sequences in the protamine-condensed and histone-condensed sperm chromatin domains, which could be potentially involved in regulatory roles after fertilization. Interestingly, recent proteomic studies have shown that sperm chromatin contains many additional proteins, in addition to the abundant histones and protamines, with specific modifications and chromatin affinity features which are also delivered to the oocyte. Both gene and protein signatures seem to be altered in infertile patients and, as such, are consistent with the potential involvement of the sperm chromatin landscape in early embryo development. This present work reviews the available information on the composition of the human sperm chromatin and its epigenetic potential, with a particular focus on recent results derived from high-throughput genomic and proteomic studies. As a complement, we provide experimental evidence for the detection of phosphorylations and acetylations in human protamine 1 using a mass spectrometry approach. The available data indicate that the sperm chromatin is much more complex than what it was previously thought, raising the possibility that it could also serve to transmit crucial paternal epigenetic information to the embryo.

  20. The discrepancy between chromatin factor location and effect.

    Science.gov (United States)

    Lenstra, Tineke L; Holstege, Frank C P

    2012-01-01

    The influence of chromatin on many cellular processes is well appreciated. Much has been learned by studying the role of chromatin remodeling and modifying complexes on individual genes. The seemingly straightforward models that inevitably arise from such studies are challenged by genome-wide analyses. Two recent studies in Saccharomyces cerevisiae provide unprecedented coverage of both the genome-wide location and the effect on gene expression for the majority of chromatin factors. Comparison of the overlap between location and expression effects reveals a large disconnect, with on average only 2.5% of occupied genes showing changes in expression. It is also interesting that only 24% of all expression effects are associated with chromatin factor occupancy. The large difference between location and effect likely reflects general properties inherent to regulation of gene expression through chromatin in yeast. Explanations for the discrepancy include gene-specific properties that exert a requirement for certain factors only on specific genes, as well as functional redundancy, whereby loss of a particular factor is compensated by others that function in a distinct but nevertheless compensatory manner. Since the majority of chromatin factor perturbations do show significant effects on specific subsets of genes, this implies the presence of different types of gene-specific properties that determine which chromatin factors a particular gene requires for proper expression. Understanding these gene-specific properties should be the focus of future studies aimed at understanding regulation of gene expression through chromatin.

  1. Chromatin architecture and gene expression in Escherichia coli

    DEFF Research Database (Denmark)

    Willenbrock, Hanni; Ussery, David

    2004-01-01

    Two recent genome-scale analyses underscore the importance of DNA topology and chromatin structure in regulating transcription in Escherichia coli.......Two recent genome-scale analyses underscore the importance of DNA topology and chromatin structure in regulating transcription in Escherichia coli....

  2. A Quantitative Proteomic Analysis of In Vitro Assembled Chromatin*

    Science.gov (United States)

    Völker-Albert, Moritz Carl; Pusch, Miriam Caroline; Fedisch, Andreas; Schilcher, Pierre; Schmidt, Andreas; Imhof, Axel

    2016-01-01

    The structure of chromatin is critical for many aspects of cellular physiology and is considered to be the primary medium to store epigenetic information. It is defined by the histone molecules that constitute the nucleosome, the positioning of the nucleosomes along the DNA and the non-histone proteins that associate with it. These factors help to establish and maintain a largely DNA sequence-independent but surprisingly stable structure. Chromatin is extensively disassembled and reassembled during DNA replication, repair, recombination or transcription in order to allow the necessary factors to gain access to their substrate. Despite such constant interference with chromatin structure, the epigenetic information is generally well maintained. Surprisingly, the mechanisms that coordinate chromatin assembly and ensure proper assembly are not particularly well understood. Here, we use label free quantitative mass spectrometry to describe the kinetics of in vitro assembled chromatin supported by an embryo extract prepared from preblastoderm Drosophila melanogaster embryos. The use of a data independent acquisition method for proteome wide quantitation allows a time resolved comparison of in vitro chromatin assembly. A comparison of our in vitro data with proteomic studies of replicative chromatin assembly in vivo reveals an extensive overlap showing that the in vitro system can be used for investigating the kinetics of chromatin assembly in a proteome-wide manner. PMID:26811354

  3. Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging.

    Science.gov (United States)

    Tatavosian, Roubina; Zhen, Chao Yu; Duc, Huy Nguyen; Balas, Maggie M; Johnson, Aaron M; Ren, Xiaojun

    2015-11-20

    Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging*♦

    Science.gov (United States)

    Tatavosian, Roubina; Zhen, Chao Yu; Duc, Huy Nguyen; Balas, Maggie M.; Johnson, Aaron M.; Ren, Xiaojun

    2015-01-01

    Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes. PMID:26381410

  5. Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation

    Science.gov (United States)

    Tropberger, Philipp; Mercier, Alexandre; Robinson, Margaret; Zhong, Weidong; Ganem, Don E.; Holdorf, Meghan

    2015-01-01

    Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide and is a major risk factor for liver failure and hepatocellular carcinoma. Current antiviral therapy inhibits cytoplasmic HBV genomic replication, but is not curative because it does not directly affect nuclear HBV closed circular DNA (cccDNA), the genomic form that templates viral transcription and sustains viral persistence. Novel approaches that directly target cccDNA regulation would therefore be highly desirable. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications (PTMs). Here, using a new cccDNA ChIP-Seq approach, we report, to our knowledge, the first genome-wide maps of PTMs in cccDNA-containing chromatin from de novo infected HepG2 cells, primary human hepatocytes, and from HBV-infected liver tissue. We find high levels of PTMs associated with active transcription enriched at specific sites within the HBV genome and, surprisingly, very low levels of PTMs linked to transcriptional repression even at silent HBV promoters. We show that transcription and active PTMs in HBV chromatin are reduced by the activation of an innate immunity pathway, and that this effect can be recapitulated with a small molecule epigenetic modifying agent, opening the possibility that chromatin-based regulation of cccDNA transcription could be a new therapeutic approach to chronic HBV infection. PMID:26438841

  6. Combgap Promotes Ovarian Niche Development and Chromatin Association of EcR-Binding Regions in BR-C.

    Directory of Open Access Journals (Sweden)

    Anna Hitrik

    2016-11-01

    Full Text Available The development of niches for tissue-specific stem cells is an important aspect of stem cell biology. Determination of niche size and niche numbers during organogenesis involves precise control of gene expression. How this is achieved in the context of a complex chromatin landscape is largely unknown. Here we show that the nuclear protein Combgap (Cg supports correct ovarian niche formation in Drosophila by controlling ecdysone-Receptor (EcR- mediated transcription and long-range chromatin contacts in the broad locus (BR-C. Both cg and BR-C promote ovarian growth and the development of niches for germ line stem cells. BR-C levels were lower when Combgap was either reduced or over-expressed, indicating an intricate regulation of the BR-C locus by Combgap. Polytene chromosome stains showed that Cg co-localizes with EcR, the major regulator of BR-C, at the BR-C locus and that EcR binding to chromatin was sensitive to changes in Cg levels. Proximity ligation assay indicated that the two proteins could reside in the same complex. Finally, chromatin conformation analysis revealed that EcR-bound regions within BR-C, which span ~30 KBs, contacted each other. Significantly, these contacts were stabilized in an ecdysone- and Combgap-dependent manner. Together, these results highlight Combgap as a novel regulator of chromatin structure that promotes transcription of ecdysone target genes and ovarian niche formation.

  7. Integration of Kinase and Calcium Signaling at the Level of Chromatin Underlies Inducible Gene Activation in T Cells.

    Science.gov (United States)

    Brignall, Ruth; Cauchy, Pierre; Bevington, Sarah L; Gorman, Bethany; Pisco, Angela O; Bagnall, James; Boddington, Christopher; Rowe, William; England, Hazel; Rich, Kevin; Schmidt, Lorraine; Dyer, Nigel P; Travis, Mark A; Ott, Sascha; Jackson, Dean A; Cockerill, Peter N; Paszek, Pawel

    2017-10-15

    TCR signaling pathways cooperate to activate the inducible transcription factors NF-κB, NFAT, and AP-1. In this study, using the calcium ionophore ionomycin and/or PMA on Jurkat T cells, we show that the gene expression program associated with activation of TCR signaling is closely related to specific chromatin landscapes. We find that calcium and kinase signaling cooperate to induce chromatin remodeling at ∼2100 chromatin regions, which demonstrate enriched binding motifs for inducible factors and correlate with target gene expression. We found that these regions typically function as inducible enhancers. Many of these elements contain composite NFAT/AP-1 sites, which typically support cooperative binding, thus further reinforcing the need for cooperation between calcium and kinase signaling in the activation of genes in T cells. In contrast, treatment with PMA or ionomycin alone induces chromatin remodeling at far fewer regions (∼600 and ∼350, respectively), which mostly represent a subset of those induced by costimulation. This suggests that the integration of TCR signaling largely occurs at the level of chromatin, which we propose plays a crucial role in regulating T cell activation. Copyright © 2017 The Authors.

  8. Chromatin Dynamics during Nucleotide Excision Repair: Histones on the Move

    Directory of Open Access Journals (Sweden)

    Sophie E. Polo

    2012-09-01

    Full Text Available It has been a long-standing question how DNA damage repair proceeds in a nuclear environment where DNA is packaged into chromatin. Several decades of analysis combining in vitro and in vivo studies in various model organisms ranging from yeast to human have markedly increased our understanding of the mechanisms underlying chromatin disorganization upon damage detection and re-assembly after repair. Here, we review the methods that have been developed over the years to delineate chromatin alterations in response to DNA damage by focusing on the well-characterized Nucleotide Excision Repair (NER pathway. We also highlight how these methods have provided key mechanistic insight into histone dynamics coupled to repair in mammals, raising new issues about the maintenance of chromatin integrity. In particular, we discuss how NER factors and central players in chromatin dynamics such as histone modifiers, nucleosome remodeling factors, and histone chaperones function to mobilize histones during repair.

  9. The chromatin regulatory code: Beyond a histone code

    Science.gov (United States)

    Lesne, A.

    2006-03-01

    In this commentary on the contribution by Arndt Benecke in this issue, I discuss why the notion of “chromatin code” introduced and elaborated in this paper is to be preferred to that of “histone code”. Speaking of a code as regards nucleosome conformation and histone tail post-translational modifications only makes sense within the chromatin fiber, where their physico-chemical features can be translated into regulatory programs at the genome level, by means of a complex, multi-level interplay with the fiber architecture and dynamics settled in the course of Evolution. In particular, this chromatin code presumably exploits allosteric transitions of the chromatin fiber. The chromatin structure dependence of its translation suggests two alternative modes of transcription initiation regulation, also proposed in the paper by A. Benecke in this issue for interpreting strikingly bimodal micro-array data.

  10. Rapid genome-scale mapping of chromatin accessibility in tissue

    DEFF Research Database (Denmark)

    Grøntved, Lars; Bandle, Russell; John, Sam

    2012-01-01

    BACKGROUND: The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant....... RESULTS: Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh). The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent...... positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions...

  11. Chromatin Dynamics during Nucleotide Excision Repair: Histones on the Move

    Science.gov (United States)

    Adam, Salomé; Polo, Sophie E.

    2012-01-01

    It has been a long-standing question how DNA damage repair proceeds in a nuclear environment where DNA is packaged into chromatin. Several decades of analysis combining in vitro and in vivo studies in various model organisms ranging from yeast to human have markedly increased our understanding of the mechanisms underlying chromatin disorganization upon damage detection and re-assembly after repair. Here, we review the methods that have been developed over the years to delineate chromatin alterations in response to DNA damage by focusing on the well-characterized Nucleotide Excision Repair (NER) pathway. We also highlight how these methods have provided key mechanistic insight into histone dynamics coupled to repair in mammals, raising new issues about the maintenance of chromatin integrity. In particular, we discuss how NER factors and central players in chromatin dynamics such as histone modifiers, nucleosome remodeling factors, and histone chaperones function to mobilize histones during repair. PMID:23109890

  12. An in vitro assay to study the recruitment and substrate specificity of chromatin modifying enzymes

    Directory of Open Access Journals (Sweden)

    Vermeulen Michiel

    2004-01-01

    Full Text Available Post-translational modifications of core histones play an important role in regulating fundamental biological processes such as DNA repair, transcription and replication. In this paper, we describe a novel assay that allows sequential targeting of distinct histone modifying enzymes to immobilized nucleosomal templates using recombinant chimeric targeting molecules. The assay can be used to study the histone substrate specificity of chromatin modifying enzymes as well as whether and how certain enzymes affect each other's histone modifying activities. As such the assay can help to understand how a certain histone code is established and interpreted.

  13. Effect of DNA groove binder distamycin A upon chromatin structure.

    Directory of Open Access Journals (Sweden)

    Parijat Majumder

    Full Text Available BACKGROUND: Distamycin A is a prototype minor groove binder, which binds to B-form DNA, preferentially at A/T rich sites. Extensive work in the past few decades has characterized the binding at the level of double stranded DNA. However, effect of the same on physiological DNA, i.e. DNA complexed in chromatin, has not been well studied. Here we elucidate from a structural perspective, the interaction of distamycin with soluble chromatin, isolated from Sprague-Dawley rat. METHODOLOGY/PRINCIPAL FINDINGS: Chromatin is a hierarchical assemblage of DNA and protein. Therefore, in order to characterize the interaction of the same with distamycin, we have classified the system into various levels, according to the requirements of the method adopted, and the information to be obtained. Isothermal titration calorimetry has been employed to characterize the binding at the levels of chromatin, chromatosome and chromosomal DNA. Thermodynamic parameters obtained thereof, identify enthalpy as the driving force for the association, with comparable binding affinity and free energy for chromatin and chromosomal DNA. Reaction enthalpies at different temperatures were utilized to evaluate the change in specific heat capacity (ΔCp, which, in turn, indicated a possible binding associated structural change. Ligand induced structural alterations have been monitored by two complementary methods--dynamic light scattering, and transmission electron microscopy. They indicate compaction of chromatin. Using transmission electron microscopy, we have visualized the effect of distamycin upon chromatin architecture at di- and trinucleosome levels. Our results elucidate the simultaneous involvement of linker bending and internucleosomal angle contraction in compaction process induced by distamycin. CONCLUSIONS/SIGNIFICANCE: We summarize here, for the first time, the thermodynamic parameters for the interaction of distamycin with soluble chromatin, and elucidate its effect on

  14. Chromatin insulators: lessons from the fly

    Science.gov (United States)

    Gurudatta, B. V.

    2009-01-01

    Chromatin insulators are DNA–protein complexes with broad functions in nuclear biology. Drosophila has at least five different types of insulators; recent results suggest that these different insulators share some components that may allow them to function through common mechanisms. Data from genome-wide localization studies of insulator proteins indicate a possible functional specialization, with different insulators playing distinct roles in nuclear biology. Cells have developed mechanisms to control insulator activity by recruiting specialized proteins or by covalent modification of core components. Current results suggest that insulators set up cell-specific blueprints of nuclear organization that may contribute to the establishment of different patterns of gene expression during cell differentiation and development. PMID:19752045

  15. The pioneer factor OCT4 requires the chromatin remodeller BRG1 to support gene regulatory element function in mouse embryonic stem cells.

    Science.gov (United States)

    King, Hamish W; Klose, Robert J

    2017-03-13

    Pioneer transcription factors recognise and bind their target sequences in inaccessible chromatin to establish new transcriptional networks throughout development and cellular reprogramming. During this process, pioneer factors establish an accessible chromatin state to facilitate additional transcription factor binding, yet it remains unclear how different pioneer factors achieve this. Here, we discover that the pluripotency-associated pioneer factor OCT4 binds chromatin to shape accessibility, transcription factor co-binding, and regulatory element function in mouse embryonic stem cells. Chromatin accessibility at OCT4-bound sites requires the chromatin remodeller BRG1, which is recruited to these sites by OCT4 to support additional transcription factor binding and expression of the pluripotency-associated transcriptome. Furthermore, the requirement for BRG1 in shaping OCT4 binding reflects how these target sites are used during cellular reprogramming and early mouse development. Together this reveals a distinct requirement for a chromatin remodeller in promoting the activity of the pioneer factor OCT4 and regulating the pluripotency network.

  16. Vitamin D-dependent chromatin association of CTCF in human monocytes.

    Science.gov (United States)

    Neme, Antonio; Seuter, Sabine; Carlberg, Carsten

    2016-11-01

    CCCTC-binding factor (CTCF) is a transcription factor being involved in 3D chromatin organization and displays a highly conserved genome-wide binding pattern. In this study, we report the cistrome of CTCF in THP-1 human monocytes and confirm that from the 40,078 CTCF binding sites nearly 85% are identical with those found in K562 monocytes. Quadruplicate chromatin immunoprecipitation sequencing (ChIP-seq) demonstrated that at 2130 loci the association strenght of CTCF with genomic DNA was significantly (p<0.05) modulated by stimulation with the natural vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Some 55% of these CTCF sites contribute to DNA looping and mark the anchors of 587 putative topologically associating domains (TADs) containing at least one VDR binding site and one 1,25(OH)2D3 target gene. These TADs can explain the regulatory scenarios of up to 70% of all 1,25(OH)2D3 target genes. A self-organizing map approach subdivided the vitamin D-sensitive CTCF sites into seven classes that can be distinguished by participation in DNA loop formation, binding to open chromatin, carrying binding motifs for CTCF or its relative BORIS, overlap with transcription start site (TSS) regions and binding of VDR. These variant molecular profiles suggest different mechanisms of the 1,25(OH)2D3-dependent action of CTCF. The co-location of VDR and 1,25(OH)2D3-dependent CTCF sites increases in the context of accessible chromatin and TSS regions but does not show any significant correlation with classical DNA binding mechanisms of CTCF. In conclusion, vitamin D-sensitive CTCF sites provide further mechanistic details to the epigenome-wide understanding of 1,25(OH)2D3-mediated gene regulation. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. PREDICTION OF CHROMATIN STATES USING DNA SEQUENCE PROPERTIES

    KAUST Repository

    Bahabri, Rihab R.

    2013-06-01

    Activities of DNA are to a great extent controlled epigenetically through the internal struc- ture of chromatin. This structure is dynamic and is influenced by different modifications of histone proteins. Various combinations of epigenetic modification of histones pinpoint to different functional regions of the DNA determining the so-called chromatin states. How- ever, the characterization of chromatin states by the DNA sequence properties remains largely unknown. In this study we aim to explore whether DNA sequence patterns in the human genome can characterize different chromatin states. Using DNA sequence motifs we built binary classifiers for each chromatic state to eval- uate whether a given genomic sequence is a good candidate for belonging to a particular chromatin state. Of four classification algorithms (C4.5, Naive Bayes, Random Forest, and SVM) used for this purpose, the decision tree based classifiers (C4.5 and Random Forest) yielded best results among those we evaluated. Our results suggest that in general these models lack sufficient predictive power, although for four chromatin states (insulators, het- erochromatin, and two types of copy number variation) we found that presence of certain motifs in DNA sequences does imply an increased probability that such a sequence is one of these chromatin states.

  18. Sm-ChIPi: Single-Molecule Chromatin Immunoprecipitation Imaging.

    Science.gov (United States)

    Tatavosian, Roubina; Ren, Xiaojun

    2018-01-01

    Epigenetic complexes regulate chromatin dynamics via binding to and assembling on chromatin. However, the mechanisms of chromatin binding and assembly of epigenetic complexes within cells remain incompletely understood, partly due to technical challenges. Here, we present a new approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) that enables to assess the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was developed based on chromatin immunoprecipitation followed by single-molecule fluorescence microscopy imaging. In this method, an epigenetic complex subunit fused with a gene coding for a fluorescent protein is stably expressed in its corresponding knockout cells. Nucleosomes associated with epigenetic complexes are isolated from cells at native conditions and incubated with biotinylated antibodies. The resulting complexes are immobilized on a quartz slide that had been passivated and functionalized with NeutrAvidin. Image stacks are then acquired by using single-molecule TIRF microscopy. The individual spots imaged by TIRF microscopy represent single protein-nucleosome complexes. The number of copies of the protein complexes on a nucleosome is inferred from the fluorescence photobleaching measurements. Sm-ChIPi is a sensitive and direct method that can quantify the cellular assembly stoichiometry of epigenetic complexes on chromatin.

  19. The specificity and topology of chromatin interaction pathways in yeast.

    Science.gov (United States)

    Lenstra, Tineke L; Benschop, Joris J; Kim, Taesoo; Schulze, Julia M; Brabers, Nathalie A C H; Margaritis, Thanasis; van de Pasch, Loes A L; van Heesch, Sebastiaan A A C; Brok, Mariel O; Groot Koerkamp, Marian J A; Ko, Cheuk W; van Leenen, Dik; Sameith, Katrin; van Hooff, Sander R; Lijnzaad, Philip; Kemmeren, Patrick; Hentrich, Thomas; Kobor, Michael S; Buratowski, Stephen; Holstege, Frank C P

    2011-05-20

    Packaging of DNA into chromatin has a profound impact on gene expression. To understand how changes in chromatin influence transcription, we analyzed 165 mutants of chromatin machinery components in Saccharomyces cerevisiae. mRNA expression patterns change in 80% of mutants, always with specific effects, even for loss of widespread histone marks. The data are assembled into a network of chromatin interaction pathways. The network is function based, has a branched, interconnected topology, and lacks strict one-to-one relationships between complexes. Chromatin pathways are not separate entities for different gene sets, but share many components. The study evaluates which interactions are important for which genes and predicts additional interactions, for example between Paf1C and Set3C, as well as a role for Mediator in subtelomeric silencing. The results indicate the presence of gene-dependent effects that go beyond context-dependent binding of chromatin factors and provide a framework for understanding how specificity is achieved through regulating chromatin. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Tracking the mechanical dynamics of human embryonic stem cell chromatin

    Directory of Open Access Journals (Sweden)

    Hinde Elizabeth

    2012-12-01

    Full Text Available Abstract Background A plastic chromatin structure has emerged as fundamental to the self-renewal and pluripotent capacity of embryonic stem (ES cells. Direct measurement of chromatin dynamics in vivo is, however, challenging as high spatiotemporal resolution is required. Here, we present a new tracking-based method which can detect high frequency chromatin movement and quantify the mechanical dynamics of chromatin in live cells. Results We use this method to study how the mechanical properties of chromatin movement in human embryonic stem cells (hESCs are modulated spatiotemporally during differentiation into cardiomyocytes (CM. Notably, we find that pluripotency is associated with a highly discrete, energy-dependent frequency of chromatin movement that we refer to as a ‘breathing’ state. We find that this ‘breathing’ state is strictly dependent on the metabolic state of the cell and is progressively silenced during differentiation. Conclusions We thus propose that the measured chromatin high frequency movements in hESCs may represent a hallmark of pluripotency and serve as a mechanism to maintain the genome in a transcriptionally accessible state. This is a result that could not have been observed without the high spatial and temporal resolution provided by this novel tracking method.

  1. Determinants of Sir2-Mediated, Silent Chromatin Cohesion.

    Science.gov (United States)

    Chen, Yu-Fan; Chou, Chia-Ching; Gartenberg, Marc R

    2016-08-01

    Cohesin associates with distinct sites on chromosomes to mediate sister chromatid cohesion. Single cohesin complexes are thought to bind by encircling both sister chromatids in a topological embrace. Transcriptionally repressed chromosomal domains in the yeast Saccharomyces cerevisiae represent specialized sites of cohesion where cohesin binds silent chromatin in a Sir2-dependent fashion. In this study, we investigated the molecular basis for Sir2-mediated cohesion. We identified a cluster of charged surface residues of Sir2, collectively termed the EKDK motif, that are required for cohesin function. In addition, we demonstrated that Esc8, a Sir2-interacting factor, is also required for silent chromatin cohesion. Esc8 was previously shown to associate with Isw1, the enzymatic core of ISW1 chromatin remodelers, to form a variant of the ISW1a chromatin remodeling complex. When ESC8 was deleted or the EKDK motif was mutated, cohesin binding at silenced chromatin domains persisted but cohesion of the domains was abolished. The data are not consistent with cohesin embracing both sister chromatids within silent chromatin domains. Transcriptional silencing remains largely intact in strains lacking ESC8 or bearing EKDK mutations, indicating that silencing and cohesion are separable functions of Sir2 and silent chromatin. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. Anti-chromatin antibodies in juvenile rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    V. Gerloni

    2011-09-01

    Full Text Available Objective: to evaluate the prevalence and clinical significance of anti-chromatin antibodies (Abs in juvenile rheumatoid arthritis (JRA. Methods: IgG anti-chromatin Abs were detected by an enzyme-linked immunosorbent assay (ELISA, in sera of 94 children with JRA (10 children with systemic, 38 with polyarticular and 46 with oligoarticular disease onset. As control group, 33 age- and-sex-matched healthy children (HC were also examined. Results: Abs to chromatin were detected in 24/94 (25,5% of children suffering from JRA. Particularly, the higher prevalence of anti-chromatin Abs has been found in children with oligoarticular (30,4% and polyarticular (23,7% onset JRA. In these groups Abs titers were significantly higher compared to systemic JRA and HC (p=0.003. Anti-chromatin Abs were observed more frequently in patients with oligoarticular disease and chronic uveitis (21,7%. Furthermore, higher levels of anti-chromatin Abs has been found in all the patients treated with anti-TNFα therapy (p<0.0001. Conclusions: our results confirm previous data about the prevalence of anti-chromatin Abs in JRA. These Abs were significantly higher in the group of patients with oligoarticular onset with past or present hystory of ocular involvement and in the group with polyarticular JRA treated with biologic therapy. A long-term follow-up study could be useful to evaluate the potential utility of these autoantibodies.

  3. Insight into the machinery that oils chromatin dynamics.

    Science.gov (United States)

    Wright, Roni H G; Fernandez-Fuentes, Narcis; Oliva, Baldomero; Beato, Miguel

    2016-11-01

    The packaging of genetic information in form of chromatin within the nucleus provides cells with the ability to store and protect massive amounts of information within a compact space. Storing information within chromatin allows selective access to specific DNA sequences by regulating the various levels of chromatin structure from nucleosomes, to chromatin fibers, loops and topological associating domains (TADs) using mechanisms that are being progressively unravelled. However, a relatively unexplored aspect is the energetic cost of changing the chromatin configuration to gain access to DNA information. Among the enzymes responsible for regulating chromatin access are the ATP-dependent chromatin remodellers that act on nucleosomes and use the energy of ATP hydrolysis to make chromatin DNA more accessible. It is assumed that the ATP used by these enzymes is provided by the mitochondria or by cytoplasmic glycolysis. We hypothesize that though this may be the case for cells in steady state, when gene expression has to be globally reprogramed in response to externals signals or stress conditions, the cell directs energy production to the cell nucleus, where rapid chromatin reorganization is needed for cell survival. We discovered that in response to hormones a nuclear ATP synthesis mechanism is activated that utilizing ADP-ribose and pyrophosphate as substrates. 1 This extra view aims to put this process within its historical context, to describe the enzymatic steps in detail, to propose a possible structure of the ATP synthesising enzyme, and to shed light on how this may link to other reactions within the cell providing a perspective for future lines of investigation.

  4. Altered oncomodules underlie chromatin regulatory factors driver mutations.

    Science.gov (United States)

    Frigola, Joan; Iturbide, Ane; Lopez-Bigas, Nuria; Peiro, Sandra; Gonzalez-Perez, Abel

    2016-05-24

    Chromatin regulatory factors (CRFs), are known to be involved in tumorigenesis in several cancer types. Nevertheless, the molecular mechanisms through which driver alterations of CRFs cause tumorigenesis remain unknown. Here, we developed a CRFs Oncomodules Discovery approach, which mines several sources of cancer genomics and perturbaomics data. The approach prioritizes sets of genes significantly miss-regulated in primary tumors (oncomodules) bearing mutations of driver CRFs. We applied the approach to eleven TCGA tumor cohorts and uncovered oncomodules potentially associated to mutations of five driver CRFs in three cancer types. Our results revealed, for example, the potential involvement of the mTOR pathway in the development of tumors with loss-of-function mutations of MLL2 in head and neck squamous cell carcinomas. The experimental validation that MLL2 loss-of-function increases the sensitivity of cancer cell lines to mTOR inhibition lends further support to the validity of our approach. The potential oncogenic modules detected by our approach may guide experiments proposing ways to indirectly target driver mutations of CRFs.

  5. Cracking the chromatin code: Precise rule of nucleosome positioning

    Science.gov (United States)

    Trifonov, Edward N.

    2011-03-01

    Various aspects of packaging DNA in eukaryotic cells are outlined in physical rather than biological terms. The informational and physical nature of packaging instructions encoded in DNA sequences is discussed with the emphasis on signal processing difficulties - very low signal-to-noise ratio and high degeneracy of the nucleosome positioning signal. As the author has been contributing to the field from its very onset in 1980, the review is mostly focused at the works of the author and his colleagues. The leading concept of the overview is the role of deformational properties of DNA in the nucleosome positioning. The target of the studies is to derive the DNA bendability matrix describing where along the DNA various dinucleotide elements should be positioned, to facilitate its bending in the nucleosome. Three different approaches are described leading to derivation of the DNA deformability sequence pattern, which is a simplified linear presentation of the bendability matrix. All three approaches converge to the same unique sequence motif CGRAAATTTYCG or, in binary form, YRRRRRYYYYYR, both representing the chromatin code.

  6. Individual Bromodomains of Polybromo-1 Contribute to Chromatin Association and Tumor Suppression in Clear Cell Renal Carcinoma.

    Science.gov (United States)

    Porter, Elizabeth G; Dykhuizen, Emily C

    2017-02-17

    The architecture of chromatin is governed, in part, by ATP-dependent chromatin remodelers. These multiprotein complexes contain targeting domains that recognize post-translational marks on histones. One such targeting domain is the bromodomain (BD), which recognizes acetyl-lysines and recruits proteins to sites of acetylation across the genome. Polybromo1 (PBRM1), a subunit of the Polybromo-associated BRG1- or hBRM-associated factors (PBAF) chromatin remodeler, contains six tandem BDs and is frequently mutated in clear cell renal cell carcinoma (ccRCC). Mutations in the PBRM1 gene often lead to the loss of protein expression; however, missense mutations in PBRM1 have been identified and tend to cluster in the BDs, particularly BD2 and BD4, suggesting that individual BDs are critical for PBRM1 function. To study the role of these six BDs, we inactivated each of the six BDs of PBRM1 and re-expressed these mutants in Caki2 cells (ccRCC cells with the loss of function mutation in PBRM1). Four of the six BDs abrogated PBRM1 tumor suppressor function, gene regulation, and chromatin affinity with the degree of importance correlating strongly to the rate of missense mutations in patients. Furthermore, we identified BD2 as the most critical for PBRM1 and confirmed BD2-mediated association to histone H3 peptides acetylated at lysine 14 (H3K14Ac), validating the importance of this specific acetylation mark for PBRM1 binding. From these data, we conclude that four of the BDs act together to target PBRM1 to sites on chromatin; when a single BD is mutated, PBRM1 no longer controls gene expression properly, leading to increased cell proliferation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. DNA methylation-based chromatin compartments and ChIP-seq profiles reveal transcriptional drivers of prostate carcinogenesis.

    Science.gov (United States)

    Simmonds, Poppy; Loomis, Erick; Curry, Edward

    2017-06-07

    Profiles of DNA methylation of many tissues relevant in human disease have been obtained from microarrays and are publicly available. These can be used to generate maps of chromatin compartmentalization, demarcating open and closed chromatin across the genome. Additionally, large sets of genome-wide transcription factor binding profiles have been made available thanks to ChIP-seq technology. We have identified genomic regions with altered chromatin compartmentalization in prostate adenocarcinoma tissue relative to normal prostate tissue, using DNA methylation microarray data from The Cancer Genome Atlas. DNA binding profiles from the Encyclopedia of DNA Elements (ENCODE) ChIP-seq studies have been systematically screened to find transcription factors with inferred DNA binding sites located in discordantly open/closed chromatin in malignant tissue (compared with non-cancer control tissue). We have combined this with tests for corresponding up-/downregulation of the transcription factors' putative target genes to obtain an integrated measure of cancer-specific regulatory activity to identify likely transcriptional drivers of prostate cancer. Generally, we find that the degree to which transcription factors preferentially bind regions of chromatin that become more accessible during prostate carcinogenesis is significantly associated to the level of systematic upregulation of their targets, at the level of gene expression. Our approach has yielded 11 transcription factors that show strong cancer-specific transcriptional activation of targets, including the novel candidates KAT2A and TRIM28, alongside established drivers of prostate cancer MYC, ETS1, GABP and YY1. This approach to integrated epigenetic and transcriptional profiling using publicly available data represents a cheap and powerful technique for identifying potential drivers of human disease. In our application to prostate adenocarcinoma data, the fact that well-known drivers are amongst the top candidates

  8. Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Johanna Sebald

    Full Text Available The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster.

  9. A Transcription Factor Pulse Can Prime Chromatin for Heritable Transcriptional Memory.

    Science.gov (United States)

    Iberg-Badeaux, Aimee; Collombet, Samuel; Laurent, Benoit; van Oevelen, Chris; Chin, Kuo-Kai; Thieffry, Denis; Graf, Thomas; Shi, Yang

    2017-02-15

    Short-term and long-term transcriptional memory is the phenomenon whereby the kinetics or magnitude of gene induction is enhanced following a prior induction period. Short-term memory persists within one cell generation or in postmitotic cells, while long-term memory can survive multiple rounds of cell division. We have developed a tissue culture model to study the epigenetic basis for long-term transcriptional memory (LTTM) and subsequently used this model to better understand the epigenetic mechanisms that enable heritable memory of temporary stimuli. We find that a pulse of transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) induces LTTM on a subset of target genes that survives nine cell divisions. The chromatin landscape at genes that acquire LTTM is more repressed than at those genes that do not exhibit memory, akin to a latent state. We show through chromatin immunoprecipitation (ChIP) and chemical inhibitor studies that RNA polymerase II (Pol II) elongation is important for establishing memory in this model but that Pol II itself is not retained as part of the memory mechanism. More generally, our work reveals that a transcription factor involved in lineage specification can induce LTTM and that failure to rerepress chromatin is one epigenetic mechanism underlying transcriptional memory. Copyright © 2017 American Society for Microbiology.

  10. Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster.

    Science.gov (United States)

    Sebald, Johanna; Willi, Michaela; Schoberleitner, Ines; Krogsdam, Anne; Orth-Höller, Dorothea; Trajanoski, Zlatko; Lusser, Alexandra

    2016-01-01

    The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster.

  11. The Use of Chromatin Insulators to Improve the Expression and Safety of Integrating Gene Transfer Vectors

    Science.gov (United States)

    2011-01-01

    Abstract The therapeutic application of recombinant retroviruses and other integrating gene transfer vectors has been limited by problems of vector expression and vector-mediated genotoxicity. These problems arise in large part from the interactions between vector sequences and the genomic environment surrounding sites of integration. Strides have been made in overcoming both of these problems through the modification of deleterious vector sequences, the inclusion of better enhancers and promoters, and the use of alternative virus systems. However, these modifications often add other restrictions on vector design, which in turn can further limit therapeutic applications. As an alternative, several groups have been investigating a class of DNA regulatory elements known as chromatin insulators. These elements provide a means of blocking the interaction between an integrating vector and the target cell genome in a manner that is independent of the vector transgene, regulatory elements, or virus of origin. This review outlines the background, rationale, and evidence for using chromatin insulators to improve the expression and safety of gene transfer vectors. Also reviewed are topological factors that constrain the use of insulators in integrating gene transfer vectors, alternative sources of insulators, and the role of chromatin insulators as one of several components for optimal vector design. PMID:21247248

  12. Thyroid hormone receptor actions on transcription in amphibia: The roles of histone modification and chromatin disruption

    Directory of Open Access Journals (Sweden)

    Shi Yun-Bo

    2012-12-01

    Full Text Available Abstract Thyroid hormone (T3 plays diverse roles in adult organ function and during vertebrate development. The most important stage of mammalian development affected by T3 is the perinatal period when plasma T3 level peaks. Amphibian metamorphosis resembles this mammalian postembryonic period and is absolutely dependent on T3. The ability to easily manipulate this process makes it an ideal model to study the molecular mechanisms governing T3 action during vertebrate development. T3 functions mostly by regulating gene expression through T3 receptors (TRs. Studies in vitro, in cell cultures and reconstituted frog oocyte transcription system have revealed that TRs can both activate and repress gene transcription in a T3-dependent manner and involve chromatin disruption and histone modifications. These changes are accompanied by the recruitment of diverse cofactor complexes. More recently, genetic studies in mouse and frog have provided strong evidence for a role of cofactor complexes in T3 signaling in vivo. Molecular studies on amphibian metamorphosis have also revealed that developmental gene regulation by T3 involves histone modifications and the disruption of chromatin structure at the target genes as evidenced by the loss of core histones, arguing that chromatin remodeling is an important mechanism for gene activation by liganded TR during vertebrate development.

  13. Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure.

    Science.gov (United States)

    Krufczik, Matthias; Sievers, Aaron; Hausmann, Annkathrin; Lee, Jin-Ho; Hildenbrand, Georg; Schaufler, Wladimir; Hausmann, Michael

    2017-05-07

    Immunostaining and fluorescence in situ hybridization (FISH) are well established methods for specific labelling of chromatin in the cell nucleus. COMBO-FISH (combinatorial oligonucleotide fluorescence in situ hybridization) is a FISH method using computer designed oligonucleotide probes specifically co-localizing at given target sites. In combination with super resolution microscopy which achieves spatial resolution far beyond the Abbe Limit, it allows new insights into the nano-scaled structure and organization of the chromatin of the nucleus. To avoid nano-structural changes of the chromatin, the COMBO-FISH labelling protocol was optimized omitting heat treatment for denaturation of the target. As an example, this protocol was applied to ALU elements-dispersed short stretches of DNA which appear in different kinds in large numbers in primate genomes. These ALU elements seem to be involved in gene regulation, genomic diversity, disease induction, DNA repair, etc. By computer search, we developed a unique COMBO-FISH probe which specifically binds to ALU consensus elements and combined this DNA-DNA labelling procedure with heterochromatin immunostainings in formaldehyde-fixed cell specimens. By localization microscopy, the chromatin network-like arrangements of ALU oligonucleotide repeats and heterochromatin antibody labelling sites were simultaneously visualized and quantified. This novel approach which simultaneously combines COMBO-FISH and immunostaining was applied to chromatin analysis on the nanoscale after low-linear-energy-transfer (LET) radiation exposure at different doses. Dose-correlated curves were obtained from the amount of ALU representing signals, and the chromatin re-arrangements during DNA repair after irradiation were quantitatively studied on the nano-scale. Beyond applications in radiation research, the labelling strategy of immunostaining and COMBO-FISH with localization microscopy will also offer new potentials for analyses of subcellular

  14. MORC-1 Integrates Nuclear RNAi and Transgenerational Chromatin Architecture to Promote Germline Immortality.

    Science.gov (United States)

    Weiser, Natasha E; Yang, Danny X; Feng, Suhua; Kalinava, Natallia; Brown, Kristen C; Khanikar, Jayshree; Freeberg, Mallory A; Snyder, Martha J; Csankovszki, Györgyi; Chan, Raymond C; Gu, Sam G; Montgomery, Taiowa A; Jacobsen, Steven E; Kim, John K

    2017-05-22

    Germline-expressed endogenous small interfering RNAs (endo-siRNAs) transmit multigenerational epigenetic information to ensure fertility in subsequent generations. In Caenorhabditis elegans, nuclear RNAi ensures robust inheritance of endo-siRNAs and deposition of repressive H3K9me3 marks at target loci. How target silencing is maintained in subsequent generations is poorly understood. We discovered that morc-1 is essential for transgenerational fertility and acts as an effector of endo-siRNAs. Unexpectedly, morc-1 is dispensable for siRNA inheritance but is required for target silencing and maintenance of siRNA-dependent chromatin organization. A forward genetic screen identified mutations in met-1, which encodes an H3K36 methyltransferase, as potent suppressors of morc-1(-) and nuclear RNAi mutant phenotypes. Further analysis of nuclear RNAi and morc-1(-) mutants revealed a progressive, met-1-dependent enrichment of H3K36me3, suggesting that robust fertility requires repression of MET-1 activity at nuclear RNAi targets. Without MORC-1 and nuclear RNAi, MET-1-mediated encroachment of euchromatin leads to detrimental decondensation of germline chromatin and germline mortality. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. The Drosophila Huntington's disease gene ortholog dhtt influences chromatin regulation during development.

    Science.gov (United States)

    Dietz, Kevin N; Di Stefano, Luisa; Maher, Robert C; Zhu, Hui; Macdonald, Marcy E; Gusella, James F; Walker, James A

    2015-01-15

    Huntington's disease is an autosomal dominant neurodegenerative disorder caused by a CAG expansion mutation in HTT, the gene encoding huntingtin. Evidence from both human genotype-phenotype relationships and mouse model systems suggests that the mutation acts by dysregulating some normal activity of huntingtin. Recent work in the mouse has revealed a role for huntingtin in epigenetic regulation during development. Here, we examine the role of the Drosophila huntingtin ortholog (dhtt) in chromatin regulation in the development of the fly. Although null dhtt mutants display no overt phenotype, we found that dhtt acts as a suppressor of position-effect variegation (PEV), suggesting that it influences chromatin organization. We demonstrate that dhtt affects heterochromatin spreading in a PEV model by modulating histone H3K9 methylation levels at the heterochromatin-euchromatin boundary. To gain mechanistic insights into how dhtt influences chromatin function, we conducted a candidate genetic screen using RNAi lines targeting known PEV modifier genes. We found that dhtt modifies phenotypes caused by knockdown of a number of key epigenetic regulators, including chromatin-associated proteins, histone demethylases (HDMs) and methyltransferases. Notably, dhtt strongly modifies phenotypes resulting from loss of the HDM dLsd1, in both the ovary and wing, and we demonstrate that dhtt appears to act as a facilitator of dLsd1 function in regulating global histone H3K4 methylation levels. These findings suggest that a fundamental aspect of huntingtin function in heterochromatin/euchromatin organization is evolutionarily conserved across phyla. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. Assessing the model transferability for prediction of transcription factor binding sites based on chromatin accessibility.

    Science.gov (United States)

    Liu, Sheng; Zibetti, Cristina; Wan, Jun; Wang, Guohua; Blackshaw, Seth; Qian, Jiang

    2017-07-27

    Computational prediction of transcription factor (TF) binding sites in different cell types is challenging. Recent technology development allows us to determine the genome-wide chromatin accessibility in various cellular and developmental contexts. The chromatin accessibility profiles provide useful information in prediction of TF binding events in various physiological conditions. Furthermore, ChIP-Seq analysis was used to determine genome-wide binding sites for a range of different TFs in multiple cell types. Integration of these two types of genomic information can improve the prediction of TF binding events. We assessed to what extent a model built upon on other TFs and/or other cell types could be used to predict the binding sites of TFs of interest. A random forest model was built using a set of cell type-independent features such as specific sequences recognized by the TFs and evolutionary conservation, as well as cell type-specific features derived from chromatin accessibility data. Our analysis suggested that the models learned from other TFs and/or cell lines performed almost as well as the model learned from the target TF in the cell type of interest. Interestingly, models based on multiple TFs performed better than single-TF models. Finally, we proposed a universal model, BPAC, which was generated using ChIP-Seq data from multiple TFs in various cell types. Integrating chromatin accessibility information with sequence information improves prediction of TF binding.The prediction of TF binding is transferable across TFs and/or cell lines suggesting there are a set of universal "rules". A computational tool was developed to predict TF binding sites based on the universal "rules".

  17. Chromatin (dis)organization and cancer: BUR-binding proteins as biomarkers for cancer.

    Science.gov (United States)

    Galande, Sanjeev

    2002-06-01

    Malignant transformation of cells is associated with changes in gene expression. Gross alterations in chromatin organization may be involved in such gene dysregulation, as well as the involvement of specific transcription factors. Specialized genomic DNA segments that exhibit high affinity to the nuclear matrix in vitro have been designated as matrix/scaffold attachment regions (MARs/SARs). MARs are postulated to anchor chromatin onto the nuclear matrix, thereby organizing genomic DNA into topologically distinct loop domains that are important in replication and transcription. In support of this notion, MARs often colocalize or exist in close proximity to regulatory sequences including enhancers. Base unpairing regions (BURs) are typically 100-150 bp regions within MARs, possess an intrinsic propensity to unwind under negative superhelical strain, and are considered to be hallmark of MARs. To investigate a potential mechanism that could lead to significant alterations in gene expression in cancer cells, this review focuses on a group of chromatin-associated proteins that specifically recognize double stranded BURs. Several important proteins have been identified from cancer cells as BUR-binding proteins, including poly (ADP-ribose) polymerase (PARP-1), Ku autoantigen, SAF-A, HMG-I(Y), nucleolin and p53. Many of these proteins are dramatically upregulated in malignancy of the breast. Increase in the amount of these BUR-binding proteins, some of which are known to interact with each other, may not only provide an architectural core but also recruit functional multi-molecular complexes at the base of chromatin loops to affect multiple distant genes. Experimental strategies by which these proteins can be exploited as carcinoma-specific diagnostic markers and as targets for antineoplastic therapy are discussed.

  18. Effect of saffron on rat sperm chromatin integrity.

    Science.gov (United States)

    Mardani, Mohammad; Vaez, Ahmad; Razavi, Shahnaz

    2014-05-01

    Currently, relation between reactive oxygen species (ROS) ROS concentration and semen quality was indicated. Saffron has traditionally been not only considered as a food additive but also as a medicinal herb, which has a good antioxidant properties. The aim of this study was to evaluate the protection potency of saffron and vitamin E on sperm chromatin integrity. Thirty adult male Wistar rats divided equally into saffron (100 mg/kg), vitamin E (100 mg/kg) and control (0.5cc distilled water /day) groups. After 60 days, cauda epididymis dissected and sperm cells were used for analysis of sperm chromatin packaging by chromomycin A3 (CMA3) staining, and sperm chromatin susceptibility to acid denaturation by acridine orange (AO) staining. The mean percentage of CMA3 positive sperm was significantly decreased in saffron and vitamin E groups relative to control group (psaffron and vitamin E groups as compared with control group (psaffron can protect sperm against DNA damage and chromatin anomalies.

  19. Insights into Chromatin Structure and Dynamics in Plants

    Directory of Open Access Journals (Sweden)

    Stefanie Rosa

    2013-11-01

    Full Text Available The packaging of chromatin into the nucleus of a eukaryotic cell requires an extraordinary degree of compaction and physical organization. In recent years, it has been shown that this organization is dynamically orchestrated to regulate responses to exogenous stimuli as well as to guide complex cell-type-specific developmental programs. Gene expression is regulated by the compartmentalization of functional domains within the nucleus, by distinct nucleosome compositions accomplished via differential modifications on the histone tails and through the replacement of core histones by histone variants. In this review, we focus on these aspects of chromatin organization and discuss novel approaches such as live cell imaging and photobleaching as important tools likely to give significant insights into our understanding of the very dynamic nature of chromatin and chromatin regulatory processes. We highlight the contribution plant studies have made in this area showing the potential advantages of plants as models in understanding this fundamental aspect of biology.

  20. Neutron scattering studies on chromatin higher-order structure

    Energy Technology Data Exchange (ETDEWEB)

    Graziano, V.; Gerchman, S.E.; Schneider, D.K.; Ramakrishnan, V. [Brookhaven National Laboratory, Upton, NY (United States)

    1994-12-31

    We have been engaged in studies of the structure and condensation of chromatin into the 30nm filament using small-angle neutron scattering. We have also used deuterated histone H1 to determine its location in the chromatin 30nm filament. Our studies indicate that chromatin condenses with increasing ionic strength to a limiting structure that has a mass per unit length of 6-7 nucleosomes/11 nm. They also show that the linker histone H1/H5 is located in the interior of the chromatin filament, in a position compatible with its binding to the inner face of the nucleosome. Analysis of the mass per unit length as a function of H5 stoichiometry suggests that 5-7 contiguous nucleosomes need to have H5 bound before a stable higher order structure can exist.

  1. The centromere: chromatin foundation for the kinetochore machinery

    National Research Council Canada - National Science Library

    Fukagawa, Tatsuo; Earnshaw, William C

    2014-01-01

    Since discovery of the centromere-specific histone H3 variant CENP-A, centromeres have come to be defined as chromatin structures that establish the assembly site for the complex kinetochore machinery...

  2. Of giraffes' necks and the inheritance of chromatin states.

    Science.gov (United States)

    Pirrotta, Vincenzo

    2017-05-26

    New work reports that both derepressed and hyper-repressed chromatin states in animals can be transmitted to progeny for many generations. Transmission depends on genomic architecture and histone modifications.

  3. Commentary: Tone up your chromatin and stay young

    Indian Academy of Sciences (India)

    01/0005-0011. Keywords. Aging; chromatin; epigenetics; histones. Author Affiliations. Navneet K Matharu1 Rakesh K Mishra1. Centre for Cellular and Molecular Biology, CSIR Hyderabad 500 007, India. Dates. Early published: 14 March 2011 ...

  4. Transcription Through Chromatin-Link to Diseases and Therapeutics

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 9; Issue 11. Transcription Through Chromatin – Link to Diseases ... Medical Sciences, Madras University, Chennai. Transcription and Disease Laboratory Molecular Biology and Genetics Unit Jawaharlal Nehru Centre for Advanced Scientific Research.

  5. Structure of plant nuclear and ribosomal DNA containing chromatin.

    Science.gov (United States)

    Leber, B; Hemleben, V

    1979-11-10

    Digestion of plant chromatin from Brassica pekinensis and Matthiola incana with staphylococcus nuclease leads to a DNA repeat of 175 plus or minus 8 and a core size of 140 base pairs. DNase I digestion results in multiples of 10 bases. Ribosomal RNN genes were studied as a model system for active plant chromatin because of their great redundancy and their high transcriptional activity in growing and differentiating tissues. The actively transcribed genes were identified by nascent RNA of ribosomal origin still attached to its matrix DNA. Hybridization techniques were used to demonstrate that even transcriptionally active gene sequences are present in nuclease generated chromatin subunits. Comparison of the DNase I kinetics of chromatin digestion with the amount of ribosomal RNA genes which is available for hybridization at the given times indicated that ribosomal RNA genes are digested, but not preferentially degraded by DNase I.

  6. Nascent Connections: R-Loops and Chromatin Patterning.

    Science.gov (United States)

    Chédin, Frédéric

    2016-12-01

    RNA molecules, such as long noncoding RNAs (lncRNAs), have critical roles in regulating gene expression, chromosome architecture, and the modification states of chromatin. Recent developments suggest that RNA also influences gene expression and chromatin patterns through the interaction of nascent transcripts with their DNA template via the formation of co-transcriptional R-loop structures. R-loop formation over specific, conserved, hotspots occurs at thousands of genes in mammalian genomes and represents an important and dynamic feature of mammalian chromatin. Here, focusing primarily on mammalian systems, I describe the accumulating connections and possible mechanisms linking R-loop formation and chromatin patterning. The possible contribution of aberrant R-loops to pathological conditions is also discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. FACT facilitates chromatin transcription by RNA polymerases I and III

    DEFF Research Database (Denmark)

    Birch, Joanna L; Tan, Bertrand C-M; Panov, Kostya I

    2009-01-01

    Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle....... The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results...... in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III...

  8. Computational analysis of promoter elements and chromatin features in yeast.

    Science.gov (United States)

    Wyrick, John J

    2012-01-01

    Regulatory elements in promoter sequences typically function as binding sites for transcription factor proteins and thus are critical determinants of gene transcription. There is growing evidence that chromatin features, such as histone modifications or nucleosome positions, also have important roles in transcriptional regulation. Recent functional genomics and computational studies have yielded extensive datasets cataloging transcription factor binding sites (TFBS) and chromatin features, such as nucleosome positions, throughout the yeast genome. However, much of this data can be difficult to navigate or analyze efficiently. This chapter describes practical methods for the visualization, data mining, and statistical analysis of yeast promoter elements and chromatin features using two Web-accessible bioinformatics databases: ChromatinDB and Ceres.

  9. Discovery of transcription factors and regulatory regions driving in vivo tumor development by ATAC-seq and FAIRE-seq open chromatin profiling.

    Directory of Open Access Journals (Sweden)

    Kristofer Davie

    2015-02-01

    Full Text Available Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs. When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling

  10. Chromatin Insulators: Linking genome organization to cellular function

    Science.gov (United States)

    Phillips-Cremins, Jennifer E.; Corces, Victor G.

    2013-01-01

    A growing body of evidence suggests that insulators have a primary role in orchestrating the topological arrangement of higher-order chromatin architecture. Insulator-mediated long-range interactions can influence the epigenetic status of the genome and, in certain contexts, may have important effects on gene expression. Here we discuss higher-order chromatin organization as a unifying mechanism for diverse insulator actions across the genome. PMID:23706817

  11. Unsupervised pattern discovery in human chromatin structure through genomic segmentation.

    Science.gov (United States)

    Hoffman, Michael M; Buske, Orion J; Wang, Jie; Weng, Zhiping; Bilmes, Jeff A; Noble, William Stafford

    2012-03-18

    We trained Segway, a dynamic Bayesian network method, simultaneously on chromatin data from multiple experiments, including positions of histone modifications, transcription-factor binding and open chromatin, all derived from a human chronic myeloid leukemia cell line. In an unsupervised fashion, we identified patterns associated with transcription start sites, gene ends, enhancers, transcriptional regulator CTCF-binding regions and repressed regions. Software and genome browser tracks are at http://noble.gs.washington.edu/proj/segway/.

  12. ATP-Dependent Chromatin Remodeling Factors and Their Roles in Affecting Nucleosome Fiber Composition

    Directory of Open Access Journals (Sweden)

    Alexandra Lusser

    2011-10-01

    Full Text Available ATP-dependent chromatin remodeling factors of the SNF2 family are key components of the cellular machineries that shape and regulate chromatin structure and function. Members of this group of proteins have broad and heterogeneous functions ranging from controlling gene activity, facilitating DNA damage repair, promoting homologous recombination to maintaining genomic stability. Several chromatin remodeling factors are critical components of nucleosome assembly processes, and recent reports have identified specific functions of distinct chromatin remodeling factors in the assembly of variant histones into chromatin. In this review we will discuss the specific roles of ATP-dependent chromatin remodeling factors in determining nucleosome composition and, thus, chromatin fiber properties.

  13. The telomere binding protein TRF2 induces chromatin compaction.

    Science.gov (United States)

    Baker, Asmaa M; Fu, Qiang; Hayward, William; Victoria, Samuel; Pedroso, Ilene M; Lindsay, Stuart M; Fletcher, Terace M

    2011-04-19

    Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

  14. Characterizing the molecular architectures of chromatin-modifying complexes.

    Science.gov (United States)

    Setiaputra, Dheva T; Yip, Calvin K

    2017-11-01

    Eukaryotic cells package their genome in the form of a DNA-protein complex known as chromatin. This organization not only condenses the genome to fit within the confines of the nucleus, but also provides a platform for a cell to regulate accessibility to different gene sequences. The basic packaging element of chromatin is the nucleosome, which consists of 146 base pairs of DNA wrapped around histone proteins. One major means that a cell regulates chromatin structure is by depositing post-translational modifications on nucleosomal histone proteins, and thereby altering internucleosomal interactions and/or binding to different chromatin associated factors. These chromatin modifications are often catalyzed by multi-subunit enzyme complexes, whose large size, sophisticated composition, and inherent conformational flexibility pose significant technical challenges to their biochemical and structural characterization. Multiple structural approaches including nuclear magnetic resonance spectroscopy, X-ray crystallography, single-particle electron microscopy, and crosslinking coupled to mass spectrometry are often used synergistically to probe the overall architecture, subunit organization, and catalytic mechanisms of these macromolecular assemblies. In this review, we highlight several recent chromatin-modifying complexes studies that embodies this multipronged structural approach, and explore common themes amongst them. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Expression and chromatin structures of cellulolytic enzyme gene regulated by heterochromatin protein 1.

    Science.gov (United States)

    Zhang, Xiujun; Qu, Yinbo; Qin, Yuqi

    2016-01-01

    (FPA) increase), but also in the industry strain RE-10 (~20-30 % FPA increase). HepA is required for chromatin condensation of prominent cellulase genes. However, the opening of chromatin mediated by the deletion of hepA was not positively correlated with cellulolytic enzyme gene activation. HepA is actually a positive regulator for cellulolytic enzyme gene expression and could be a promising target for genetic modification to improve cellulolytic enzyme synthesis.

  16. Interphase Chromosome Conformation and Chromatin-Chromatin Interactions in Human Epithelial Cells Cultured Under Different Gravity Conditions

    Science.gov (United States)

    Zhang, Ye; Wong, Michael; Hada, Megumi; Wu, Honglu

    2015-01-01

    Microgravity has been shown to alter global gene expression patterns and protein levels both in cultured cells and animal models. It has been suggested that the packaging of chromatin fibers in the interphase nucleus is closely related to genome function, and the changes in transcriptional activity are tightly correlated with changes in chromatin folding. This study explores the changes of chromatin conformation and chromatin-chromatin interactions in the simulated microgravity environment, and investigates their correlation to the expression of genes located at different regions of the chromosome. To investigate the folding of chromatin in interphase under various culture conditions, human epithelial cells, fibroblasts, and lymphocytes were fixed in the G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome as separate colors. After images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multi-mega base pair scale. In order to determine the effects of microgravity on chromosome conformation and orientation, measures such as distance between homologous pairs, relative orientation of chromosome arms about a shared midpoint, and orientation of arms within individual chromosomes were all considered as potentially impacted by simulated microgravity conditions. The studies revealed non-random folding of chromatin in interphase, and suggested an association of interphase chromatin folding with radiation-induced chromosome aberration hotspots. Interestingly, the distributions of genes with expression changes over chromosome 3 in cells cultured under microgravity environment are apparently clustered on specific loci and chromosomes. This data provides important insights into how mammalian cells respond to microgravity at molecular level.

  17. Initial high-resolution microscopic mapping of active and inactive regulatory sequences proves non-random 3D arrangements in chromatin domain clusters.

    Science.gov (United States)

    Cremer, Marion; Schmid, Volker J; Kraus, Felix; Markaki, Yolanda; Hellmann, Ines; Maiser, Andreas; Leonhardt, Heinrich; John, Sam; Stamatoyannopoulos, John; Cremer, Thomas

    2017-08-07

    The association of active transcription regulatory elements (TREs) with DNAse I hypersensitivity (DHS[+]) and an 'open' local chromatin configuration has long been known. However, the 3D topography of TREs within the nuclear landscape of individual cells in relation to their active or inactive status has remained elusive. Here, we explored the 3D nuclear topography of active and inactive TREs in the context of a recently proposed model for a functionally defined nuclear architecture, where an active and an inactive nuclear compartment (ANC-INC) form two spatially co-aligned and functionally interacting networks. Using 3D structured illumination microscopy, we performed 3D FISH with differently labeled DNA probe sets targeting either sites with DHS[+], apparently active TREs, or DHS[-] sites harboring inactive TREs. Using an in-house image analysis tool, DNA targets were quantitatively mapped on chromatin compaction shaped 3D nuclear landscapes. Our analyses present evidence for a radial 3D organization of chromatin domain clusters (CDCs) with layers of increasing chromatin compaction from the periphery to the CDC core. Segments harboring active TREs are significantly enriched at the decondensed periphery of CDCs with loops penetrating into interchromatin compartment channels, constituting the ANC. In contrast, segments lacking active TREs (DHS[-]) are enriched toward the compacted interior of CDCs (INC). Our results add further evidence in support of the ANC-INC network model. The different 3D topographies of DHS[+] and DHS[-] sites suggest positional changes of TREs between the ANC and INC depending on their functional state, which might provide additional protection against an inappropriate activation. Our finding of a structural organization of CDCs based on radially arranged layers of different chromatin compaction levels indicates a complex higher-order chromatin organization beyond a dichotomic classification of chromatin into an 'open,' active and 'closed

  18. Phenotypic plasticity in Drosophila pigmentation caused by temperature sensitivity of a chromatin regulator network.

    Directory of Open Access Journals (Sweden)

    Jean-Michel Gibert

    2007-02-01

    Full Text Available Phenotypic plasticity is the ability of a genotype to produce contrasting phenotypes in different environments. Although many examples have been described, the responsible mechanisms are poorly understood. In particular, it is not clear how phenotypic plasticity is related to buffering, the maintenance of a constant phenotype against genetic or environmental variation. We investigate here the genetic basis of a particularly well described plastic phenotype: the abdominal pigmentation in female Drosophila melanogaster. Cold temperature induces a dark pigmentation, in particular in posterior segments, while higher temperature has the opposite effect. We show that the homeotic gene Abdominal-B (Abd-B has a major role in the plasticity of pigmentation in the abdomen. Abd-B plays opposite roles on melanin production through the regulation of several pigmentation enzymes. This makes the control of pigmentation very unstable in the posterior abdomen, and we show that the relative spatio-temporal expression of limiting pigmentation enzymes in this region of the body is thermosensitive. Temperature acts on melanin production by modulating a chromatin regulator network, interacting genetically with the transcription factor bric-à-brac (bab, a target of Abd-B and Hsp83, encoding the chaperone Hsp90. Genetic disruption of this chromatin regulator network increases the effect of temperature and the instability of the pigmentation pattern in the posterior abdomen. Colocalizations on polytene chromosomes suggest that BAB and these chromatin regulators cooperate in the regulation of many targets, including several pigmentation enzymes. We show that they are also involved in sex comb development in males and that genetic destabilization of this network is also strongly modulated by temperature for this phenotype. Thus, we propose that phenotypic plasticity of pigmentation is a side effect reflecting a global impact of temperature on epigenetic mechanisms

  19. Systematic Epigenomic Analysis Reveals Chromatin States Associated with Melanoma Progression

    Directory of Open Access Journals (Sweden)

    Petko Fiziev

    2017-04-01

    Full Text Available The extent and nature of epigenomic changes associated with melanoma progression is poorly understood. Through systematic epigenomic profiling of 35 epigenetic modifications and transcriptomic analysis, we define chromatin state changes associated with melanomagenesis by using a cell phenotypic model of non-tumorigenic and tumorigenic states. Computation of specific chromatin state transitions showed loss of histone acetylations and H3K4me2/3 on regulatory regions proximal to specific cancer-regulatory genes in important melanoma-driving cell signaling pathways. Importantly, such acetylation changes were also observed between benign nevi and malignant melanoma human tissues. Intriguingly, only a small fraction of chromatin state transitions correlated with expected changes in gene expression patterns. Restoration of acetylation levels on deacetylated loci by histone deacetylase (HDAC inhibitors selectively blocked excessive proliferation in tumorigenic cells and human melanoma cells, suggesting functional roles of observed chromatin state transitions in driving hyperproliferative phenotype. Through these results, we define functionally relevant chromatin states associated with melanoma progression.

  20. The Circadian NAD+ Metabolism: Impact on Chromatin Remodeling and Aging

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    Yasukazu Nakahata

    2016-01-01

    Full Text Available Gene expression is known to be a stochastic phenomenon. The stochastic gene expression rate is thought to be altered by topological change of chromosome and/or by chromatin modifications such as acetylation and methylation. Changes in mechanical properties of chromosome/chromatin by soluble factors, mechanical stresses from the environment, or metabolites determine cell fate, regulate cellular functions, or maintain cellular homeostasis. Circadian clock, which drives the expression of thousands of genes with 24-hour rhythmicity, has been known to be indispensable for maintaining cellular functions/homeostasis. During the last decade, it has been demonstrated that chromatin also undergoes modifications with 24-hour rhythmicity and facilitates the fine-tuning of circadian gene expression patterns. In this review, we cover data which suggests that chromatin structure changes in a circadian manner and that NAD+ is the key metabolite for circadian chromatin remodeling. Furthermore, we discuss the relationship among circadian clock, NAD+ metabolism, and aging/age-related diseases. In addition, the interventions of NAD+ metabolism for the prevention and treatment of aging and age-related diseases are also discussed.

  1. Circadian Rhythms and Memory Formation: Regulation by Chromatin Remodeling

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    Saurabh eSahar

    2012-03-01

    Full Text Available Epigenetic changes, such as DNA methylation or histone modification, can remodel the chromatin and regulate gene expression. Remodeling of chromatin provides an efficient mechanism of transducing signals, such as light or nutrient availability, to regulate gene expression. CLOCK:BMAL1 mediated activation of clock-controlled genes (CCGs is coupled to circadian changes in histone modification at their promoters. Several chromatin modifiers, such as the deacetylases SIRT1 and HDAC3 or methyltransferase MLL1, have been shown to be recruited to the promoters of the CCGs in a circadian manner. Interestingly, the central element of the core clock machinery, the transcription factor CLOCK, also possesses histone acetyltransferase activity. Rhythmic expression of the CCGs is abolished in the absence of these chromatin modifiers. Recent research has demonstrated that chromatin remodeling is at the cross-roads of circadian rhythms and regulation of metabolism and aging. It would be of interest to identify if similar pathways exist in the epigenetic regulation of memory formation.

  2. SMC complexes orchestrate the mitotic chromatin interaction landscape.

    Science.gov (United States)

    Kakui, Yasutaka; Uhlmann, Frank

    2017-09-21

    Chromatin is a very long DNA-protein complex that controls the expression and inheritance of the genetic information. Chromatin is stored within the nucleus in interphase and further compacted into chromosomes during mitosis. This process, known as chromosome condensation, is essential for faithful segregation of genomic DNA into daughter cells. Condensin and cohesin, members of the structural maintenance of chromosomes (SMC) family, are fundamental for chromosome architecture, both for establishment of chromatin structure in the interphase nucleus and for the formation of condensed chromosomes in mitosis. These ring-shaped SMC complexes are thought to regulate the interactions between DNA strands by topologically entrapping DNA. How this activity shapes chromosomes is not yet understood. Recent high throughput chromosome conformation capture studies revealed how chromatin is reorganized during the cell cycle and have started to explore the role of SMC complexes in mitotic chromatin architecture. Here, we summarize these findings and discuss the conserved nature of chromosome condensation in eukaryotes. We highlight the unexpected finding that condensin-dependent intra-chromosomal interactions in mitosis increase within a distinctive distance range that is characteristic for an organism, while longer and shorter-range interactions are suppressed. This reveals important molecular insight into chromosome architecture.

  3. Supervised learning method for predicting chromatin boundary associated insulator elements.

    Science.gov (United States)

    Bednarz, Paweł; Wilczyński, Bartek

    2014-12-01

    In eukaryotic cells, the DNA material is densely packed inside the nucleus in the form of a DNA-protein complex structure called chromatin. Since the actual conformation of the chromatin fiber defines the possible regulatory interactions between genes and their regulatory elements, it is very important to understand the mechanisms governing folding of chromatin. In this paper, we show that supervised methods for predicting chromatin boundary elements are much more effective than the currently popular unsupervised methods. Using boundary locations from published Hi-C experiments and modEncode tracks as features, we can tell the insulator elements from randomly selected background sequences with great accuracy. In addition to accurate predictions of the training boundary elements, our classifiers make new predictions. Many of them correspond to the locations of known insulator elements. The key features used for predicting boundary elements do not depend on the prediction method. Because of its miniscule size, chromatin state cannot be measured directly, we need to rely on indirect measurements, such as ChIP-Seq and fill in the gaps with computational models. Our results show that currently, at least in the model organisms, where we have many measurements including ChIP-Seq and Hi-C, we can make accurate predictions of insulator positions.

  4. Plant programmed cell death from a chromatin point of view.

    Science.gov (United States)

    Latrasse, D; Benhamed, M; Bergounioux, C; Raynaud, C; Delarue, M

    2016-10-01

    Programmed cell death (PCD) is a ubiquitous genetically regulated process consisting of the activation of finely controlled signalling pathways that lead to cellular suicide. PCD can be part of a developmental programme (dPCD) or be triggered by environmental conditions (ePCD). In plant cells, as in animal cells, extensive chromatin condensation and degradation of the nuclear DNA are among the most conspicuous features of cells undergoing PCD. Changes in chromatin condensation could either reflect the structural changes required for internucleosomal fragmentation of nuclear DNA or relate to large-scale chromatin rearrangements associated with a major transcriptional switch occurring during cell death. The aim of this review is to give an update on plant PCD processes from a chromatin point of view. The first part will be dedicated to chromatin conformational changes associated with cell death observed in various developmental and physiological conditions, whereas the second part will be devoted to histone dynamics and DNA modifications associated with critical changes in genome expression during the cell death process. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  5. Systematic Epigenomic Analysis Reveals Chromatin States Associated with Melanoma Progression.

    Science.gov (United States)

    Fiziev, Petko; Akdemir, Kadir C; Miller, John P; Keung, Emily Z; Samant, Neha S; Sharma, Sneha; Natale, Christopher A; Terranova, Christopher J; Maitituoheti, Mayinuer; Amin, Samirkumar B; Martinez-Ledesma, Emmanuel; Dhamdhere, Mayura; Axelrad, Jacob B; Shah, Amiksha; Cheng, Christine S; Mahadeshwar, Harshad; Seth, Sahil; Barton, Michelle C; Protopopov, Alexei; Tsai, Kenneth Y; Davies, Michael A; Garcia, Benjamin A; Amit, Ido; Chin, Lynda; Ernst, Jason; Rai, Kunal

    2017-04-25

    The extent and nature of epigenomic changes associated with melanoma progression is poorly understood. Through systematic epigenomic profiling of 35 epigenetic modifications and transcriptomic analysis, we define chromatin state changes associated with melanomagenesis by using a cell phenotypic model of non-tumorigenic and tumorigenic states. Computation of specific chromatin state transitions showed loss of histone acetylations and H3K4me2/3 on regulatory regions proximal to specific cancer-regulatory genes in important melanoma-driving cell signaling pathways. Importantly, such acetylation changes were also observed between benign nevi and malignant melanoma human tissues. Intriguingly, only a small fraction of chromatin state transitions correlated with expected changes in gene expression patterns. Restoration of acetylation levels on deacetylated loci by histone deacetylase (HDAC) inhibitors selectively blocked excessive proliferation in tumorigenic cells and human melanoma cells, suggesting functional roles of observed chromatin state transitions in driving hyperproliferative phenotype. Through these results, we define functionally relevant chromatin states associated with melanoma progression. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Structure of Nascent Chromatin Is Essential for Hematopoietic Lineage Specification.

    Science.gov (United States)

    Petruk, Svetlana; Mariani, Samanta A; De Dominici, Marco; Porazzi, Patrizia; Minieri, Valentina; Cai, Jingli; Iacovitti, Lorraine; Flomenberg, Neal; Calabretta, Bruno; Mazo, Alexander

    2017-04-11

    The role of chromatin structure in lineage commitment of multipotent hematopoietic progenitors (HPCs) is presently unclear. We show here that CD34 + HPCs possess a post-replicative chromatin globally devoid of the repressive histone mark H3K27me3. This H3K27-unmodified chromatin is required for recruitment of lineage-determining transcription factors (TFs) C/EBPα, PU.1, and GATA-1 to DNA just after DNA replication upon cytokine-induced myeloid or erythroid commitment. Blocking DNA replication or increasing H3K27me3 levels prevents recruitment of these TFs to DNA and suppresses cytokine-induced erythroid or myeloid differentiation. However, H3K27me3 is rapidly associated with nascent DNA in more primitive human and murine HPCs. Treatment of these cells with instructive cytokines leads to a significant delay in accumulation of H3K27me3 in nascent chromatin due to activity of the H3K27me3 demethylase UTX. Thus, HPCs utilize special mechanisms of chromatin modification for recruitment of specific TFs to DNA during early stages of lineage specification. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. Structure of Nascent Chromatin Is Essential for Hematopoietic Lineage Specification

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    Svetlana Petruk

    2017-04-01

    Full Text Available The role of chromatin structure in lineage commitment of multipotent hematopoietic progenitors (HPCs is presently unclear. We show here that CD34+ HPCs possess a post-replicative chromatin globally devoid of the repressive histone mark H3K27me3. This H3K27-unmodified chromatin is required for recruitment of lineage-determining transcription factors (TFs C/EBPα, PU.1, and GATA-1 to DNA just after DNA replication upon cytokine-induced myeloid or erythroid commitment. Blocking DNA replication or increasing H3K27me3 levels prevents recruitment of these TFs to DNA and suppresses cytokine-induced erythroid or myeloid differentiation. However, H3K27me3 is rapidly associated with nascent DNA in more primitive human and murine HPCs. Treatment of these cells with instructive cytokines leads to a significant delay in accumulation of H3K27me3 in nascent chromatin due to activity of the H3K27me3 demethylase UTX. Thus, HPCs utilize special mechanisms of chromatin modification for recruitment of specific TFs to DNA during early stages of lineage specification.

  8. HAT2 mediates histone H4K4 acetylation and affects micrococcal nuclease sensitivity of chromatin in Leishmania donovani.

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    Pravin K Jha

    Full Text Available Histone post-translational modifications (PTMs such as acetylation and methylation are known to affect chromatin higher order structures. Primary targets of these modifications include basic residues present at N-terminus tail region of core histones. Four histone acetyltransferase (HAT genes have been identified in trypanosomatids. HAT1, HAT3 and HAT4 of Leishmania donovani have been partially characterized. However, there is no report about HAT2 of Leishmania donovani. Lysine residues present on the N-terminal tail of Leishmania donovani histone H4 are conserved in other trypanosomatids and humans. PTMs of lysines modulate various functions at chromatin level. The four histone acetyltransferases encoded in Leishmania genome were over-expressed to analyse their functional activity. All four HATs were found actively acetylating core histones H3/H4. Similar to L. donovani HAT3 and HAT4, HAT2 was found to be a member of MYST family protein and have SAS2 type domain. Over-expression of HAT2 significantly increases acetylation of H4K4. To analyse the effect of HAT2 over-expression on chromatin accessibility, micrococcal nuclease digestion assay was performed. MNase digestion resulted in a higher proportion of the mononucleosomes and dinucleosomes in HAT2 over-expressing cells as compared to WT L. donovani cells. Acetylation of lysine-4 neutralizes the amino terminal region of histone H4. This weakens its interaction with neighbouring nucleosomes and the linker DNA. HAT2 over-expression in L. donovani resulted in highly accessible chromatin suggesting chromatin decondensation. HAT2 may have an important role to play in global regulation of transcription in L. donovani. Better understanding of these epigenetic determinants of parasite would help in designing novel therapeutic strategies.

  9. Chromatin status of apoptosis genes correlates with sensitivity to chemo-, immune- and radiation therapy in colorectal cancer cell lines.

    Science.gov (United States)

    Benard, Anne; Janssen, Connie M; van den Elsen, Peter J; van Eggermond, Marja C J A; Hoon, Dave S B; van de Velde, Cornelis J H; Kuppen, Peter J K

    2014-12-01

    The apoptosis pathway of programmed cell death is frequently deregulated in cancer. An intact apoptosis pathway is required for proper response to anti-cancer treatment. We investigated the chromatin status of key apoptosis genes in the apoptosis pathway in colorectal cancer cell lines in relation to apoptosis induced by chemo-, immune- or radiation therapy. Using chromatin immunoprecipitation (ChIP), we measured the presence of transcription-activating histone modifications H3Ac and H3K4me3 and silencing modifications H3K9me3 and H3K27me3 at the gene promoter regions of key apoptosis genes Bax, Bcl2, Caspase-9, Fas (CD95) and p53. Cell lines DLD1, SW620, Colo320, Caco2, Lovo and HT29 were treated with cisplatin, anti-Fas or radiation. The apoptotic response was measured by flow cytometry using propidium iodide and annexin V-FITC. The chromatin status of the apoptosis genes reflected the activation status of the intrinsic (Bax, Bcl2, Caspase-9 and p53) and extrinsic (Fas) pathways. An active intrinsic apoptotic pathway corresponded to sensitivity to cisplatin and radiation treatment of cell lines DLD1, SW620 and Colo320. An active Fas promoter corresponded to an active extrinsic apoptotic pathway in cell line DLD1. mRNA expression data correlated with the chromatin status of the apoptosis genes as measured by ChIP. In conclusion, the results presented in this study indicate that the balance between activating and silencing histone modifications, reflecting the chromatin status of apoptosis genes, can be used to predict the response of tumor cells to different anti-cancer therapies and could provide a novel target to sensitize tumors to obtain adequate treatment responses.

  10. A role for CARM1-mediated histone H3 arginine methylation in protecting histone acetylation by releasing corepressors from chromatin.

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    Jing Wu

    Full Text Available Arginine methylation broadly occurs in histones and has been linked to transcriptional regulation, cell cycle regulation and DNA repair. While numerous proteins (histone code effectors that specifically recognize or read the methylated lysine residues in core histones have been identified, little is known for effectors specific for methylated arginines in histones. In this study, we attempted to identify effector(s recognizing asymmetrically methylated R17 and R26 in H3, which are catalyzed by CARM1/PRMT4, through an unbiased biochemical approach. Although we have yet to identify such effector using this approach, we find that these modifications function cooperatively with histone acetylation to inhibit the binding of the nucleosome remodeling and deacetylase complex (NuRD and TIF1 family corepressors to H3 tail in vitro. In support of this finding, we show that overexpression of CARM1 in 293 T cells leads to reduced association of NuRD with chromatin, whereas knockdown of CARM1 in HeLa cells leads to increased association of NuRD with chromatin and decreased level of histone acetylation. Furthermore, in the Carm1-/- MEF cells there is an increased association of NuRD and TIF1β with chromatin and a global decrease in histone acetylation. By chromatin immunoprecipitation assay, we show that overexpression of CARM1 results in reduced association of NuRD complex and TIF1β with an episomal reporter and that CARM1 is required in MEF cells for LPS-induced dissociation of NuRD from a NF-κb target gene. Taking together, our study provides evidence for a role of CARM1-mediated arginine methylation in regulation of histone acetylation and transcription: facilitating transcription by discharging corepressors from chromatin.

  11. Interactions of chromatin context, binding site sequence content, and sequence evolution in stress-induced p53 occupancy and transactivation.

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    Dan Su

    2015-01-01

    Full Text Available Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs and gene transactivation from a large pool of potential p53 REs (p53REs. To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 hypersensitivity, DHS, ENCODE chromatin states, p53RE sequence, and evolutionary conservation. We observed that the inducible expression of p53-regulated genes was associated with the steady-state chromatin status of the cell. Most highly inducible p53-regulated genes were suppressed at baseline and marked by repressive histone modifications or displayed CTCF binding. Comparison of p53RE sequences residing in different chromatin contexts demonstrated that weaker p53REs resided in open promoters, while stronger p53REs were located within enhancers and repressed chromatin. p53 occupancy was strongly correlated with similarity of the target DNA sequences to the p53RE consensus, but surprisingly, inversely correlated with pre-existing nucleosome accessibility (DHS and evolutionary conservation at the p53RE. Occupancy by p53 of REs that overlapped transposable element (TE repeats was significantly higher (p<10-7 and correlated with stronger p53RE sequences (p<10-110 relative to nonTE-associated p53REs, particularly for MLT1H, LTR10B, and Mer61 TEs. However, binding at these elements was generally not associated with transactivation of adjacent genes. Occupied p53REs located in L2-like TEs were unique in displaying highly negative PhyloP scores (predicted fast-evolving and being associated with altered H3K4me3 and DHS levels. These results underscore the systematic interaction between chromatin status and p53

  12. Osmotic Challenge Drives Rapid and Reversible Chromatin Condensation in Chondrocytes

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    Irianto, Jerome; Swift, Joe; Martins, Rui P.; McPhail, Graham D.; Knight, Martin M.; Discher, Dennis E.; Lee, David A.

    2013-01-01

    Changes in extracellular osmolality have been shown to alter gene expression patterns and metabolic activity of various cell types, including chondrocytes. However, mechanisms by which physiological or pathological changes in osmolality impact chondrocyte function remain unclear. Here we use quantitative image analysis, electron microscopy, and a DNase I assay to show that hyperosmotic conditions (>400 mOsm/kg) induce chromatin condensation, while hypoosmotic conditions (100 mOsm/kg) cause decondensation. Large density changes (p condensation and decondensation during a daily loading cycle. The effect of changes in osmolality on nuclear morphology (p condensation (p condensation and osmolality was accurately modeled by a polymer gel model which, along with the rapid nature of the chromatin condensation (<20 s), reveals the basic physicochemical nature of the process. Alterations in chromatin structure are expected to influence gene expression and thereby regulate chondrocyte activity in response to osmotic changes. PMID:23442954

  13. chromswitch: A flexible method to detect chromatin state switches.

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    Jessa, Selin; Kleinman, Claudia L

    2018-02-09

    Chromatin state plays a major role in controlling gene expression, and comparative analysis of ChIP-seq data is key to understanding epigenetic regulation. We present chromswitch, an R/Bioconductor package to integrate epigenomic data in a defined window of interest to detect an overall switch in chromatin state. Chromswitch accurately classifies a benchmarking dataset, and when applied genome-wide, the tool successfully detects chromatin changes that result in brain-specific expression. Chromswitch is implemented as an R package available from Bioconductor at https://bioconductor.org/packages/chromswitch. claudia.kleinman@mcgill.ca. Supplementary material are available online. All data and code for the analysis presented in this paper are available at https://doi.org/10.5281/zenodo.1101260.

  14. Sliding and peeling of histone during chromatin remodelling

    CERN Document Server

    Garai, Ashok; Chowdhury, Debashish

    2011-01-01

    ATP-dependent chromatin remodeling enzymes (CRE) are bio-molecular motors in eukaryotic cells. These are driven by a chemical fuel, namely, adenosine triphosphate (ATP). CREs actively participate in many cellular processes that require accessibility of specific stretches of DNA which are packaged as chromatin. The basic unit of chromatin is a nucleosome where 146 bp $\\sim$ 50 nm of a double stranded DNA (dsDNA) is wrapped around a spool formed by histone proteins. We investigate the mechanism of peeling of the histone spool, and its complete detachment, from the dsDNA by a CRE. Our two-state model of a CRE captures effectively two distinct chemical (or conformational) states in the mechano-chemical cycle of each ATP-dependent CRE. We calculate the mean times for histone detachment. Our predictions on the ATP-dependence of the measurable quantities can be tested by carrying out {\\it in-vitro} experiments.

  15. Structural plasticity of single chromatin fibers revealed by torsional manipulation

    CERN Document Server

    Bancaud, Aurelien; Barbi, Maria; Wagner, Gaudeline; Allemand, Jean-Francois; Mozziconacci, Julien; Lavelle, Christophe; Croquette, Vincent; Victor, Jean-Marc; Prunell, Ariel; Viovy, Jean-Louis

    2006-01-01

    Magnetic tweezers are used to study the mechanical response under torsion of single nucleosome arrays reconstituted on tandem repeats of 5S positioning sequences. Regular arrays are extremely resilient and can reversibly accommodate a large amount of supercoiling without much change in length. This behavior is quantitatively described by a molecular model of the chromatin 3-D architecture. In this model, we assume the existence of a dynamic equilibrium between three conformations of the nucleosome, which are determined by the crossing status of the entry/exit DNAs (positive, null or negative). Torsional strain, in displacing that equilibrium, extensively reorganizes the fiber architecture. The model explains a number of long-standing topological questions regarding DNA in chromatin, and may provide the ground to better understand the dynamic binding of most chromatin-associated proteins.

  16. UV-Induced DNA Damage and Mutagenesis in Chromatin.

    Science.gov (United States)

    Mao, Peng; Wyrick, John J; Roberts, Steven A; Smerdon, Michael J

    2017-01-01

    UV radiation induces photolesions that distort the DNA double helix and, if not repaired, can cause severe biological consequences, including mutagenesis or cell death. In eukaryotes, both the formation and repair of UV damage occur in the context of chromatin, in which genomic DNA is packaged with histones into nucleosomes and higher order chromatin structures. Here, we review how chromatin impacts the formation of UV photoproducts in eukaryotic cells. We describe the initial discovery that nucleosomes and other DNA binding proteins induce characteristic "photofootprints" during the formation of UV photoproducts. We also describe recent progress in genomewide methods for mapping UV damage, which echoes early biochemical studies, and highlights the role of nucleosomes and transcription factors in UV damage formation and repair at unprecedented resolution. Finally, we discuss our current understanding of how the distribution and repair of UV-induced DNA damage influence mutagenesis in human skin cancers. © 2016 The American Society of Photobiology.

  17. Epigenetic response to environmental stress: Assembly of BRG1-G9a/GLP-DNMT3 repressive chromatin complex on Myh6 promoter in pathologically stressed hearts.

    Science.gov (United States)

    Han, Pei; Li, Wei; Yang, Jin; Shang, Ching; Lin, Chiou-Hong; Cheng, Wei; Hang, Calvin T; Cheng, Hsiu-Ling; Chen, Chen-Hao; Wong, Johnson; Xiong, Yiqin; Zhao, Mingming; Drakos, Stavros G; Ghetti, Andrea; Li, Dean Y; Bernstein, Daniel; Chen, Huei-Sheng Vincent; Quertermous, Thomas; Chang, Ching-Pin

    2016-07-01

    Chromatin structure is determined by nucleosome positioning, histone modifications, and DNA methylation. How chromatin modifications are coordinately altered under pathological conditions remains elusive. Here we describe a stress-activated mechanism of concerted chromatin modification in the heart. In mice, pathological stress activates cardiomyocytes to express Brg1 (nucleosome-remodeling factor), G9a/Glp (histone methyltransferase), and Dnmt3 (DNA methyltransferase). Once activated, Brg1 recruits G9a and then Dnmt3 to sequentially assemble repressive chromatin-marked by H3K9 and CpG methylation-on a key molecular motor gene (Myh6), thereby silencing Myh6 and impairing cardiac contraction. Disruption of Brg1, G9a or Dnmt3 erases repressive chromatin marks and de-represses Myh6, reducing stress-induced cardiac dysfunction. In human hypertrophic hearts, BRG1-G9a/GLP-DNMT3 complex is also activated; its level correlates with H3K9/CpG methylation, Myh6 repression, and cardiomyopathy. Our studies demonstrate a new mechanism of chromatin assembly in stressed hearts and novel therapeutic targets for restoring Myh6 and ventricular function. The stress-induced Brg1-G9a-Dnmt3 interactions and sequence of repressive chromatin assembly on Myh6 illustrates a molecular mechanism by which the heart epigenetically responds to environmental signals. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel. Copyright © 2016. Published by Elsevier B.V.

  18. The telomere binding protein TRF2 induces chromatin compaction.

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    Asmaa M Baker

    2011-04-01

    Full Text Available Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

  19. Rapid genome-scale mapping of chromatin accessibility in tissue

    Science.gov (United States)

    2012-01-01

    Background The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on large amounts of purified nuclei as starting material. This complicates analysis of trace clinical tissue samples that are often stored frozen. We have developed an alternative nuclease based procedure to bypass nuclear preparation to interrogate nuclease accessible regions in frozen tissue samples. Results Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh). The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergence between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases and accordingly accessibility of CpG islands is probed more efficiently using TACh. Conclusion The TACh methodology identifies accessible chromatin derived from frozen tissue samples. We propose that this simple, robust approach can be applied across a broad range of

  20. Rapid genome-scale mapping of chromatin accessibility in tissue

    Directory of Open Access Journals (Sweden)

    Grøntved Lars

    2012-06-01

    Full Text Available Abstract Background The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on large amounts of purified nuclei as starting material. This complicates analysis of trace clinical tissue samples that are often stored frozen. We have developed an alternative nuclease based procedure to bypass nuclear preparation to interrogate nuclease accessible regions in frozen tissue samples. Results Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh. The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergence between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases and accordingly accessibility of CpG islands is probed more efficiently using TACh. Conclusion The TACh methodology identifies accessible chromatin derived from frozen tissue samples. We propose that this simple, robust approach can be applied

  1. Chromatin structure and evolution in the human genome

    Directory of Open Access Journals (Sweden)

    Dunlop Malcolm G

    2007-05-01

    Full Text Available Abstract Background Evolutionary rates are not constant across the human genome but genes in close proximity have been shown to experience similar levels of divergence and selection. The higher-order organisation of chromosomes has often been invoked to explain such phenomena but previously there has been insufficient data on chromosome structure to investigate this rigorously. Using the results of a recent genome-wide analysis of open and closed human chromatin structures we have investigated the global association between divergence, selection and chromatin structure for the first time. Results In this study we have shown that, paradoxically, synonymous site divergence (dS at non-CpG sites is highest in regions of open chromatin, primarily as a result of an increased number of transitions, while the rates of other traditional measures of mutation (intergenic, intronic and ancient repeat divergence as well as SNP density are highest in closed regions of the genome. Analysis of human-chimpanzee divergence across intron-exon boundaries indicates that although genes in relatively open chromatin generally display little selection at their synonymous sites, those in closed regions show markedly lower divergence at their fourfold degenerate sites than in neighbouring introns and intergenic regions. Exclusion of known Exonic Splice Enhancer hexamers has little affect on the divergence observed at fourfold degenerate sites across chromatin categories; however, we show that closed chromatin is enriched with certain classes of ncRNA genes whose RNA secondary structure may be particularly important. Conclusion We conclude that, overall, non-CpG mutation rates are lowest in open regions of the genome and that regions of the genome with a closed chromatin structure have the highest background mutation rate. This might reflect lower rates of DNA damage or enhanced DNA repair processes in regions of open chromatin. Our results also indicate that dS is a poor

  2. Chromatin structure and pre-mRNA processing work together.

    Science.gov (United States)

    Jimeno-González, Silvia; Reyes, José C

    2016-05-26

    Chromatin is the natural context for transcription elongation. However, the elongating RNA polymerase II (RNAPII) is forced to pause by the positioned nucleosomes present in gene bodies. Here, we briefly discuss the current results suggesting that those pauses could serve as a mechanism to coordinate transcription elongation with pre-mRNA processing. Further, histone post-translational modifications have been found to regulate the recruitment of factors involved in pre-mRNA processing. This view highlights the important regulatory role of the chromatin context in the whole process of the mature mRNA synthesis.

  3. The importance of topoisomerases for chromatin regulated genes

    DEFF Research Database (Denmark)

    Fredsøe, Jacob Christian; Pedersen, Jakob Madsen; Rødgaard, Morten Terpager

    2013-01-01

    DNA topoisomerases are enzymes, which function to relieve torsional stress in the DNA helix by introducing transient breaks into the DNA molecule. By use of Saccharomyces cerevisiae and microarray technology we have previously shown that topoisomerases are required for the activation of chromatin...... topoisomerases for optimal activation, but in contrast to the PHO5 gene, topoisomerases are not required for chromatin remodeling of the GAL1/10 promoter region, indicating a different role of the enzymes. We are currently performing a detailed investigation of the GAL genes to elucidate the precise role...

  4. Genomic and chromatin signals underlying transcription start-site selection

    DEFF Research Database (Denmark)

    Valen, Eivind; Sandelin, Albin Gustav

    2011-01-01

    . In recent years substantial progress has been made towards this goal, spurred by the possibility of applying genome-wide, sequencing-based analysis. We now have a large collection of high-resolution datasets identifying locations of TSSs, protein-DNA interactions, and chromatin features over whole genomes......; the field is now faced with the daunting challenge of translating these descriptive maps into quantitative and predictive models describing the underlying biology. We review here the genomic and chromatin features that underlie TSS selection and usage, focusing on the differences between the major classes...

  5. Brd2 gene disruption causes ‘metabolically healthy’ obesity: Epigenetic and chromatin-based mechanisms that uncouple obesity from Type 2 diabetes

    Science.gov (United States)

    Wang, Fangnian; Deeney, Jude T.; Denis, Gerald V.

    2014-01-01

    Disturbed body energy balance can lead to obesity and obesity-driven diseases such as Type 2 diabetes, which have reached an epidemic level. Evidence indicates that obesity induced inflammation is a major cause of insulin resistance and Type 2 diabetes. Environmental factors, such as nutrients, affect body energy balance through epigenetic or chromatin-based mechanisms. As a bromodomain and external domain family transcription regulator, Brd2 regulates expression of many genes through interpretation of chromatin codes, and participates in the regulation of body energy balance and immune function. In the severely obese state, Brd2 knockdown in mice prevented obesity-induced inflammatory responses, protected animals from Type 2 diabetes, and thus uncoupled obesity from diabetes. Brd2 provides an important model for investigation of the function of transcription regulators and the development of obesity and diabetes; it also provides a possible target to treat obesity and diabetes through modulation of the function of a chromatin code reader. PMID:23374712

  6. Transcription and chromatin organization of a housekeeping gene cluster containing an integrated β-globin locus control region

    NARCIS (Netherlands)

    D. Noordermeer (Daan); M.R. Branco (Miguel); E. Splinter (Erik); P. Klous (Petra); W.F.J. van IJcken (Wilfred); S.M.A. Swagemakers (Sigrid); M. Koutsourakis (Manousos); P.J. van der Spek (Peter); A. Pombo (Ana); W.L. de Laat (Wouter)

    2008-01-01

    textabstractThe activity of locus control regions (LCR) has been correlated with chromatin decondensation, spreading of active chromatin marks, locus repositioning away from its chromosome territory (CT), increased association with transcription factories, and long-range interactions via chromatin

  7. Inactivation of chromatin remodeling factors sensitizes cells to selective cytotoxic stress

    Directory of Open Access Journals (Sweden)

    Freeman MD

    2014-11-01

    Full Text Available Miles D Freeman, Tryphon Mazu, Jana S Miles, Selina Darling-Reed, Hernan Flores-Rozas College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL, USA Abstract: The SWI/SNF chromatin-remodeling complex plays an essential role in several cellular processes including cell proliferation, differentiation, and DNA repair. Loss of normal function of the SWI/SNF complex because of mutations in its subunits correlates with tumorigenesis in humans. For many of these cancers, cytotoxic chemotherapy is the primary, and sometimes the only, therapeutic alternative. Among the antineoplastic agents, anthracyclines are a common treatment option. Although effective, resistance to these agents usually develops and serious dose-related toxicity, namely, chronic cardiotoxicity, limits its use. Previous work from our laboratory showed that a deletion of the SWI/SNF factor SNF2 resulted in hypersensitivity to doxorubicin. We further investigated the contribution of other chromatin remodeling complex components in the response to cytotoxic chemotherapy. Our results indicate that, of the eight SWI/SNF strains tested, snf2, taf14, and swi3 were the most sensitive and displayed distinct sensitivity to different cytotoxic agents, while snf5 displayed resistance. Our experimental results indicate that the SWI/SNF complex plays a critical role in protecting cells from exposure to cytotoxic chemotherapy and other cytotoxic agents. Our findings may prove useful in the development of a strategy aimed at targeting these genes to provide an alternative by hypersensitizing cancer cells to chemotherapeutic agents. Keywords: chromatin remodeling, cancer, DNA damage/repair, heat-shock response, oxidative stress

  8. Chromatin landscaping in algae reveals novel regulation pathway for biofuels production

    Energy Technology Data Exchange (ETDEWEB)

    Ngan, Chew Yee; Wong, Chee-Hong; Choi, Cindy; Pratap, Abhishek; Han, James; Wei, Chia-Lin

    2013-02-19

    The diminishing reserve of fossil fuels calls for the development of biofuels. Biofuels are produced from renewable resources, including photosynthetic organisms, generating clean energy. Microalgae is one of the potential feedstock for biofuels production. It grows easily even in waste water, and poses no competition to agricultural crops for arable land. However, little is known about the algae lipid biosynthetic regulatory mechanisms. Most studies relied on the homology to other plant model organisms, in particular Arabidopsis or through low coverage expression analysis to identify key enzymes. This limits the discovery of new components in the biosynthetic pathways, particularly the genetic regulators and effort to maximize the production efficiency of algal biofuels. Here we report an unprecedented and de novo approach to dissect the algal lipid pathways through disclosing the temporal regulations of chromatin states during lipid biosynthesis. We have generated genome wide chromatin maps in chlamydomonas genome using ChIP-seq targeting 7 histone modifications and RNA polymerase II in a time-series manner throughout conditions activating lipid biosynthesis. To our surprise, the combinatory profiles of histone codes uncovered new regulatory mechanism in gene expression in algae. Coupled with matched RNA-seq data, chromatin changes revealed potential novel regulators and candidate genes involved in the activation of lipid accumulations. Genetic perturbation on these candidate regulators further demonstrated the potential to manipulate the regulatory cascade for lipid synthesis efficiency. Exploring epigenetic landscape in microalgae shown here provides powerful tools needed in improving biofuel production and new technology platform for renewable energy generation, global carbon management, and environmental survey.

  9. HTLV-1 Tax mediated downregulation of miRNAs associated with chromatin remodeling factors in T cells with stably integrated viral promoter.

    Directory of Open Access Journals (Sweden)

    Saifur Rahman

    Full Text Available RNA interference (RNAi is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1 transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR using a CD4(+ T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type.

  10. A Poly-ADP-Ribose Trigger Releases the Auto-Inhibition of a Chromatin Remodeling Oncogene

    DEFF Research Database (Denmark)

    Singh, Hari R; Nardozza, Aurelio P; Möller, Ingvar R

    2017-01-01

    DNA damage triggers chromatin remodeling by mechanisms that are poorly understood. The oncogene and chromatin remodeler ALC1/CHD1L massively decompacts chromatin in vivo yet is inactive prior to DNA-damage-mediated PARP1 induction. We show that the interaction of the ALC1 macrodomain with the ATP......DNA damage triggers chromatin remodeling by mechanisms that are poorly understood. The oncogene and chromatin remodeler ALC1/CHD1L massively decompacts chromatin in vivo yet is inactive prior to DNA-damage-mediated PARP1 induction. We show that the interaction of the ALC1 macrodomain...... cancer mutants disrupt ALC1's auto-inhibition and activate chromatin remodeling. Our data show that the NAD+-metabolite and nucleic acid PAR triggers ALC1 to drive chromatin relaxation. Modular allostery in this oncogene tightly controls its robust, DNA-damage-dependent activation....

  11. Bridging the dynamics and organization of chromatin domains by mathematical modeling.

    Science.gov (United States)

    Shinkai, Soya; Nozaki, Tadasu; Maeshima, Kazuhiro; Togashi, Yuichi

    2017-07-04

    The genome is 3-dimensionally organized in the cell, and the mammalian genome DNA is partitioned into submegabase-sized chromatin domains. Genome functions are regulated within and across the domains according to their organization, whereas the chromatin itself is highly dynamic. However, the details of such dynamic organization of chromatin domains in living cells remain unclear. To unify chromatin dynamics and organization, we recently demonstrated that structural information of chromatin domains in living human cells can be extracted from analyses of the subdiffusive nucleosome movement using mathematical modeling. Our mathematical analysis suggested that as the chromatin domain becomes smaller and more compact, nucleosome movement becomes increasingly restricted. Here, we show the implication of these results for bridging the gap between chromatin dynamics and organization, and provide physical insight into chromatin domains as efficient units to conduct genome functions in the thermal noisy environment of the cell.

  12. Histone lysine demethylases as targets for anticancer therapy

    DEFF Research Database (Denmark)

    Højfeldt, Jonas W; Agger, Karl; Helin, Kristian

    2013-01-01

    It has recently been demonstrated that the genes controlling the epigenetic programmes that are required for maintaining chromatin structure and cell identity include genes that drive human cancer. This observation has led to an increased awareness of chromatin-associated proteins as potentially ...... lysine demethylase class of enzymes in the development of cancers, discuss the potential and challenges of therapeutically targeting them, and highlight emerging small-molecule inhibitors of these enzymes....

  13. Chromatin regulation landscape of embryonic stem cell identity.

    Science.gov (United States)

    Lee, Yun Hwa; Wu, Qiang

    2011-04-01

    ES cells (embryonic stem cells) derived from the ICM (inner cell mass) of blastocysts are pluripotent and are capable of giving rise to most cell types. The ES cell identity is mainly maintained by the Oct4 (octamer-binding transcription factor 4) and Nanog transcriptional networks. Recently, a tremendous amount of work has focused on deciphering how ES cell identity is regulated epigenetically. It has been shown that histone methylation/demethylation, histone acetylation/deacetylation, histone variants and chromatin remodelling play crucial roles in ES cell maintenance and differentiation. Moreover, perturbation of those chromatin regulators results in loss of ES cell identity or aberrant differentiation. Therefore, it is important to fully understand the chromatin regulation landscape of ES cells. The knowledge gained will help us to harness the unique characteristics of ES cells for stem cell-related therapy and regenerative medicine. In the present review, we will discuss recent proceedings that provide novel insights into chromatin regulation of ES cell identity.

  14. Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization

    Directory of Open Access Journals (Sweden)

    Peterhansel Christoph

    2007-09-01

    Full Text Available Abstract Background Chromatin remodeling, histone modifications and other chromatin-related processes play a crucial role in gene regulation. A very useful technique to study these processes is chromatin immunoprecipitation (ChIP. ChIP is widely used for a few model systems, including Arabidopsis, but establishment of the technique for other organisms is still remarkably challenging. Furthermore, quantitative analysis of the precipitated material and normalization of the data is often underestimated, negatively affecting data quality. Results We developed a robust ChIP protocol, using maize (Zea mays as a model system, and present a general strategy to systematically optimize this protocol for any type of tissue. We propose endogenous controls for active and for repressed chromatin, and discuss various other controls that are essential for successful ChIP experiments. We experienced that the use of quantitative PCR (QPCR is crucial for obtaining high quality ChIP data and we explain why. The method of data normalization has a major impact on the quality of ChIP analyses. Therefore, we analyzed different normalization strategies, resulting in a thorough discussion of the advantages and drawbacks of the various approaches. Conclusion Here we provide a robust ChIP protocol and strategy to optimize the protocol for any type of tissue; we argue that quantitative real-time PCR (QPCR is the best method to analyze the precipitates, and present comprehensive insights into data normalization.

  15. DNA damage, chromatin, and transcription: the trinity of aging.

    Science.gov (United States)

    Burgess, Rebecca C; Misteli, Tom; Oberdoerffer, Philipp

    2012-12-01

    Aging brings about numerous cellular defects. Amongst the most prominent are elevated levels of persistent DNA damage, changes to chromatin structure and epigenetic modifications, and alterations of global transcription programs. These are not independent events and recent work begins to shed light on the intricate interplay between these aging-related defects. Published by Elsevier Ltd.

  16. CTCF-mediated chromatin loops enclose inducible gene regulatory domains

    NARCIS (Netherlands)

    Oti, M.O.; Falck, J.; Huynen, M.A.; Zhou, Huiqing

    2016-01-01

    BACKGROUND: The CCTC-binding factor (CTCF) protein is involved in genome organization, including mediating three-dimensional chromatin interactions. Human patient lymphocytes with mutations in a single copy of the CTCF gene have reduced expression of enhancer-associated genes involved in response to

  17. Trichostatin A induced histone acetylation causes decondensation of interphase chromatin.

    NARCIS (Netherlands)

    T.A. Knoch (Tobias); M. Wachsmuth (Malte); M. Frank-Stöhr (Monika); M. Stöhr (Michael); C.P. Bacher (Christian); K. Rippe (Karsten)

    2004-01-01

    textabstractThe effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a

  18. Regulation of chromatin structure by poly(ADP-ribosylation

    Directory of Open Access Journals (Sweden)

    Sascha eBeneke

    2012-09-01

    Full Text Available The interaction of DNA with proteins in the context of chromatin has to be tightly regulated to achieve so different tasks as packaging, transcription, replication and repair. The very rapid and transient post-translational modification of proteins by poly(ADP-ribose has been shown to take part in all four. Originally identified as immediate cellular answer to a variety of genotoxic stresses, already early data indicated the ability of this highly charged nucleic acid-like polymer to modulate nucleosome structure, the basic unit of chromatin. At the same time the enzyme responsible for synthesizing poly(ADP-ribose, the zinc-finger protein poly(ADP-ribose polymerase-1 (PARP1, was shown to control transcription initiation as basic factor TFIIC within the RNA-polymerase II machinery. Later research focused more on PARP-mediated regulation of DNA repair and cell death, but in the last few years, transcription as well as chromatin modulation has re-appeared on the scene. This review will discuss the impact of PARP1 on transcription and transcription factors, its implication in chromatin remodeling for DNA repair and probably also replication, and its role in controlling epigenetic events such as DNA methylation and the functionality of the insulator protein CCCTC-binding factor.

  19. Role of chromatin factors in Arabidopsis root stem cell maintenance

    NARCIS (Netherlands)

    Kornet, N.G.

    2008-01-01

    Stem cells replenish the cells present in an organism throughout its lifetime and sustain growth. They have unique characteristics: the capability to self-renew and the potential to differentiate into several cell types. Recently, it has become clear that chromatin factors support these unique

  20. Interaction of maize chromatin-associated HMG proteins with mononucleosomes

    DEFF Research Database (Denmark)

    Lichota, J.; Grasser, Klaus D.

    2003-01-01

    maize HMGA and five different HMGB proteins with mononucleosomes (containing approx. 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin. The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA...

  1. Chromatin remodelers in the DNA double strand break response

    NARCIS (Netherlands)

    Smeenk, Godelieve

    2012-01-01

    During my PhD project, I studied the role of several chromatin remodelers in the DNA double strand break (DSB) response. We discovered that both CHD4 and SMARCA5 are required for ubiquitin signaling through the E3 ubiquitin ligases RNF8 and RNF168, which is a central signaling event in the response

  2. Frequent involvement of chromatin remodeler alterations in gastric field cancerization.

    Science.gov (United States)

    Takeshima, Hideyuki; Niwa, Tohru; Takahashi, Takamasa; Wakabayashi, Mika; Yamashita, Satoshi; Ando, Takayuki; Inagawa, Yuki; Taniguchi, Hirokazu; Katai, Hitoshi; Sugiyama, Toshiro; Kiyono, Tohru; Ushijima, Toshikazu

    2015-02-01

    A field for cancerization, or a field defect, is formed by the accumulation of genetic and epigenetic alterations in normal-appearing tissues, and is involved in various cancers, especially multiple cancers. Epigenetic alterations are frequently present in chronic inflammation-exposed tissues, but information on individual genes involved in the formation of a field defect is still fragmental. Here, using non-cancerous gastric tissues of cancer patients, we isolated 16 aberrantly methylated genes, and identified chromatin remodelers ACTL6B and SMARCA1 as novel genes frequently methylated in non-cancerous tissues. SMARCA1 was expressed at high levels in normal gastric tissues, but was frequently silenced by aberrant methylation in gastric cancer cells. Moreover, somatic mutations of additional chromatin remodelers, such as ARID1A, SMARCA2, and SMARCA4, were found in 30% of gastric cancers. Mutant allele frequency suggested that the majority of cancer cells harbored a mutation when present. Depletion of a chromatin remodeler, SMARCA1 or SMARCA2, in cancer cell lines promoted their growth. These results showed that epigenetic and genetic alterations of chromatin remodelers are induced at an early stage of carcinogenesis and are frequently involved in the formation of a field defect. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Generation of bivalent chromatin domains during cell fate decisions

    Directory of Open Access Journals (Sweden)

    De Gobbi Marco

    2011-06-01

    Full Text Available Abstract Background In self-renewing, pluripotent cells, bivalent chromatin modification is thought to silence (H3K27me3 lineage control genes while 'poising' (H3K4me3 them for subsequent activation during differentiation, implying an important role for epigenetic modification in directing cell fate decisions. However, rather than representing an equivalently balanced epigenetic mark, the patterns and levels of histone modifications at bivalent genes can vary widely and the criteria for identifying this chromatin signature are poorly defined. Results Here, we initially show how chromatin status alters during lineage commitment and differentiation at a single well characterised bivalent locus. In addition we have determined how chromatin modifications at this locus change with gene expression in both ensemble and single cell analyses. We also show, on a global scale, how mRNA expression may be reflected in the ratio of H3K4me3/H3K27me3. Conclusions While truly 'poised' bivalently modified genes may exist, the original hypothesis that all bivalent genes are epigenetically premarked for subsequent expression might be oversimplistic. In fact, from the data presented in the present work, it is equally possible that many genes that appear to be bivalent in pluripotent and multipotent cells may simply be stochastically expressed at low levels in the process of multilineage priming. Although both situations could be considered to be forms of 'poising', the underlying mechanisms and the associated implications are clearly different.

  4. Environmental car exhaust pollution damages human sperm chromatin and DNA.

    Science.gov (United States)

    Calogero, A E; La Vignera, S; Condorelli, R A; Perdichizzi, A; Valenti, D; Asero, P; Carbone, U; Boggia, B; De Rosa, N; Lombardi, G; D'Agata, R; Vicari, L O; Vicari, E; De Rosa, M

    2011-06-01

    The adverse role of traffic pollutants on male fertility is well known. Aim of this study was to evaluate their effects on sperm chromatin/DNA integrity. To accomplish this, 36 men working at motorway tollgates and 32 unexposed healthy men (controls) were enrolled. All of them were interviewed about their lifestyle. Hormone, semen samples, and environmental and biological markers of pollution were evaluated. Sperm chromatin and DNA integrity were evaluated by flow cytometry following propidium iodide staining and TUNEL assay, respectively. LH, FSH, and testosterone serum levels were within the normal range in tollgate workers. Sperm concentration, total sperm count, total and progressive motility, and normal forms were significantly lower in these men compared with controls. Motorway tollgate workers had a significantly higher percentage of spermatozoa with damaged chromatin and DNA fragmentation, a late sign of apoptosis, compared with controls. A significant direct correlation was found between spermatozoa with damaged chromatin or fragmented DNA and the length of occupational exposure, suggesting a time-dependent relationship. This study showed that car exhaust exposure has a genotoxic effect on human spermatozoa. This may be of relevant importance not only for the reproductive performance of the men exposed, but also for the offspring health.

  5. Epigenetic mechanisms in mood disorders: targeting neuroplasticity.

    Science.gov (United States)

    Fass, D M; Schroeder, F A; Perlis, R H; Haggarty, S J

    2014-04-04

    Developing novel therapeutics and diagnostic tools based upon an understanding of neuroplasticity is critical in order to improve the treatment and ultimately the prevention of a broad range of nervous system disorders. In the case of mood disorders, such as major depressive disorder (MDD) and bipolar disorder (BPD), where diagnoses are based solely on nosology rather than pathophysiology, there exists a clear unmet medical need to advance our understanding of the underlying molecular mechanisms and to develop fundamentally new mechanism experimental medicines with improved efficacy. In this context, recent preclinical molecular, cellular, and behavioral findings have begun to reveal the importance of epigenetic mechanisms that alter chromatin structure and dynamically regulate patterns of gene expression that may play a critical role in the pathophysiology of mood disorders. Here, we will review recent advances involving the use of animal models in combination with genetic and pharmacological probes to dissect the underlying molecular mechanisms and neurobiological consequence of targeting this chromatin-mediated neuroplasticity. We discuss evidence for the direct and indirect effects of mood stabilizers, antidepressants, and antipsychotics, among their many other effects, on chromatin-modifying enzymes and on the epigenetic state of defined genomic loci, in defined cell types and in specific regions of the brain. These data, as well as findings from patient-derived tissue, have also begun to reveal alterations of epigenetic mechanisms in the pathophysiology and treatment of mood disorders. We summarize growing evidence supporting the notion that selectively targeting chromatin-modifying complexes, including those containing histone deacetylases (HDACs), provides a means to reversibly alter the acetylation state of neuronal chromatin and beneficially impact neuronal activity-regulated gene transcription and mood-related behaviors. Looking beyond current knowledge

  6. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Biosciences. Kamalvishnu P Gottimukkala. Articles written in Journal of Biosciences. Volume 36 Issue 3 August 2011 pp 461-469 Articles. N-terminal PDZ-like domain of chromatin organizer SATB1 contributes towards its function as transcription regulator · Dimple Notani Praveena L Ramanujam ...

  7. Human genome replication proceeds through four chromatin states.

    Directory of Open Access Journals (Sweden)

    Hanna Julienne

    Full Text Available Advances in genomic studies have led to significant progress in understanding the epigenetically controlled interplay between chromatin structure and nuclear functions. Epigenetic modifications were shown to play a key role in transcription regulation and genome activity during development and differentiation or in response to the environment. Paradoxically, the molecular mechanisms that regulate the initiation and the maintenance of the spatio-temporal replication program in higher eukaryotes, and in particular their links to epigenetic modifications, still remain elusive. By integrative analysis of the genome-wide distributions of thirteen epigenetic marks in the human cell line K562, at the 100 kb resolution of corresponding mean replication timing (MRT data, we identify four major groups of chromatin marks with shared features. These states have different MRT, namely from early to late replicating, replication proceeds though a transcriptionally active euchromatin state (C1, a repressive type of chromatin (C2 associated with polycomb complexes, a silent state (C3 not enriched in any available marks, and a gene poor HP1-associated heterochromatin state (C4. When mapping these chromatin states inside the megabase-sized U-domains (U-shaped MRT profile covering about 50% of the human genome, we reveal that the associated replication fork polarity gradient corresponds to a directional path across the four chromatin states, from C1 at U-domains borders followed by C2, C3 and C4 at centers. Analysis of the other genome half is consistent with early and late replication loci occurring in separate compartments, the former correspond to gene-rich, high-GC domains of intermingled chromatin states C1 and C2, whereas the latter correspond to gene-poor, low-GC domains of alternating chromatin states C3 and C4 or long C4 domains. This new segmentation sheds a new light on the epigenetic regulation of the spatio-temporal replication program in human and

  8. Human Genome Replication Proceeds through Four Chromatin States

    Science.gov (United States)

    Julienne, Hanna; Zoufir, Azedine; Audit, Benjamin; Arneodo, Alain

    2013-01-01

    Advances in genomic studies have led to significant progress in understanding the epigenetically controlled interplay between chromatin structure and nuclear functions. Epigenetic modifications were shown to play a key role in transcription regulation and genome activity during development and differentiation or in response to the environment. Paradoxically, the molecular mechanisms that regulate the initiation and the maintenance of the spatio-temporal replication program in higher eukaryotes, and in particular their links to epigenetic modifications, still remain elusive. By integrative analysis of the genome-wide distributions of thirteen epigenetic marks in the human cell line K562, at the 100 kb resolution of corresponding mean replication timing (MRT) data, we identify four major groups of chromatin marks with shared features. These states have different MRT, namely from early to late replicating, replication proceeds though a transcriptionally active euchromatin state (C1), a repressive type of chromatin (C2) associated with polycomb complexes, a silent state (C3) not enriched in any available marks, and a gene poor HP1-associated heterochromatin state (C4). When mapping these chromatin states inside the megabase-sized U-domains (U-shaped MRT profile) covering about 50% of the human genome, we reveal that the associated replication fork polarity gradient corresponds to a directional path across the four chromatin states, from C1 at U-domains borders followed by C2, C3 and C4 at centers. Analysis of the other genome half is consistent with early and late replication loci occurring in separate compartments, the former correspond to gene-rich, high-GC domains of intermingled chromatin states C1 and C2, whereas the latter correspond to gene-poor, low-GC domains of alternating chromatin states C3 and C4 or long C4 domains. This new segmentation sheds a new light on the epigenetic regulation of the spatio-temporal replication program in human and provides a

  9. The Adenomatous Polyposis Coli Protein Contributes to Normal Compaction of Mitotic Chromatin

    Science.gov (United States)

    Dikovskaya, Dina; Khoudoli, Guennadi; Newton, Ian P.; Chadha, Gaganmeet S.; Klotz, Daniel; Visvanathan, Ashwat; Lamond, Angus; Swedlow, Jason R.; Näthke, Inke S.

    2012-01-01

    The tumour suppressor Adenomatous Polyposis Coli (APC) is required for proper mitosis; however, the exact role of APC in mitosis is not understood. Using demembranated sperm chromatin exposed to meiotic Xenopus egg extract and HeLa cells expressing fluorescently labelled histones, we established that APC contributes to chromatin compaction. Sperm chromatin in APC-depleted Xenopus egg extract frequently formed tight round or elongated structures. Such abnormally compacted chromatin predominantly formed spindles with low microtubule content. Furthermore, in mitotic HeLa cells expressing GFP- and mCherry-labelled H2B histones, depletion of APC caused a decrease in the donor fluorescence lifetime of neighbouring fluorophores, indicative of excessive chromatin compaction. Profiling the chromatin-associated proteome of sperm chromatin incubated with Xenopus egg extracts revealed temporal APC-dependent changes in the abundance of histones, closely mirrored by chromatin-associated Topoisomerase IIa, condensin I complex and Kif4. In the absence of APC these factors initially accumulated on chromatin, but then decreased faster than in controls. We also found and validated significant APC-dependent changes in chromatin modifiers Set-a and Rbbp7. Both were decreased on chromatin in APC-depleted extract; in addition, the kinetics of association of Set-a with chromatin was altered in the absence of APC. PMID:22719865

  10. Citrullination regulates pluripotency and histone H1 binding to chromatin

    Science.gov (United States)

    Christophorou, Maria A.; Castelo-Branco, Gonçalo; Halley-Stott, Richard P.; Oliveira, Clara Slade; Loos, Remco; Radzisheuskaya, Aliaksandra; Mowen, Kerri A.; Bertone, Paul; Silva, José C. R.; Zernicka-Goetz, Magdalena; Nielsen, Michael L.; Gurdon, John B.; Kouzarides, Tony

    2014-03-01

    Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.

  11. The ING tumor suppressors in cellular senescence and chromatin

    Directory of Open Access Journals (Sweden)

    Ludwig Susann

    2011-07-01

    Full Text Available Abstract The Inhibitor of Growth (ING proteins represent a type II tumor suppressor family comprising five conserved genes, ING1 to ING5. While ING1, ING2 and ING3 proteins are stable components of the mSIN3a-HDAC complexes, the association of ING1, ING4 and ING5 with HAT protein complexes was also reported. Among these the ING1 and ING2 have been analyzed more deeply. Similar to other tumor suppressor factors the ING proteins are also involved in many cellular pathways linked to cancer and cell proliferation such as cell cycle regulation, cellular senescence, DNA repair, apoptosis, inhibition of angiogenesis and modulation of chromatin. A common structural feature of ING factors is the conserved plant homeodomain (PHD, which can bind directly to the histone mark trimethylated lysine of histone H3 (H3K4me3. PHD mutants lose the ability to undergo cellular senescence linking chromatin mark recognition with cellular senescence. ING1 and ING2 are localized in the cell nucleus and associated with chromatin modifying enzymes, linking tumor suppression directly to chromatin regulation. In line with this, the expression of ING1 in tumors is aberrant or identified point mutations are mostly localized in the PHD finger and affect histone binding. Interestingly, ING1 protein levels increase in replicative senescent cells, latter representing an efficient pathway to inhibit cancer proliferation. In association with this, suppression of p33ING1 expression prolongs replicative life span and is also sufficient to bypass oncogene-induced senescence. Recent analyses of ING1- and ING2-deficient mice confirm a tumor suppressive role of ING1 and ING2 and also indicate an essential role of ING2 in meiosis. Here we summarize the activity of ING1 and ING2 as tumor suppressors, chromatin factors and in development.

  12. Epigenetic Heterogeneity of B-Cell Lymphoma: DNA Methylation, Gene Expression and Chromatin States

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    Lydia Hopp

    2015-09-01

    Full Text Available Mature B-cell lymphoma is a clinically and biologically highly diverse disease. Its diagnosis and prognosis is a challenge due to its molecular heterogeneity and diverse regimes of biological dysfunctions, which are partly driven by epigenetic mechanisms. We here present an integrative analysis of DNA methylation and gene expression data of several lymphoma subtypes. Our study confirms previous results about the role of stemness genes during development and maturation of B-cells and their dysfunction in lymphoma locking in more proliferative or immune-reactive states referring to B-cell functionalities in the dark and light zone of the germinal center and also in plasma cells. These dysfunctions are governed by widespread epigenetic effects altering the promoter methylation of the involved genes, their activity status as moderated by histone modifications and also by chromatin remodeling. We identified four groups of genes showing characteristic expression and methylation signatures among Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma and multiple myeloma. These signatures are associated with epigenetic effects such as remodeling from transcriptionally inactive into active chromatin states, differential promoter methylation and the enrichment of targets of transcription factors such as EZH2 and SUZ12.

  13. Chromatin remodeling regulates catalase expression during cancer cells adaptation to chronic oxidative stress.

    Science.gov (United States)

    Glorieux, Christophe; Sandoval, Juan Marcelo; Fattaccioli, Antoine; Dejeans, Nicolas; Garbe, James C; Dieu, Marc; Verrax, Julien; Renard, Patricia; Huang, Peng; Calderon, Pedro Buc

    2016-10-01

    Regulation of ROS metabolism plays a major role in cellular adaptation to oxidative stress in cancer cells, but the molecular mechanism that regulates catalase, a key antioxidant enzyme responsible for conversion of hydrogen peroxide to water and oxygen, remains to be elucidated. Therefore, we investigated the transcriptional regulatory mechanism controlling catalase expression in three human mammary cell lines: the normal mammary epithelial 250MK primary cells, the breast adenocarcinoma MCF-7 cells and an experimental model of MCF-7 cells resistant against oxidative stress resulting from chronic exposure to H 2 O 2 (Resox), in which catalase was overexpressed. Here we identify a novel promoter region responsible for the regulation of catalase expression at -1518/-1226 locus and the key molecules that interact with this promoter and affect catalase transcription. We show that the AP-1 family member JunB and retinoic acid receptor alpha (RARα) mediate catalase transcriptional activation and repression, respectively, by controlling chromatin remodeling through a histone deacetylases-dependent mechanism. This regulatory mechanism plays an important role in redox adaptation to chronic exposure to H 2 O 2 in breast cancer cells. Our study suggests that cancer adaptation to oxidative stress may be regulated by transcriptional factors through chromatin remodeling, and reveals a potential new mechanism to target cancer cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Important biological information uncovered in previously unaligned reads from chromatin immunoprecipitation experiments (ChIP-Seq)

    Science.gov (United States)

    Ouma, Wilberforce Zachary; Mejia-Guerra, Maria Katherine; Yilmaz, Alper; Pareja-Tobes, Pablo; Li, Wei; Doseff, Andrea I.; Grotewold, Erich

    2015-01-01

    Establishing the architecture of gene regulatory networks (GRNs) relies on chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) methods that provide genome-wide transcription factor binding sites (TFBSs). ChIP-Seq furnishes millions of short reads that, after alignment, describe the genome-wide binding sites of a particular TF. However, in all organisms investigated an average of 40% of reads fail to align to the corresponding genome, with some datasets having as much as 80% of reads failing to align. We describe here the provenance of previously unaligned reads in ChIP-Seq experiments from animals and plants. We show that a substantial portion corresponds to sequences of bacterial and metazoan origin, irrespective of the ChIP-Seq chromatin source. Unforeseen was the finding that 30%–40% of unaligned reads were actually alignable. To validate these observations, we investigated the characteristics of the previously unaligned reads corresponding to TAL1, a human TF involved in lineage specification of hemopoietic cells. We show that, while unmapped ChIP-Seq read datasets contain foreign DNA sequences, additional TFBSs can be identified from the previously unaligned ChIP-Seq reads. Our results indicate that the re-evaluation of previously unaligned reads from ChIP-Seq experiments will significantly contribute to TF target identification and determination of emerging properties of GRNs. PMID:25727450

  15. Chromatin conformation in living cells: support for a zig-zag model of the 30 nm chromatin fiber

    Science.gov (United States)

    Rydberg, B.; Holley, W. R.; Mian, I. S.; Chatterjee, A.

    1998-01-01

    A new method was used to probe the conformation of chromatin in living mammalian cells. The method employs ionizing radiation and is based on the concept that such radiation induces correlated breaks in DNA strands that are in spatial proximity. Human dermal fibroblasts in G0 phase of the cell cycle and Chinese hamster ovary cells in mitosis were irradiated by X-rays or accelerated ions. Following lysis of the cells, DNA fragments induced by correlated breaks were end-labeled and separated according to size on denaturing polyacrylamide gels. A characteristic peak was obtained for a fragment size of 78 bases, which is the size that corresponds to one turn of DNA around the nucleosome. Additional peaks between 175 and 450 bases reflect the relative position of nearest-neighbor nucleosomes. Theoretical calculations that simulate the indirect and direct effect of radiation on DNA demonstrate that the fragment size distributions are closely related to the chromatin structure model used. Comparison of the experimental data with theoretical results support a zig-zag model of the chromatin fiber rather than a simple helical model. Thus, radiation-induced damage analysis can provide information on chromatin structure in the living cell. Copyright 1998 Academic Press.

  16. Chromatin analyses of Zymoseptoria tritici: Methods for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq).

    Science.gov (United States)

    Soyer, Jessica L; Möller, Mareike; Schotanus, Klaas; Connolly, Lanelle R; Galazka, Jonathan M; Freitag, Michael; Stukenbrock, Eva H

    2015-06-01

    The presence or absence of specific transcription factors, chromatin remodeling machineries, chromatin modification enzymes, post-translational histone modifications and histone variants all play crucial roles in the regulation of pathogenicity genes. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) provides an important tool to study genome-wide protein-DNA interactions to help understand gene regulation in the context of native chromatin. ChIP-seq is a convenient in vivo technique to identify, map and characterize occupancy of specific DNA fragments with proteins against which specific antibodies exist or which can be epitope-tagged in vivo. We optimized existing ChIP protocols for use in the wheat pathogen Zymoseptoria tritici and closely related sister species. Here, we provide a detailed method, underscoring which aspects of the technique are organism-specific. Library preparation for Illumina sequencing is described, as this is currently the most widely used ChIP-seq method. One approach for the analysis and visualization of representative sequence is described; improved tools for these analyses are constantly being developed. Using ChIP-seq with antibodies against H3K4me2, which is considered a mark for euchromatin or H3K9me3 and H3K27me3, which are considered marks for heterochromatin, the overall distribution of euchromatin and heterochromatin in the genome of Z. tritici can be determined. Our ChIP-seq protocol was also successfully applied to Z. tritici strains with high levels of melanization or aberrant colony morphology, and to different species of the genus (Z. ardabiliae and Z. pseudotritici), suggesting that our technique is robust. The methods described here provide a powerful framework to study new aspects of chromatin biology and gene regulation in this prominent wheat pathogen. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Comparative Analysis of Chromatin Binding by Sex Comb on Midleg (SCM) and Other Polycomb Group Repressors at a Drosophila Hox Gene▿

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    Wang, Liangjun; Jahren, Neal; Miller, Ellen L.; Ketel, Carrie S.; Mallin, Daniel R.; Simon, Jeffrey A.

    2010-01-01

    Sex Comb on Midleg (SCM) is a transcriptional repressor in the Polycomb group (PcG), but its molecular role in PcG silencing is not known. Although SCM can interact with Polycomb repressive complex 1 (PRC1) in vitro, biochemical studies have indicated that SCM is not a core constituent of PRC1 or PRC2. Nevertheless, SCM is just as critical for Drosophila Hox gene silencing as canonical subunits of these well-characterized PcG complexes. To address functional relationships between SCM and other PcG components, we have performed chromatin immunoprecipitation studies using cultured Drosophila Schneider line 2 (S2) cells and larval imaginal discs. We find that SCM associates with a Polycomb response element (PRE) upstream of the Ubx gene which also binds PRC1, PRC2, and the DNA-binding PcG protein Pleiohomeotic (PHO). However, SCM is retained at this Ubx PRE despite genetic disruption or knockdown of PHO, PRC1, or PRC2, suggesting that SCM chromatin targeting does not require prior association of these other PcG components. Chromatin immunoprecipitations (IPs) to test the consequences of SCM genetic disruption or knockdown revealed that PHO association is unaffected, but reduced levels of PRE-bound PRC2 and PRC1 were observed. We discuss these results in light of current models for recruitment of PcG complexes to chromatin targets. PMID:20351181

  18. Comparative analysis of chromatin binding by Sex Comb on Midleg (SCM) and other polycomb group repressors at a Drosophila Hox gene.

    Science.gov (United States)

    Wang, Liangjun; Jahren, Neal; Miller, Ellen L; Ketel, Carrie S; Mallin, Daniel R; Simon, Jeffrey A

    2010-06-01

    Sex Comb on Midleg (SCM) is a transcriptional repressor in the Polycomb group (PcG), but its molecular role in PcG silencing is not known. Although SCM can interact with Polycomb repressive complex 1 (PRC1) in vitro, biochemical studies have indicated that SCM is not a core constituent of PRC1 or PRC2. Nevertheless, SCM is just as critical for Drosophila Hox gene silencing as canonical subunits of these well-characterized PcG complexes. To address functional relationships between SCM and other PcG components, we have performed chromatin immunoprecipitation studies using cultured Drosophila Schneider line 2 (S2) cells and larval imaginal discs. We find that SCM associates with a Polycomb response element (PRE) upstream of the Ubx gene which also binds PRC1, PRC2, and the DNA-binding PcG protein Pleiohomeotic (PHO). However, SCM is retained at this Ubx PRE despite genetic disruption or knockdown of PHO, PRC1, or PRC2, suggesting that SCM chromatin targeting does not require prior association of these other PcG components. Chromatin immunoprecipitations (IPs) to test the consequences of SCM genetic disruption or knockdown revealed that PHO association is unaffected, but reduced levels of PRE-bound PRC2 and PRC1 were observed. We discuss these results in light of current models for recruitment of PcG complexes to chromatin targets.

  19. Allelic reprogramming of 3D chromatin architecture during early mammalian development.

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    Du, Zhenhai; Zheng, Hui; Huang, Bo; Ma, Rui; Wu, Jingyi; Zhang, Xianglin; He, Jing; Xiang, Yunlong; Wang, Qiujun; Li, Yuanyuan; Ma, Jing; Zhang, Xu; Zhang, Ke; Wang, Yang; Zhang, Michael Q; Gao, Juntao; Dixon, Jesse R; Wang, Xiaowo; Zeng, Jianyang; Xie, Wei

    2017-07-12

    In mammals, chromatin organization undergoes drastic reprogramming after fertilization. However, the three-dimensional structure of chromatin and its reprogramming in preimplantation development remain poorly understood. Here, by developing a low-input Hi-C (genome-wide chromosome conformation capture) approach, we examined the reprogramming of chromatin organization during early development in mice. We found that oocytes in metaphase II show homogeneous chromatin folding that lacks detectable topologically associating domains (TADs) and chromatin compartments. Strikingly, chromatin shows greatly diminished higher-order structure after fertilization. Unexpectedly, the subsequent establishment of chromatin organization is a prolonged process that extends through preimplantation development, as characterized by slow consolidation of TADs and segregation of chromatin compartments. The two sets of parental chromosomes are spatially separated from each other and display distinct compartmentalization in zygotes. Such allele separation and allelic compartmentalization can be found as late as the 8-cell stage. Finally, we show that chromatin compaction in preimplantation embryos can partially proceed in the absence of zygotic transcription and is a multi-level hierarchical process. Taken together, our data suggest that chromatin may exist in a markedly relaxed state after fertilization, followed by progressive maturation of higher-order chromatin architecture during early development.

  20. Widespread Chromatin Accessibility at Repetitive Elements Links Stem Cells with Human Cancer

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    Nicholas C. Gomez

    2016-11-01

    Full Text Available Chromatin regulation is critical for differentiation and disease. However, features linking the chromatin environment of stem cells with disease remain largely unknown. We explored chromatin accessibility in embryonic and multipotent stem cells and unexpectedly identified widespread chromatin accessibility at repetitive elements. Integrating genomic and biochemical approaches, we demonstrate that these sites of increased accessibility are associated with well-positioned nucleosomes marked by distinct histone modifications. Differentiation is accompanied by chromatin remodeling at repetitive elements associated with altered expression of genes in relevant developmental pathways. Remarkably, we found that the chromatin environment of Ewing sarcoma, a mesenchymally derived tumor, is shared with primary mesenchymal stem cells (MSCs. Accessibility at repetitive elements in MSCs offers a permissive environment that is exploited by the critical oncogene responsible for this cancer. Our data demonstrate that stem cells harbor a unique chromatin landscape characterized by accessibility at repetitive elements, a feature associated with differentiation and oncogenesis.

  1. Bar-shaped nuclear chromatin in conjunctival samples: with cytologic features and ultrastructural correlation.

    Science.gov (United States)

    Kobayashi, T K; Ishida, Y; Kaneko, C; Tsubota, K

    1998-01-01

    To study the cytomorphologic features of bar-shaped chromatin in conjunctival samples and assess whether there are morphologic similarities between nuclear grooves seen in thyroid papillary carcinoma and bar-shaped chromatin. A total of 10 conjunctival samples from five volunteers were studied cytologically. Of the 10 samples, 2 showed barshaped chromatin. This chromatin was studied using a light microscope, scanning electron microscope (SEM) and transmission electron microscope (TEM). On SEM, bar-shaped chromatin was observed as a fissure or shallow cracks. On TEM the barshaped chromatin existed both in the center of the nuclei and in the nuclear membrane and cytoplasm. It was surrounded by outer and inner membranes. It seems that bar-shaped chromatin is formed by the nuclear membrane and that these changes are essentially the same configuration as cytoplasmic invaginations commonly present in papillary carcinoma of the thyroid.

  2. Increased chromatin plasticity supports enhanced metastatic potential of mouse melanoma cells.

    Science.gov (United States)

    Maizels, Yael; Elbaz, Adi; Hernandez-Vicens, Rosari; Sandrusy, Oshrat; Rosenberg, Anna; Gerlitz, Gabi

    2017-08-15

    Metastasis formation is strongly dependent on the migration capabilities of tumor cells. Recently it has become apparent that nuclear structure and morphology affect the cellular ability to migrate. Previously we found that migration of melanoma cells is both associated with and dependent on global chromatin condensation. Therefore, we anticipated that tumor progression would be associated with increased chromatin condensation. Interestingly, the opposite has been reported for melanoma. In trying to resolve this contradiction, we show that during growth conditions, tumor progression is associated with global chromatin de-condensation that is beneficial for faster proliferation. However, upon induction of migration, in both low- and high-metastatic mouse melanoma cells chromatin undergoes condensation to support cell migration. Our results reveal that throughout tumor progression induction of chromatin condensation by migration signals is maintained, whereas the organization of chromatin during growth conditions is altered. Thus, tumor progression is associated with an increase in chromatin dynamics. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Radiation-induced DNA damage and chromatin structure

    Science.gov (United States)

    Rydberg, B.; Chatterjee, A. (Principal Investigator)

    2001-01-01

    DNA lesions induced by ionizing radiation in cells are clustered and not randomly distributed. For low linear energy transfer (LET) radiation this clustering occurs mainly on the small scales of DNA molecules and nucleosomes. For example, experimental evidence suggests that both strands of DNA on the nucleosomal surface can be damaged in single events and that this damage occurs with a 10-bp modulation because of protection by histones. For high LET radiation, clustering also occurs on a larger scale and depends on chromatin organization. A particularly significant clustering occurs when an ionizing particle traverses the 30 nm chromatin fiber with generation of heavily damaged DNA regions with an average size of about 2 kbp. On an even larger scale, high LET radiation can produce several DNA double-strand breaks in closer proximity than expected from randomness. It is suggested that this increases the probability of misrejoining of DNA ends and generation of lethal chromosome aberrations.

  4. Chromatin priming of genes in development: Concepts, mechanisms and consequences.

    Science.gov (United States)

    Bonifer, Constanze; Cockerill, Peter N

    2017-05-01

    During ontogeny, cells progress through multiple alternate differentiation states by activating distinct gene regulatory networks. In this review, we highlight the important role of chromatin priming in facilitating gene activation during lineage specification and in maintaining an epigenetic memory of previous gene activation. We show that chromatin priming is part of a hugely diverse repertoire of regulatory mechanisms that genes use to ensure that they are expressed at the correct time, in the correct cell type, and at the correct level, but also that they react to signals. We also emphasize how increasing our knowledge of these principles could inform our understanding of developmental failure and disease. Copyright © 2017 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  5. Chromatin structure and transposable elements in organismal aging

    Directory of Open Access Journals (Sweden)

    Jason G. Wood

    2013-12-01

    Full Text Available Epigenetic regulatory mechanisms are increasingly appreciated as central to a diverse array of biological processes, including aging. An association between heterochromatic silencing and longevity has long been recognized in yeast, and in more recent years evidence has accumulated of age-related chromatin changes in C. elegans, Drosophila, and mouse model systems, as well as in the tissue culture-based replicative senescence model of cell aging. In addition, a number of studies have linked expression of transposable elements (TEs, as well as changes in the RNAi pathways that cells use to combat TEs, to the aging process. This review summarizes the recent evidence linking chromatin structure and function to aging, with a particular focus on the relationship of heterochromatin structure to organismal aging.

  6. Chromatin Conformation Capture-Based Analysis of Nuclear Architecture.

    Science.gov (United States)

    Grob, Stefan; Grossniklaus, Ueli

    2017-01-01

    Nuclear organization and higher-order chromosome structure in interphase nuclei are thought to have important effects on fundamental biological processes, including chromosome condensation, replication, and transcription. Until recently, however, nuclear organization could only be analyzed microscopically. The development of chromatin conformation capture (3C)-based techniques now allows a detailed look at chromosomal architecture from the level of individual loci to the entire genome. Here we provide a robust Hi-C protocol, allowing the analysis of nuclear organization in nuclei from different wild-type and mutant plant tissues. This method is quantitative and provides a highly efficient and comprehensive way to study chromatin organization during plant development, in response to different environmental stimuli, and in mutants disrupting a variety of processes, including epigenetic pathways regulating gene expression.

  7. Epitope tagging of endogenous proteins for genome-wide chromatin immunoprecipitation analysis.

    Science.gov (United States)

    Wang, Zhenghe

    2009-01-01

    The development of chromatin immunoprecipitation methods coupled with DNA microarray (ChIP-chip) technology has enabled genome-wide identification of cis-DNA regulatory elements to which transcription factors bind. Nonetheless, the ChIP-chip technology requires antibodies with extremely high affinity and specificity for the target transcription factors. Unfortunately, such antibodies are not available for most human transcription factors. In principle, this problem can be circumvented by utilizing ectopically expressed epitope-tagged proteins recognizable by well-characterized antibodies. However, such expression is no longer endogenous. To surmount this problem, we have successfully developed a facile method to knock in a 3xFlag epitope into the endogenous gene loci of transcription factors. The knock-in approach provides a general solution for the study of proteins for which antibodies are substandard or not available.

  8. Epitope tagging of endogenous proteins for genome wide Chromatin immunoprecipitation analysis

    Science.gov (United States)

    Wang, Zhenghe

    2010-01-01

    The development of chromatin immunoprecipitation methods coupled with DNA microarray (ChIP-chip) technology has enabled genome-wide identification of cis-DNA regulatory elements to which transcription factors bind. Nonetheless, the ChIP-chip technology requires antibodies with extremely high affinity and specificity for the target transcription factors. Unfortunately, such antibodies are not available for most human transcription factors. In principle, this problem can be circumvented by utilizing ectopically expressed epitope-tagged proteins recognizable by well-characterized antibodies. However, such expression is no longer endogenous. To surmount this problem, we have successfully developed a facile method to knock-in a 3xFlag epitope into the endogenous gene loci of transcription factors. The knock-in approach provides a general solution for the study of proteins for which antibodies are substandard or not available. PMID:19588087

  9. Chromatin condensation of Xist genomic loci during oogenesis in mice.

    Science.gov (United States)

    Fukuda, Atsushi; Mitani, Atsushi; Miyashita, Toshiyuki; Umezawa, Akihiro; Akutsu, Hidenori

    2015-12-01

    Repression of maternal Xist (Xm-Xist) during preimplantation in mouse embryos is essential for establishing imprinted X chromosome inactivation. Nuclear transplantation (NT) studies using nuclei derived from non-growing (ng) and full-grown (fg) oocytes have indicated that maternal-specific repressive modifications are imposed on Xm-Xist during oogenesis, as well as on autosomal imprinted genes. Recent studies have revealed that histone H3 lysine 9 trimethylation (H3K9me3) enrichments on Xm-Xist promoter regions are involved in silencing at the preimplantation stages. However, whether H3K9me3 is imposed on Xm-Xist during oogenesis is not known. Here, we dissected the chromatin states in ng and fg oocytes and early preimplantation stage embryos. Chromatin immunoprecipitation experiments against H3K9me3 revealed that there was no significant enrichment within the Xm-Xist region during oogenesis. However, NT embryos with ng nuclei (ngNT) showed extensive Xm-Xist derepression and H3K9me3 hypomethylation of the promoter region at the 4-cell stage, which corresponds to the onset of paternal Xist expression. We also found that the chromatin state at the Xist genomic locus became markedly condensed as oocyte growth proceeded. Although the condensed Xm-Xist genomic locus relaxed during early preimplantation phases, the extent of the relaxation across Xm-Xist loci derived from normally developed oocytes was significantly smaller than those of paternal-Xist and ngNT-Xist genomic loci. Furthermore, Xm-Xist from 2-cell metaphase nuclei became derepressed following NT. We propose that chromatin condensation is associated with imprinted Xist repression and that skipping of the condensation step by NT leads to Xist activation during the early preimplantation phase. © 2015. Published by The Company of Biologists Ltd.

  10. H2A.Z acidic patch couples chromatin dynamics to regulation of gene expression programs during ESC differentiation.

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    Vidya Subramanian

    Full Text Available The histone H2A variant H2A.Z is essential for embryonic development and for proper control of developmental gene expression programs in embryonic stem cells (ESCs. Divergent regions of amino acid sequence of H2A.Z likely determine its functional specialization compared to core histone H2A. For example, H2A.Z contains three divergent residues in the essential C-terminal acidic patch that reside on the surface of the histone octamer as an uninterrupted acidic patch domain; however, we know little about how these residues contribute to chromatin structure and function. Here, we show that the divergent amino acids Gly92, Asp97, and Ser98 in the H2A.Z C-terminal acidic patch (H2A.Z(AP3 are critical for lineage commitment during ESC differentiation. H2A.Z is enriched at most H3K4me3 promoters in ESCs including poised, bivalent promoters that harbor both activating and repressive marks, H3K4me3 and H3K27me3 respectively. We found that while H2A.Z(AP3 interacted with its deposition complex and displayed a highly similar distribution pattern compared to wild-type H2A.Z, its enrichment levels were reduced at target promoters. Further analysis revealed that H2A.Z(AP3 was less tightly associated with chromatin, suggesting that the mutant is more dynamic. Notably, bivalent genes in H2A.Z(AP3 ESCs displayed significant changes in expression compared to active genes. Moreover, bivalent genes in H2A.Z(AP3 ESCs gained H3.3, a variant associated with higher nucleosome turnover, compared to wild-type H2A.Z. We next performed single cell imaging to measure H2A.Z dynamics. We found that H2A.Z(AP3 displayed higher mobility in chromatin compared to wild-type H2A.Z by fluorescent recovery after photobleaching (FRAP. Moreover, ESCs treated with the transcriptional inhibitor flavopiridol resulted in a decrease in the H2A.Z(AP3 mobile fraction and an increase in its occupancy at target genes indicating that the mutant can be properly incorporated into chromatin

  11. Shaping Chromatin in the Nucleus: The Bricks and the Architects.

    Science.gov (United States)

    Sitbon, David; Podsypanina, Katrina; Yadav, Tejas; Almouzni, Geneviève

    2017-12-05

    Chromatin organization in the nucleus provides a vast repertoire of information in addition to that encoded genetically. Understanding how this organization impacts genome stability and influences cell fate and tumorigenesis is an area of rapid progress. Considering the nucleosome, the fundamental unit of chromatin structure, the study of histone variants (the bricks) and their selective loading by histone chaperones (the architects) is particularly informative. Here, we report recent advances in understanding how relationships between histone variants and their chaperones contribute to tumorigenesis using cell lines and Xenopus development as model systems. In addition to their role in histone deposition, we also document interactions between histone chaperones and other chromatin factors that govern higher-order structure and control DNA metabolism. We highlight how a fine-tuned assembly line of bricks (H3.3 and CENP-A) and architects (HIRA, HJURP, and DAXX) is key in adaptation to developmental and pathological changes. An example of this conceptual advance is the exquisite sensitivity displayed by p53-null tumor cells to modulation of HJURP, the histone chaperone for CENP-A (CenH3 variant). We discuss how these findings open avenues for novel therapeutic paradigms in cancer care. © 2017 Sitbon et al.; Published by Cold Spring Harbor Laboratory Press.

  12. Biphasic Chromatin Structure and FISH Signals Reflect Intranuclear Order

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    Jyoti P. Chaudhuri

    2005-01-01

    Full Text Available Background and Aim: One of the two parental allelic genes may selectively be expressed, regulated by imprinting, X-inactivation or by other less known mechanisms. This study aims to reflect on such genetic mechanisms. Materials and Methods: Slides from short term cultures or direct smears of blood, bone marrow and amniotic fluids were hybridized with FISH probes singly, combined or sequentially. Two to three hundred cells were examined from each preparation. Results and Aignificance: A small number of cells (up to about 5%, more frequent in leukemia cases, showed the twin features: (1 nuclei with biphasic chromatin, one part decondensed and the other condensed; and (2 homologous FISH signals distributed equitably in those two regions. The biphasic chromatin structure with equitable distribution of the homologous FISH signals may correspond to the two sets of chromosomes, supporting observations on ploidywise intranuclear order. The decondensed chromatin may relate to enhanced transcriptions or advanced replications. Conclusions: Transcriptions of only one of the two parental genomes cause allelic exclusion. Genomes may switch with alternating monoallelic expression of biallelic genes as an efficient genetic mechanism. If genomes fail to switch, allelic exclusion may lead to malignancy. Similarly, a genome-wide monoallelic replication may tilt the balance of heterozygosity resulting in aneusomy, initiating early events in malignant transformation and in predicting cancer mortality.

  13. Footprint traversal by adenosine-triphosphate-dependent chromatin remodeler motor

    Science.gov (United States)

    Garai, Ashok; Mani, Jesrael; Chowdhury, Debashish

    2012-04-01

    Adenosine-triphosphate (ATP)-dependent chromatin remodeling enzymes (CREs) are biomolecular motors in eukaryotic cells. These are driven by a chemical fuel, namely, ATP. CREs actively participate in many cellular processes that require accessibility of specific segments of DNA which are packaged as chromatin. The basic unit of chromatin is a nucleosome where 146 bp ˜ 50 nm of a double-stranded DNA (dsDNA) is wrapped around a spool formed by histone proteins. The helical path of histone-DNA contact on a nucleosome is also called “footprint.” We investigate the mechanism of footprint traversal by a CRE that translocates along the dsDNA. Our two-state model of a CRE captures effectively two distinct chemical (or conformational) states in the mechanochemical cycle of each ATP-dependent CRE. We calculate the mean time of traversal. Our predictions on the ATP dependence of the mean traversal time can be tested by carrying out in vitro experiments on mononucleosomes.

  14. Predicting chromatin architecture from models of polymer physics.

    Science.gov (United States)

    Bianco, Simona; Chiariello, Andrea M; Annunziatella, Carlo; Esposito, Andrea; Nicodemi, Mario

    2017-03-01

    We review the picture of chromatin large-scale 3D organization emerging from the analysis of Hi-C data and polymer modeling. In higher mammals, Hi-C contact maps reveal a complex higher-order organization, extending from the sub-Mb to chromosomal scales, hierarchically folded in a structure of domains-within-domains (metaTADs). The domain folding hierarchy is partially conserved throughout differentiation, and deeply correlated to epigenomic features. Rearrangements in the metaTAD topology relate to gene expression modifications: in particular, in neuronal differentiation models, topologically associated domains (TADs) tend to have coherent expression changes within architecturally conserved metaTAD niches. To identify the nature of architectural domains and their molecular determinants within a principled approach, we discuss models based on polymer physics. We show that basic concepts of interacting polymer physics explain chromatin spatial organization across chromosomal scales and cell types. The 3D structure of genomic loci can be derived with high accuracy and its molecular determinants identified by crossing information with epigenomic databases. In particular, we illustrate the case of the Sox9 locus, linked to human congenital disorders. The model in-silico predictions on the effects of genomic rearrangements are confirmed by available 5C data. That can help establishing new diagnostic tools for diseases linked to chromatin mis-folding, such as congenital disorders and cancer.

  15. Structure of centromere chromatin: from nucleosome to chromosomal architecture.

    Science.gov (United States)

    Schalch, Thomas; Steiner, Florian A

    2017-08-01

    The centromere is essential for the segregation of chromosomes, as it serves as attachment site for microtubules to mediate chromosome segregation during mitosis and meiosis. In most organisms, the centromere is restricted to one chromosomal region that appears as primary constriction on the condensed chromosome and is partitioned into two chromatin domains: The centromere core is characterized by the centromere-specific histone H3 variant CENP-A (also called cenH3) and is required for specifying the centromere and for building the kinetochore complex during mitosis. This core region is generally flanked by pericentric heterochromatin, characterized by nucleosomes containing H3 methylated on lysine 9 (H3K9me) that are bound by heterochromatin proteins. During mitosis, these two domains together form a three-dimensional structure that exposes CENP-A-containing chromatin to the surface for interaction with the kinetochore and microtubules. At the same time, this structure supports the tension generated during the segregation of sister chromatids to opposite poles. In this review, we discuss recent insight into the characteristics of the centromere, from the specialized chromatin structures at the centromere core and the pericentromere to the three-dimensional organization of these regions that make up the functional centromere.

  16. Tissue-specific regulation of chromatin insulator function.

    Science.gov (United States)

    Matzat, Leah H; Dale, Ryan K; Moshkovich, Nellie; Lei, Elissa P

    2012-01-01

    Chromatin insulators organize the genome into distinct transcriptional domains and contribute to cell type-specific chromatin organization. However, factors regulating tissue-specific insulator function have not yet been discovered. Here we identify the RNA recognition motif-containing protein Shep as a direct interactor of two individual components of the gypsy insulator complex in Drosophila. Mutation of shep improves gypsy-dependent enhancer blocking, indicating a role as a negative regulator of insulator activity. Unlike ubiquitously expressed core gypsy insulator proteins, Shep is highly expressed in the central nervous system (CNS) with lower expression in other tissues. We developed a novel, quantitative tissue-specific barrier assay to demonstrate that Shep functions as a negative regulator of insulator activity in the CNS but not in muscle tissue. Additionally, mutation of shep alters insulator complex nuclear localization in the CNS but has no effect in other tissues. Consistent with negative regulatory activity, ChIP-seq analysis of Shep in a CNS-derived cell line indicates substantial genome-wide colocalization with a single gypsy insulator component but limited overlap with intact insulator complexes. Taken together, these data reveal a novel, tissue-specific mode of regulation of a chromatin insulator.

  17. Human tRNA genes function as chromatin insulators

    Science.gov (United States)

    Raab, Jesse R; Chiu, Jonathan; Zhu, Jingchun; Katzman, Sol; Kurukuti, Sreenivasulu; Wade, Paul A; Haussler, David; Kamakaka, Rohinton T

    2012-01-01

    Insulators help separate active chromatin domains from silenced ones. In yeast, gene promoters act as insulators to block the spread of Sir and HP1 mediated silencing while in metazoans most insulators are multipartite autonomous entities. tDNAs are repetitive sequences dispersed throughout the human genome and we now show that some of these tDNAs can function as insulators in human cells. Using computational methods, we identified putative human tDNA insulators. Using silencer blocking, transgene protection and repressor blocking assays we show that some of these tDNA-containing fragments can function as barrier insulators in human cells. We find that these elements also have the ability to block enhancers from activating RNA pol II transcribed promoters. Characterization of a putative tDNA insulator in human cells reveals that the site possesses chromatin signatures similar to those observed at other better-characterized eukaryotic insulators. Enhanced 4C analysis demonstrates that the tDNA insulator makes long-range chromatin contacts with other tDNAs and ETC sites but not with intervening or flanking RNA pol II transcribed genes. PMID:22085927

  18. Tissue-Specific Regulation of Chromatin Insulator Function

    Science.gov (United States)

    Matzat, Leah H.; Dale, Ryan K.; Moshkovich, Nellie; Lei, Elissa P.

    2012-01-01

    Chromatin insulators organize the genome into distinct transcriptional domains and contribute to cell type–specific chromatin organization. However, factors regulating tissue-specific insulator function have not yet been discovered. Here we identify the RNA recognition motif-containing protein Shep as a direct interactor of two individual components of the gypsy insulator complex in Drosophila. Mutation of shep improves gypsy-dependent enhancer blocking, indicating a role as a negative regulator of insulator activity. Unlike ubiquitously expressed core gypsy insulator proteins, Shep is highly expressed in the central nervous system (CNS) with lower expression in other tissues. We developed a novel, quantitative tissue-specific barrier assay to demonstrate that Shep functions as a negative regulator of insulator activity in the CNS but not in muscle tissue. Additionally, mutation of shep alters insulator complex nuclear localization in the CNS but has no effect in other tissues. Consistent with negative regulatory activity, ChIP–seq analysis of Shep in a CNS-derived cell line indicates substantial genome-wide colocalization with a single gypsy insulator component but limited overlap with intact insulator complexes. Taken together, these data reveal a novel, tissue-specific mode of regulation of a chromatin insulator. PMID:23209434

  19. Novel chromatin texture features for the classification of pap smears

    Science.gov (United States)

    Bejnordi, Babak E.; Moshavegh, Ramin; Sujathan, K.; Malm, Patrik; Bengtsson, Ewert; Mehnert, Andrew

    2013-03-01

    This paper presents a set of novel structural texture features for quantifying nuclear chromatin patterns in cells on a conventional Pap smear. The features are derived from an initial segmentation of the chromatin into bloblike texture primitives. The results of a comprehensive feature selection experiment, including the set of proposed structural texture features and a range of different cytology features drawn from the literature, show that two of the four top ranking features are structural texture features. They also show that a combination of structural and conventional features yields a classification performance of 0.954±0.019 (AUC±SE) for the discrimination of normal (NILM) and abnormal (LSIL and HSIL) slides. The results of a second classification experiment, using only normal-appearing cells from both normal and abnormal slides, demonstrates that a single structural texture feature measuring chromatin margination yields a classification performance of 0.815±0.019. Overall the results demonstrate the efficacy of the proposed structural approach and that it is possible to detect malignancy associated changes (MACs) in Papanicoloau stain.

  20. Transcription-dependent association of HDAC2 with active chromatin.

    Science.gov (United States)

    Jahan, Sanzida; Sun, Jian-Min; He, Shihua; Davie, James R

    2018-02-01

    Histone deacetylase 2 (HDAC2) catalyzes deacetylation of histones at the promoter and coding regions of transcribed genes and regulates chromatin structure and transcription. To explore the role of HDAC2 and phosphorylated HDAC2 in gene regulation, we studied the location along transcribed genes, the mode of recruitment and the associated proteins with HDAC2 and HDAC2S394ph in chicken polychromatic erythrocytes. We show that HDAC2 and HDAC2S394ph are associated with transcriptionally active chromatin and located in the interchromatin channels. HDAC2S394ph was present primarly at the upstream promoter region of the transcribed CA2 and GAS41 genes, while total HDAC2 was also found within the coding region of the CA2 gene. Recruitment of HDAC2 to these genes was partially dependent upon on-going transcription. Unmodified HDAC2 was associated with RNA binding proteins and interacted with RNA bound to the initiating and elongating forms of RNA polymerase II. HDAC2S394ph was not associated with RNA polymerase II. These results highlight the differential properties of unmodified and phosphorylated HDAC2 and the organization of acetylated transcriptionally active chromatin in the chicken polychromatic erythrocyte. © 2017 Wiley Periodicals, Inc.

  1. The protein encoded by the proto-oncogene DEK changes the topology of chromatin and reduces the efficiency of DNA replication in a chromatin-specific manner

    DEFF Research Database (Denmark)

    Alexiadis, V; Waldmann, T; Andersen, Jens S.

    2000-01-01

    The structure of chromatin regulates the genetic activity of the underlying DNA sequence. We report here that the protein encoded by the proto-oncogene DEK, which is involved in acute myelogenous leukemia, induces alterations of the superhelical density of DNA in chromatin. The change in topology...

  2. ADP-ribose-specific chromatin-affinity purification for investigating genome-wide or locus-specific chromatin ADP-ribosylation.

    Science.gov (United States)

    Bisceglie, Lavinia; Bartolomei, Giody; Hottiger, Michael O

    2017-09-01

    Protein ADP-ribosylation is a structurally heterogeneous post-translational modification (PTM) that influences the physicochemical and biological properties of the modified protein. ADP-ribosylation of chromatin changes its structural properties, thereby regulating important nuclear functions. A lack of suitable antibodies for chromatin immunoprecipitation (ChIP) has so far prevented a comprehensive analysis of DNA-associated protein ADP-ribosylation. To analyze chromatin ADP-ribosylation, we recently developed a novel ADP-ribose-specific chromatin-affinity purification (ADPr-ChAP) methodology that uses the recently identified ADP-ribose-binding domains RNF146 WWE and Af1521. In this protocol, we describe how to use this robust and versatile method for genome-wide and loci-specific localization of chromatin ADP-ribosylation. ADPr-ChAP enables bioinformatic comparisons of ADP-ribosylation with other chromatin modifications and is useful for understanding how ADP-ribosylation regulates biologically important cellular processes. ADPr-ChAP takes 1 week and requires standard skills in molecular biology and biochemistry. Although not covered in detail here, this technique can also be combined with conventional ChIP or DNA analysis to define the histone marks specifically associated with the ADP-ribosylated chromatin fractions and dissect the molecular mechanism and functional role of chromatin ADP-ribosylation.

  3. Linker Histone H1.2 Directs Genome-wide Chromatin Association of the Retinoblastoma Tumor Suppressor Protein and Facilitates Its Function

    Directory of Open Access Journals (Sweden)

    Shonagh Munro

    2017-06-01

    Full Text Available The retinoblastoma tumor suppressor protein pRb is a master regulator of cellular proliferation, principally through interaction with E2F and regulation of E2F target genes. Here, we describe the H1.2 linker histone as a major pRb interaction partner. We establish that H1.2 and pRb are found in a chromatin-bound complex on diverse E2F target genes. Interrogating the global influence of H1.2 on the genome-wide distribution of pRb indicated that the E2F target genes affected by H1.2 are functionally linked to cell-cycle control, consistent with the ability of H1.2 to hinder cell proliferation and the elevated levels of chromatin-bound H1-pRb complex, which occur in growth-arrested cells. Our results define a network of E2F target genes as susceptible to the regulatory influence of H1.2, where H1.2 augments global association of pRb with chromatin, enhances transcriptional repression by pRb, and facilitates pRb-dependent cell-cycle arrest.

  4. The binding of lamin B receptor to chromatin is regulated by phosphorylation in the RS region.

    Science.gov (United States)

    Takano, Makoto; Takeuchi, Masaki; Ito, Hiromi; Furukawa, Kazuhiro; Sugimoto, Kenji; Omata, Saburo; Horigome, Tsuneyoshi

    2002-02-01

    Binding of lamin B receptor (LBR) to chromatin was studied by means of an in vitro assay system involving recombinant fragments of human LBR and Xenopus sperm chromatin. Glutathione-S-transferase (GST)-fused proteins including LBR fragments containing the N-terminal region (residues 1-53) and arginine-serine repeat-containing region (residues 54-89) bound to chromatin. The binding of GST-fusion proteins incorporating the N-terminal and arginine-serine repeat-containing regions to chromatin was suppressed by mild trypsinization of the chromatin and by pretreatment with a DNA solution. A new cell-free system for analyzing the cell cycle-dependent binding of a protein to chromatin was developed from recombinant proteins, a Xenopus egg cytosol fraction and sperm chromatin. The system was applied to analyse the binding of LBR to chromatin. It was shown that the binding of LBR fragments to chromatin was stimulated by phosphorylation in the arginine-serine repeat-containing region by a protein kinase(s) in a synthetic phase egg cytosol. However, the binding of LBR fragments was suppressed by phosphorylation at different residues in the same region by a kinase(s) in a mitotic phase cytosol. These results suggested that the cell cycle-dependent binding of LBR to chromatin is regulated by phosphorylation in the arginine-serine repeat-containing region by multiple kinases.

  5. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation

    Science.gov (United States)

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-01-01

    Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation. PMID:25870416

  6. RNA is an integral component of chromatin that contributes to its structural organization.

    Directory of Open Access Journals (Sweden)

    Antonio Rodríguez-Campos

    Full Text Available Chromatin structure is influenced by multiples factors, such as pH, temperature, nature and concentration of counterions, post-translational modifications of histones and binding of structural non-histone proteins. RNA is also known to contribute to the regulation of chromatin structure as chromatin-induced gene silencing was shown to depend on the RNAi machinery in S. pombe, plants and Drosophila. Moreover, both in Drosophila and mammals, dosage compensation requires the contribution of specific non-coding RNAs. However, whether RNA itself plays a direct structural role in chromatin is not known. Here, we report results that indicate a general structural role for RNA in eukaryotic chromatin. RNA is found associated to purified chromatin prepared from chicken liver, or cultured Drosophila S2 cells, and treatment with RNase A alters the structural properties of chromatin. Our results indicate that chromatin-associated RNAs, which account for 2%-5% of total chromatin-associated nucleic acids, are polyA(- and show a size similar to that of the DNA contained in the corresponding chromatin fragments. Chromatin-associated RNA(s are not likely to correspond to nascent transcripts as they are also found bound to chromatin when cells are treated with alpha-amanitin. After treatment with RNase A, chromatin fragments of molecular weight >3.000 bp of DNA showed reduced sedimentation through sucrose gradients and increased sensitivity to micrococcal nuclease digestion. This structural transition, which is observed both at euchromatic and heterochromatic regions, proceeds without loss of histone H1 or any significant change in core-histone composition and integrity.

  7. Sperm chromatin maturity and integrity correlated to zygote development in ICSI program.

    Science.gov (United States)

    Asmarinah; Syauqy, Ahmad; Umar, Liya Agustin; Lestari, Silvia Werdhy; Mansyur, Eliza; Hestiantoro, Andon; Paradowszka-Dogan, Agnieszka

    2016-10-01

    This study aimed to evaluate sperm chromatin maturity and integrity of that injected into good-quality oocytes in an in vitro fertilization-intra cytoplasmic sperm injection (IVF-ICSI) program. A cut-off value of sperm chromatin maturity and integrity was developed as a function of their correlation to the zygote development, i.e., embryo formation and cleavage rate. The study assessed sperm chromatin maturity using aniline blue (AB) staining, whereas toluidine blue (TB) staining was used to assess sperm chromatin integrity. Ejaculates from 59 patients undergoing ICSI and 46 fertile normozoospermic donors for determination of normal values of sperm chromatin status were used in this study. Embryo formation and cleavage rates were observed for the period of 3 days after ICSI. There was a significant difference in the percentage of sperm with mature chromatin between ejaculate from ICSI patients and fertile donor (p=0.020); while there was no significant difference in sperm chromatin integrity of both samples (p=0.120). There was no significant correlation between sperm chromatin maturity and either embryo formation or cleavage rate; as well as sperm chromatin integrity to both parameters of zygote development (p>0.05). Furthermore, we found that the cut-off value of sperm chromatin maturity and integrity of the fertile normozoospermic ejaculates were 87.2% and 80.2%, respectively. Using the cut-offs, we found that low sperm chromatin maturity at the level of chromatin integrity at the level of chromatin (AB<87% and TB<80%, respectively), could affect zygote development following ICSI. AB: aniline blue; CMA3: chromomycin A3; ICSI: intra cytoplasmic sperm injection; IVF: in vitro fertilization; PBS: phosphate buffer saline; SPSS: Statistical Package for Social Science; TB: toluidine blue; WHO: World Health Organization.

  8. H4K20me0 marks post-replicative chromatin and recruits the TONSL₋MMS22L DNA repair complex

    Energy Technology Data Exchange (ETDEWEB)

    Saredi, Giulia; Huang, Hongda; Hammond, Colin M.; Alabert, Constance; Bekker-Jensen, Simon; Forne, Ignasi; Reverón-Gómez, Nazaret; Foster, Benjamin M.; Mlejnkova, Lucie; Bartke, Till; Cejka, Petr; Mailand, Niels; Imhof, Axel; Patel, Dinshaw J.; Groth, Anja [UCopenhagen; (MSKCC); (ICL); (LMU); (Zurich)

    2016-06-22

    Here, we report that after DNA replication, chromosomal processes including DNA repair and transcription take place in the context of sister chromatids. While cell cycle regulation can guide these processes globally, mechanisms to distinguish pre- and post-replicative states locally remain unknown. In this paper we reveal that new histones incorporated during DNA replication provide a signature of post-replicative chromatin, read by the human TONSL–MMS22L1, 2, 3, 4 homologous recombination complex. We identify the TONSL ankyrin repeat domain (ARD) as a reader of histone H4 tails unmethylated at K20 (H4K20me0), which are specific to new histones incorporated during DNA replication and mark post-replicative chromatin until the G2/M phase of the cell cycle. Accordingly, TONSL–MMS22L binds new histones H3–H4 both before and after incorporation into nucleosomes, remaining on replicated chromatin until late G2/M. H4K20me0 recognition is required for TONSL–MMS22L binding to chromatin and accumulation at challenged replication forks and DNA lesions. Consequently, TONSL ARD mutants are toxic, compromising genome stability, cell viability and resistance to replication stress. Finally, together, these data reveal a histone-reader-based mechanism for recognizing the post-replicative state, offering a new angle to understand DNA repair with the potential for targeted cancer therapy.

  9. DNA repair goes hip-hop: SMARCA and CHD chromatin remodellers join the break dance.

    Science.gov (United States)

    Rother, Magdalena B; van Attikum, Haico

    2017-10-05

    Proper signalling and repair of DNA double-strand breaks (DSB) is critical to prevent genome instability and diseases such as cancer. The packaging of DNA into chromatin, however, has evolved as a mere obstacle to these DSB responses. Posttranslational modifications and ATP-dependent chromatin remodelling help to overcome this barrier by modulating nucleosome structures and allow signalling and repair machineries access to DSBs in chromatin. Here we recap our current knowledge on how ATP-dependent SMARCA- and CHD-type chromatin remodellers alter chromatin structure during the signalling and repair of DSBs and discuss how their dysfunction impacts genome stability and human disease.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Authors.

  10. Contribution of Topological Domains and Loop Formation to 3D Chromatin Organization

    Directory of Open Access Journals (Sweden)

    Vuthy Ea

    2015-07-01

    Full Text Available Recent investigations on 3D chromatin folding revealed that the eukaryote genomes are both highly compartmentalized and extremely dynamic. This review presents the most recent advances in topological domains’ organization of the eukaryote genomes and discusses the relationship to chromatin loop formation. CTCF protein appears as a central factor of these two organization levels having either a strong insulating role at TAD borders, or a weaker architectural role in chromatin loop formation. TAD borders directly impact on chromatin dynamics by restricting contacts within specific genomic portions thus confining chromatin loop formation within TADs. We discuss how sub-TAD chromatin dynamics, constrained into a recently described statistical helix conformation, can produce functional interactions by contact stabilization.

  11. Dynamic chromatin technologies: from individual molecules to epigenomic regulation in cells.

    Science.gov (United States)

    Cuvier, Olivier; Fierz, Beat

    2017-08-01

    The establishment and maintenance of chromatin states involves multiscale dynamic processes integrating transcription factor and multiprotein effector dynamics, cycles of chemical chromatin modifications, and chromatin structural organization. Recent developments in genomic technologies are emerging that are enabling a view beyond ensemble- and time-averaged properties and are revealing the importance of dynamic chromatin states for cell fate decisions, differentiation and reprogramming at the single-cell level. Concurrently, biochemical and single-molecule methodologies are providing key insights into the underlying molecular mechanisms. Combining results from defined in vitro and single-molecule studies with single-cell genomic approaches thus holds great promise for understanding chromatin-based transcriptional memory and cell fate. In this Review, we discuss recent developments in biochemical, single-molecule biophysical and single-cell genomic technologies and review how the findings from these approaches can be integrated to paint a comprehensive picture of dynamic chromatin states.

  12. Higher-order structure of the 30-nm chromatin fiber revealed by cryo-EM.

    Science.gov (United States)

    Zhu, Ping; Li, Guohong

    2016-11-01

    Genomic DNA is hierarchically packaged into chromatin in eukaryotes. As a central-level chromatin structure between nucleosomal arrays and higher order organizations, 30 nm chromatin fiber, and its dynamics play a crucial role in regulating DNA accessibility for gene transcription. However, despite extensive efforts over three decades, the higher-order structure of the 30 nm chromatin fiber remains unresolved and controversial. We have recently reconstituted the 30 nm chromatin fibers from 12 nucleosomal arrays in vitro in the presence of linker histone H1, and determined their cryo-EM structures at resolution of 11 Å (Song et al., Science 344, 376-380). Here, we briefly reviewed the higher-order structure studies of chromatin fibers, mainly focusing on the insights from the cryo-EM structures we recently solved. © 2016 IUBMB Life, 68(11):873-878, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

  13. How chromatin is remodelled during DNA repair of UV-induced DNA damage in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Shirong Yu

    2011-06-01

    Full Text Available Global genome nucleotide excision repair removes DNA damage from transcriptionally silent regions of the genome. Relatively little is known about the molecular events that initiate and regulate this process in the context of chromatin. We've shown that, in response to UV radiation-induced DNA damage, increased histone H3 acetylation at lysine 9 and 14 correlates with changes in chromatin structure, and these alterations are associated with efficient global genome nucleotide excision repair in yeast. These changes depend on the presence of the Rad16 protein. Remarkably, constitutive hyperacetylation of histone H3 can suppress the requirement for Rad7 and Rad16, two components of a global genome repair complex, during repair. This reveals the connection between histone H3 acetylation and DNA repair. Here, we investigate how chromatin structure is modified following UV irradiation to facilitate DNA repair in yeast. Using a combination of chromatin immunoprecipitation to measure histone acetylation levels, histone acetylase occupancy in chromatin, MNase digestion, or restriction enzyme endonuclease accessibility assays to analyse chromatin structure, and finally nucleotide excision repair assays to examine DNA repair, we demonstrate that global genome nucleotide excision repair drives UV-induced chromatin remodelling by controlling histone H3 acetylation levels in chromatin. The concerted action of the ATPase and C3HC4 RING domains of Rad16 combine to regulate the occupancy of the histone acetyl transferase Gcn5 on chromatin in response to UV damage. We conclude that the global genome repair complex in yeast regulates UV-induced histone H3 acetylation by controlling the accessibility of the histone acetyl transferase Gcn5 in chromatin. The resultant changes in histone H3 acetylation promote chromatin remodelling necessary for efficient repair of DNA damage. Recent evidence suggests that GCN5 plays a role in NER in human cells. Our work provides

  14. How chromatin is remodelled during DNA repair of UV-induced DNA damage in Saccharomyces cerevisiae.

    Science.gov (United States)

    Yu, Shirong; Teng, Yumin; Waters, Raymond; Reed, Simon H

    2011-06-01

    Global genome nucleotide excision repair removes DNA damage from transcriptionally silent regions of the genome. Relatively little is known about the molecular events that initiate and regulate this process in the context of chromatin. We've shown that, in response to UV radiation-induced DNA damage, increased histone H3 acetylation at lysine 9 and 14 correlates with changes in chromatin structure, and these alterations are associated with efficient global genome nucleotide excision repair in yeast. These changes depend on the presence of the Rad16 protein. Remarkably, constitutive hyperacetylation of histone H3 can suppress the requirement for Rad7 and Rad16, two components of a global genome repair complex, during repair. This reveals the connection between histone H3 acetylation and DNA repair. Here, we investigate how chromatin structure is modified following UV irradiation to facilitate DNA repair in yeast. Using a combination of chromatin immunoprecipitation to measure histone acetylation levels, histone acetylase occupancy in chromatin, MNase digestion, or restriction enzyme endonuclease accessibility assays to analyse chromatin structure, and finally nucleotide excision repair assays to examine DNA repair, we demonstrate that global genome nucleotide excision repair drives UV-induced chromatin remodelling by controlling histone H3 acetylation levels in chromatin. The concerted action of the ATPase and C3HC4 RING domains of Rad16 combine to regulate the occupancy of the histone acetyl transferase Gcn5 on chromatin in response to UV damage. We conclude that the global genome repair complex in yeast regulates UV-induced histone H3 acetylation by controlling the accessibility of the histone acetyl transferase Gcn5 in chromatin. The resultant changes in histone H3 acetylation promote chromatin remodelling necessary for efficient repair of DNA damage. Recent evidence suggests that GCN5 plays a role in NER in human cells. Our work provides important insight into

  15. Salt and divalent cations affect the flexible nature of the natural beaded chromatin structure

    DEFF Research Database (Denmark)

    Christiansen, Gunna; Griffith, J

    1977-01-01

    A natural chromatin containing simian virus 40 (SV40) DNA and histone has been used to examine changes in chromatin structure caused by various physical and chemical treatments. We find that histone H1 depleted chromatin is more compact in solutions of 0.15M NaCl or 2 mM MgCl2 than in 0.01 M NaCl...

  16. The chromatin landscape of Drosophila: comparisons between species, sexes, and chromosomes

    Science.gov (United States)

    Brown, Emily J.; Bachtrog, Doris

    2014-01-01

    The chromatin landscape is key for gene regulation, but little is known about how it differs between sexes or between species. Here, we study the sex-specific chromatin landscape of Drosophila miranda, a species with young sex chromosomes, and compare it with Drosophila melanogaster. We analyze six histone modifications in male and female larvae of D. miranda (H3K4me1, H3K4me3, H3K36me3, H4K16ac, H3K27me3, and H3K9me2), and define seven biologically meaningful chromatin states that show different enrichments for transcribed and silent genes, repetitive elements, housekeeping, and tissue-specific genes. The genome-wide distribution of both active and repressive chromatin states differs between males and females. In males, active chromatin is enriched on the X, relative to females, due to dosage compensation of the hemizygous X. Furthermore, a smaller fraction of the euchromatic portion of the genome is in a repressive chromatin state in males relative to females. However, sex-specific chromatin states appear not to explain sex-biased expression of genes. Overall, conservation of chromatin states between male and female D. miranda is comparable to conservation between D. miranda and D. melanogaster, which diverged >30 MY ago. Active chromatin states are more highly conserved across species, while heterochromatin shows very low levels of conservation. Divergence in chromatin profiles contributes to expression divergence between species, with ∼26% of genes in different chromatin states in the two species showing species-specific or species-biased expression, an enrichment of approximately threefold over null expectation. Our data suggest that heteromorphic sex chromosomes in males (that is, a hypertranscribed X and an inactivated Y) may contribute to global redistribution of active and repressive chromatin marks between chromosomes and sexes. PMID:24840603

  17. PeakAnalyzer: Genome-wide annotation of chromatin binding and modification loci

    Directory of Open Access Journals (Sweden)

    Tammoja Kairi

    2010-08-01

    Full Text Available Abstract Background Functional genomic studies involving high-throughput sequencing and tiling array applications, such as ChIP-seq and ChIP-chip, generate large numbers of experimentally-derived signal peaks across the genome under study. In analyzing these loci to determine their potential regulatory functions, areas of signal enrichment must be considered relative to proximal genes and regulatory elements annotated throughout the target genome Regions of chromatin association by transcriptional regulators should be distinguished as individual binding sites in order to enhance downstream analyses, such as the identification of known and novel consensus motifs. Results PeakAnalyzer is a set of high-performance utilities for the automated processing of experimentally-derived peak regions and annotation of genomic loci. The programs can accurately subdivide multimodal regions of signal enrichment into distinct subpeaks corresponding to binding sites or chromatin modifications, retrieve genomic sequences encompassing the computed subpeak summits, and identify positional features of interest such as intersection with exon/intron gene components, proximity to up- or downstream transcriptional start sites and cis-regulatory elements. The software can be configured to run either as a pipeline component for high-throughput analyses, or as a cross-platform desktop application with an intuitive user interface. Conclusions PeakAnalyzer comprises a number of utilities essential for ChIP-seq and ChIP-chip data analysis. High-performance implementations are provided for Unix pipeline integration along with a GUI version for interactive use. Source code in C++ and Java is provided, as are native binaries for Linux, Mac OS X and Windows systems.

  18. Mutations and modeling of the chromatin remodeler CHD8 define an emerging autism etiology

    Directory of Open Access Journals (Sweden)

    Rebecca A Barnard

    2015-12-01

    Full Text Available Autism Spectrum Disorder (ASD is a common neurodevelopmental disorder with a strong but complex genetic component. Recent family based exome-sequencing strategies have identified recurrent de novo mutations at specific genes, providing strong evidence for ASD risk, but also highlighting the extreme genetic heterogeneity of the disorder. However, disruptions in these genes converge on key molecular pathways early in development. In particular, functional enrichment analyses have found that there is a bias towards genes involved in transcriptional regulation, such as chromatin regulators. Here we review recent genetic, animal model, co-expression network, and functional genomics studies relating to the high confidence ASD risk gene, CHD8. CHD8 a chromatin remodeling factor, may serve as a master regulator of a common ASD etiology. Individuals with a CHD8 mutation show an ASD subtype that includes similar physical characteristics, such as macrocephaly and prolonged GI problems including recurrent constipation. Similarly, animal models of CHD8 disruption exhibit enlarged head circumference and reduced gut motility phenotypes. Systems biology approaches suggest CHD8 and other candidate ASD risk genes are enriched during mid-fetal development, which may represent a critical time window in ASD etiology. Transcription profiles from cell and primary tissue models of early development indicate that CHD8 may also positively regulate other candidate ASD risk genes through both direct and indirect means. However continued study is needed to elucidate the mechanism of regulation as well as identify which CHD8 targets are most relevant to ASD risk. Overall, these initial studies suggest the potential for common ASD etiologies and the development of personalized treatments in the future.

  19. Evaluation of sperm chromatin structure in boar semen

    Directory of Open Access Journals (Sweden)

    Banaszewska Dorota

    2015-06-01

    Full Text Available This study was an attempt to evaluate sperm chromatin structure in the semen of insemination boars. Preparations of semen were stained with acridine orange, aniline blue, and chromomycin A3. Abnormal protamination occurred more frequently in young individuals whose sexual development was not yet complete, but may also be an individual trait. This possibility is important to factor into the decision regarding further exploitation of insemination boars. Thus a precise assessment of abnormalities in the protamination process would seem to be expedient as a tool supplementing morphological and molecular evaluation of semen. Disruptions in nucleoprotein structure can be treated as indicators of the biological value of sperm cells.

  20. Determination of histone acetylation status by chromatin immunoprecipitation.

    Science.gov (United States)

    Galdieri, Luciano; Moon, John; Vancura, Ales

    2012-01-01

    Histone acetylation is the most studied posttranslation modification of nucleosomes. Understanding the mechanisms involved in global and promoter-specific histone acetylation will shed light on the control of transcriptional regulation. Chromatin immunoprecipitation is a powerful technique to study protein-DNA interactions in vivo. Proteins and DNA are cross-linked with formaldehyde, cells are lysed, and DNA is sheared by sonication. Protein-DNA complexes are immunoprecipitated with antibodies specific for total and acetylated histones and the relative occupancy of acetylated and total histones at selected loci is assessed by real-time PCR of the purified DNA.

  1. Mouse sperm chromatin proteins: quantitative isolation and partial characterization. [Mice

    Energy Technology Data Exchange (ETDEWEB)

    Balhorn, R.; Gledhill, B.L.; Wyrobek, A.J.

    1977-09-06

    Conditions are described that permit the quantitative extraction of chromatin proteins from the epididymal sperm of the mouse. These proteins have been isolated free of contaminating tail proteins following removal of the tails with cetyltrimethylammonium bromide (CTAB). Without this treatment, numerous acid-soluble tail proteins coextract with the nuclear proteins isolated from partially purified heads. The proteins isolated in this manner do not require prior modification with iodoacetamide and show no evidence of proteolytic degradation. In acid-urea polyacrylamide gels, 99% of the sperm protein migrates as one electrophoretic band. Evidence is presented that suggests that this single band contains two protamine-like proteins.

  2. Effects of nuclear isolation on psoralen affinity for chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Inadomi, T.; Ross, P.M. (Rockefeller Univ., New York, NY (USA))

    1989-09-29

    We have tested the effects of nuclear isolation on intercalation of TMP (a psoralen) at specific sequences and in total DNA of cultured human cells. DNA in nuclei photobound about 20% more TMP than in cells and about 10% as much as purified DNA. In contrast, a transcribed ras gene and a randomly selected polymorphic sequence each bound about 20% more TMP than total DNA in cells. However, in nuclei, as in purified DNA, both sequences were just as sensitive as total DNA. Apparently, chromatin in cells exists within diverse TMP-binding environments and some of this diversity was lost upon nuclear isolation.

  3. Effects of polyamines on higher-order folding of in situ chromatin.

    Science.gov (United States)

    Vergani, L; Mascetti, G; Nicolini, C

    1998-11-01

    Modifications of the higher-order chromatin structure induced by polyamines have been quantitatively investigated in situ through a non-invasive biophysical approach using Differential Scanning Calorimetry and Quantitative Fluorescence Microscopy. Calorimetric and intensitometric profiles have been acquired for samples of native thymocytes, alternatively suspended in buffers, with or without natural polyamines (spermine and spermidine). The results here reported show that the structure and distribution of nuclear chromatin in situ considerably change upon the ionic composition of the environment. A quantitative analysis of this data and a comparison with previous results obtained from isolated chromatin fibers was carried out. Finally, an inverse relationship between chromatin condensation and nuclear volume was observed.

  4. Chromatin Domain Organization of the TCRb Locus and Its Perturbation by Ectopic CTCF Binding.

    Science.gov (United States)

    Rawat, Pratishtha; Jalan, Manisha; Sadhu, Ananya; Kanaujia, Abhilasha; Srivastava, Madhulika

    2017-05-01

    CTCF-mediated chromatin interactions influence organization and function of mammalian genome in diverse ways. We analyzed the interactions among CTCF binding sites (CBS) at the murine TCRb locus to discern the role of CTCF-mediated interactions in the regulation of transcription and VDJ recombination. Chromosome conformation capture analysis revealed thymocyte-specific long-range intrachromosomal interactions among various CBS across the locus that were relevant for defining the limit of the enhancer Eb-regulated recombination center (RC) and for facilitating the spatial proximity of TCRb variable (V) gene segments to the RC. Ectopic CTCF binding in the RC region, effected via genetic manipulation, altered CBS-directed chromatin loops, interfered with RC establishment, and reduced the spatial proximity of the RC with Trbv segments. Changes in chromatin loop organization by ectopic CTCF binding were relatively modest but influenced transcription and VDJ recombination dramatically. Besides revealing the importance of CTCF-mediated chromatin organization for TCRb regulation, the observed chromatin loops were consistent with the emerging idea that CBS orientations influence chromatin loop organization and underscored the importance of CBS orientations for defining chromatin architecture that supports VDJ recombination. Further, our study suggests that in addition to mediating long-range chromatin interactions, CTCF influences intricate configuration of chromatin loops that govern functional interactions between elements. Copyright © 2017 American Society for Microbiology.

  5. O-GlcNAcylation and chromatin remodeling in mammals: an up-to-date overview.

    Science.gov (United States)

    Leturcq, Maïté; Lefebvre, Tony; Vercoutter-Edouart, Anne-Sophie

    2017-04-15

    Post-translational modifications of histones and the dynamic DNA methylation cycle are finely regulated by a myriad of chromatin-binding factors and chromatin-modifying enzymes. Epigenetic modifications ensure local changes in the architecture of chromatin, thus controlling in fine the accessibility of the machinery of transcription, replication or DNA repair to the chromatin. Over the past decade, the nutrient-sensor enzyme O-GlcNAc transferase (OGT) has emerged as a modulator of chromatin remodeling. In mammals, OGT acts either directly through dynamic and reversible O-GlcNAcylation of histones and chromatin effectors, or in an indirect manner through its recruitment into chromatin-bound multiprotein complexes. In particular, there is an increasing amount of evidence of a cross-talk between OGT and the DNA dioxygenase ten-eleven translocation proteins that catalyze active DNA demethylation. Conversely, the stability of OGT itself can be controlled by the histone lysine-specific demethylase 2 (LSD2). Finally, a few studies have explored the role of O-GlcNAcase (OGA) in chromatin remodeling. In this review, we summarize the recent findings on the link between OGT, OGA and chromatin regulators in mammalian cellular models, and discuss their relevance in physiological and pathological conditions. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  6. Chromatin Dynamics in Genome Stability: Roles in Suppressing Endogenous DNA Damage and Facilitating DNA Repair

    Directory of Open Access Journals (Sweden)

    Nidhi Nair

    2017-07-01

    Full Text Available Genomic DNA is compacted into chromatin through packaging with histone and non-histone proteins. Importantly, DNA accessibility is dynamically regulated to ensure genome stability. This is exemplified in the response to DNA damage where chromatin relaxation near genomic lesions serves to promote access of relevant enzymes to specific DNA regions for signaling and repair. Furthermore, recent data highlight genome maintenance roles of chromatin through the regulation of endogenous DNA-templated processes including transcription and replication. Here, we review research that shows the importance of chromatin structure regulation in maintaining genome integrity by multiple mechanisms including facilitating DNA repair and directly suppressing endogenous DNA damage.

  7. Immuno-cytogenetic manifestation of epigenetic chromatin modification marks in plants.

    Science.gov (United States)

    Sharma, Santosh Kumar; Yamamoto, Maki; Mukai, Yasuhiko

    2015-02-01

    Histone proteins and the nucleosomes along with DNA are the essential components of eukaryotic chromatin. Post-translational histone-DNA interactions and modifications eventually offer significant alteration in the chromatin environment and potentially influence diverse fundamental biological processes, some of which are known to be epigenetically inherited and constitute the "epigenetic code". Such chromatin modifications evidently uncover remarkable diversity and biological specificity associated with distinct patterns of covalent histone marks. The past few years have witnessed major breakthroughs in plant biology research by utilizing chromatin modification-specific antibodies through molecular cytogenetic tools to ascertain hallmark signatures of chromatin domains on the chromosomes. Here, we survey current information on chromosomal distribution patterns of chromatin modifications with special emphasis on histone methylation, acetylation, phosphorylation, and centromere-specific histone 3 (CENH3) marks in plants using immuno-FISH as a basic tool. Major available information has been classified under typical and comparative cytogenetic detection of chromatin modifications in plants. Further, spatial distribution of chromatin environment that exists between different cell types such as angiosperm/gymnosperm, monocot/dicot, diploid/polyploids, vegetative/generative cells, as well as different stages, i.e., mitosis versus meiosis has also been discussed in detail. Several challenges and future perspectives of molecular cytogenetics in the grooming field of plant chromatin dynamics have also been addressed.

  8. CASA derived human sperm abnormalities: correlation with chromatin packing and DNA fragmentation

    National Research Council Canada - National Science Library

    Sivanarayana, T; Krishna, Ch Ravi; Prakash, G Jaya; Krishna, K Murali; Madan, K; Rani, B Sireesha; Sudhakar, G; Raju, G A. Rama

    2012-01-01

    The present study was undertaken to evaluate the effects of morphokinetic abnormalities of human spermatozoa on chromatin packing and DNA integrity and possible beneficial effects of sperm selection...

  9. Restoring chromatin after replication: How new and old histone marks come together

    DEFF Research Database (Denmark)

    Jasencakova, Zusana; Groth, Anja

    2010-01-01

    present on new and parental histones and how they influence genome stability and restoration of epigenetically defined domains. Newly deposited histones must change their signature in the process of chromatin restoration, this may occur in a step-wise fashion involving replication-coupled processes...... replication and chromatin assembly processes in time and space. Dynamic recycling and de novo deposition of histones are fundamental for chromatin restoration. Histone post-translational modifications (PTMs) are thought to have a causal role in establishing distinct chromatin structures. Here we discuss PTMs...

  10. Evolution and genetic architecture of chromatin accessibility and function in yeast.

    Directory of Open Access Journals (Sweden)

    Caitlin F Connelly

    2014-07-01

    Full Text Available Chromatin accessibility is an important functional genomics phenotype that influences transcription factor binding and gene expression. Genome-scale technologies allow chromatin accessibility to be mapped with high-resolution, facilitating detailed analyses into the genetic architecture and evolution of chromatin structure within and between species. We performed Formaldehyde-Assisted Isolation of Regulatory Elements sequencing (FAIRE-Seq to map chromatin accessibility in two parental haploid yeast species, Saccharomyces cerevisiae and Saccharomyces paradoxus and their diploid hybrid. We show that although broad-scale characteristics of the chromatin landscape are well conserved between these species, accessibility is significantly different for 947 regions upstream of genes that are enriched for GO terms such as intracellular transport and protein localization exhibit. We also develop new statistical methods to investigate the genetic architecture of variation in chromatin accessibility between species, and find that cis effects are more common and of greater magnitude than trans effects. Interestingly, we find that cis and trans effects at individual genes are often negatively correlated, suggesting widespread compensatory evolution to stabilize levels of chromatin accessibility. Finally, we demonstrate that the relationship between chromatin accessibility and gene expression levels is complex, and a significant proportion of differences in chromatin accessibility might be functionally benign.

  11. Chromatin stiffening underlies enhanced locus mobility after DNA damage in budding yeast.

    Science.gov (United States)

    Herbert, Sébastien; Brion, Alice; Arbona, Jean-Michel; Lelek, Mickaël; Veillet, Adeline; Lelandais, Benoît; Parmar, Jyotsana; Fernández, Fabiola García; Almayrac, Etienne; Khalil, Yasmine; Birgy, Eleonore; Fabre, Emmanuelle; Zimmer, Christophe

    2017-09-01

    DNA double-strand breaks (DSBs) induce a cellular response that involves histone modifications and chromatin remodeling at the damaged site and increases chromosome dynamics both locally at the damaged site and globally in the nucleus. In parallel, it has become clear that the spatial organization and dynamics of chromosomes can be largely explained by the statistical properties of tethered, but randomly moving, polymer chains, characterized mainly by their rigidity and compaction. How these properties of chromatin are affected during DNA damage remains, however, unclear. Here, we use live cell microscopy to track chromatin loci and measure distances between loci on yeast chromosome IV in thousands of cells, in the presence or absence of genotoxic stress. We confirm that DSBs result in enhanced chromatin subdiffusion and show that intrachromosomal distances increase with DNA damage all along the chromosome. Our data can be explained by an increase in chromatin rigidity, but not by chromatin decondensation or centromeric untethering only. We provide evidence that chromatin stiffening is mediated in part by histone H2A phosphorylation. Our results support a genome-wide stiffening of the chromatin fiber as a consequence of DNA damage and as a novel mechanism underlying increased chromatin mobility. © 2017 The Authors.

  12. MRN1 implicates chromatin remodeling complexes and architectural factors in mRNA maturation

    DEFF Research Database (Denmark)

    Düring, Louis; Thorsen, Michael; Petersen, Darima

    2012-01-01

    A functional relationship between chromatin structure and mRNA processing events has been suggested, however, so far only a few involved factors have been characterized. Here we show that rsc nhp6¿¿ mutants, deficient for the function of the chromatin remodeling factor RSC and the chromatin....... Genetic interactions are observed between 2 µm-MRN1 and the splicing deficient mutants snt309¿, prp3, prp4, and prp22, and additional genetic analyses link MRN1, SNT309, NHP6A/B, SWI/SNF, and RSC supporting the notion of a role of chromatin structure in mRNA processing....

  13. Enrichment of HP1a on Drosophila chromosome 4 genes creates an alternate chromatin structure critical for regulation in this heterochromatic domain.

    Directory of Open Access Journals (Sweden)

    Nicole C Riddle

    2012-09-01

    Full Text Available Chromatin environments differ greatly within a eukaryotic genome, depending on expression state, chromosomal location, and nuclear position. In genomic regions characterized by high repeat content and high gene density, chromatin structure must silence transposable elements but permit expression of embedded genes. We have investigated one such region, chromosome 4 of Drosophila melanogaster. Using chromatin-immunoprecipitation followed by microarray (ChIP-chip analysis, we examined enrichment patterns of 20 histone modifications and 25 chromosomal proteins in S2 and BG3 cells, as well as the changes in several marks resulting from mutations in key proteins. Active genes on chromosome 4 are distinct from those in euchromatin or pericentric heterochromatin: while there is a depletion of silencing marks at the transcription start sites (TSSs, HP1a and H3K9me3, but not H3K9me2, are enriched strongly over gene bodies. Intriguingly, genes on chromosome 4 are less frequently associated with paused polymerase. However, when the chromatin is altered by depleting HP1a or POF, the RNA pol II enrichment patterns of many chromosome 4 genes shift, showing a significant decrease over gene bodies but not at TSSs, accompanied by lower expression of those genes. Chromosome 4 genes have a low incidence of TRL/GAGA factor binding sites and a low T(m downstream of the TSS, characteristics that could contribute to a low incidence of RNA polymerase pausing. Our data also indicate that EGG and POF jointly regulate H3K9 methylation and promote HP1a binding over gene bodies, while HP1a targeting and H3K9 methylation are maintained at the repeats by an independent mechanism. The HP1a-enriched, POF-associated chromatin structure over the gene bodies may represent one type of adaptation for genes embedded in repetitive DNA.

  14. 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.

  15. NERD, a plant-specific GW protein, defines an additional RNAi-dependent chromatin-based pathway in Arabidopsis.

    Science.gov (United States)

    Pontier, Dominique; Picart, Claire; Roudier, François; Garcia, Damien; Lahmy, Sylvie; Azevedo, Jacinthe; Alart, Emilie; Laudié, Michèle; Karlowski, Wojciech M; Cooke, Richard; Colot, Vincent; Voinnet, Olivier; Lagrange, Thierry

    2012-10-12

    In Arabidopsis, transcriptional gene silencing (TGS) can be triggered by 24 nt small-interfering RNAs (siRNAs) through the RNA-directed DNA methylation (RdDM) pathway. By functional analysis of NERD, a GW repeat- and PHD finger-containing protein, we demonstrate that Arabidopsis harbors a second siRNA-dependent DNA methylation pathway targeting a subset of nonconserved genomic loci. The activity of the NERD-dependent pathway differs from RdDM by the fact that it relies both on silencing-related factors previously implicated only in posttranscriptional gene silencing (PTGS), including RNA-DEPENDENT RNA POLYMERASE1/6 and ARGONAUTE2, and most likely on 21 nt siRNAs. A central role for NERD in integrating RNA silencing and chromatin signals in transcriptional silencing is supported by data showing that it binds both to histone H3 and AGO2 proteins and contributes to siRNA accumulation at a NERD-targeted locus. Our results unravel the existence of a conserved chromatin-based RNA silencing pathway encompassing both PTGS and TGS components in plants. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Transcriptional and Chromatin Dynamics of Muscle Regeneration after Severe Trauma

    Directory of Open Access Journals (Sweden)

    Carlos A. Aguilar

    2016-11-01

    Full Text Available Following injury, adult skeletal muscle undergoes a well-coordinated sequence of molecular and physiological events to promote repair and regeneration. However, a thorough understanding of the in vivo epigenomic and transcriptional mechanisms that control these reparative events is lacking. To address this, we monitored the in vivo dynamics of three histone modifications and coding and noncoding RNA expression throughout the regenerative process in a mouse model of traumatic muscle injury. We first illustrate how both coding and noncoding RNAs in tissues and sorted satellite cells are modified and regulated during various stages after trauma. Next, we use chromatin immunoprecipitation followed by sequencing to evaluate the chromatin state of cis-regulatory elements (promoters and enhancers and view how these elements evolve and influence various muscle repair and regeneration transcriptional programs. These results provide a comprehensive view of the central factors that regulate muscle regeneration and underscore the multiple levels through which both transcriptional and epigenetic patterns are regulated to enact appropriate repair and regeneration.

  17. Epigenetic Regulation of Chromatin States in Schizosaccharomyces pombe

    Science.gov (United States)

    Allshire, Robin C.; Ekwall, Karl

    2015-01-01

    This article discusses the advances made in epigenetic research using the model organism fission yeast Schizosaccharomyces pombe. S. pombe has been used for epigenetic research since the discovery of position effect variegation (PEV). This is a phenomenon in which a transgene inserted within heterochromatin is variably expressed, but can be stably inherited in subsequent cell generations. PEV occurs at centromeres, telomeres, ribosomal DNA (rDNA) loci, and mating-type regions of S. pombe chromosomes. Heterochromatin assembly in these regions requires enzymes that modify histones and the RNA interference (RNAi) machinery. One of the key histone-modifying enzymes is the lysine methyltransferase Clr4, which methylates histone H3 on lysine 9 (H3K9), a classic hallmark of heterochromatin. The kinetochore is assembled on specialized chromatin in which histone H3 is replaced by the variant CENP-A. Studies in fission yeast have contributed to our understanding of the establishment and maintenance of CENP-A chromatin and the epigenetic activation and inactivation of centromeres. PMID:26134317

  18. Epigenetic Regulation of Chromatin States in Schizosaccharomyces pombe.

    Science.gov (United States)

    Allshire, Robin C; Ekwall, Karl

    2015-07-01

    This article discusses the advances made in epigenetic research using the model organism fission yeast Schizosaccharomyces pombe. S. pombe has been used for epigenetic research since the discovery of position effect variegation (PEV). This is a phenomenon in which a transgene inserted within heterochromatin is variably expressed, but can be stably inherited in subsequent cell generations. PEV occurs at centromeres, telomeres, ribosomal DNA (rDNA) loci, and mating-type regions of S. pombe chromosomes. Heterochromatin assembly in these regions requires enzymes that modify histones and the RNA interference (RNAi) machinery. One of the key histone-modifying enzymes is the lysine methyltransferase Clr4, which methylates histone H3 on lysine 9 (H3K9), a classic hallmark of heterochromatin. The kinetochore is assembled on specialized chromatin in which histone H3 is replaced by the variant CENP-A. Studies in fission yeast have contributed to our understanding of the establishment and maintenance of CENP-A chromatin and the epigenetic activation and inactivation of centromeres. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.

  19. Histone propionylation is a mark of active chromatin.

    Science.gov (United States)

    Kebede, Adam F; Nieborak, Anna; Shahidian, Lara Zorro; Le Gras, Stephanie; Richter, Florian; Gómez, Diana Aguilar; Baltissen, Marijke P; Meszaros, Gergo; Magliarelli, Helena de Fatima; Taudt, Aaron; Margueron, Raphael; Colomé-Tatché, Maria; Ricci, Romeo; Daujat, Sylvain; Vermeulen, Michiel; Mittler, Gerhard; Schneider, Robert

    2017-12-01

    Histones are highly covalently modified, but the functions of many of these modifications remain unknown. In particular, it is unclear how histone marks are coupled to cellular metabolism and how this coupling affects chromatin architecture. We identified histone H3 Lys14 (H3K14) as a site of propionylation and butyrylation in vivo and carried out the first systematic characterization of histone propionylation. We found that H3K14pr and H3K14bu are deposited by histone acetyltransferases, are preferentially enriched at promoters of active genes and are recognized by acylation-state-specific reader proteins. In agreement with these findings, propionyl-CoA was able to stimulate transcription in an in vitro transcription system. Notably, genome-wide H3 acylation profiles were redefined following changes to the metabolic state, and deletion of the metabolic enzyme propionyl-CoA carboxylase altered global histone propionylation levels. We propose that histone propionylation, acetylation and butyrylation may act in combination to promote high transcriptional output and to couple cellular metabolism with chromatin structure and function.

  20. Insulator speckles associated with long-distance chromatin contacts.

    Science.gov (United States)

    Buxa, Melanie K; Slotman, Johan A; van Royen, Martin E; Paul, Maarten W; Houtsmuller, Adriaan B; Renkawitz, Rainer

    2016-09-15

    Nuclear foci of chromatin binding factors are, in many cases, discussed as sites of long-range chromatin interaction in the three-dimensional nuclear space. Insulator binding proteins have been shown to aggregate into insulator bodies, which are large structures not involved in insulation; however, the more diffusely distributed insulator speckles have not been analysed in this respect. Furthermore, insulator binding proteins have been shown to drive binding sites for Polycomb group proteins into Polycomb bodies. Here we find that insulator speckles, marked by the insulator binding protein dCTCF, and Polycomb bodies show differential association with the insulator protein CP190. They differ in number and three-dimensional location with only 26% of the Polycomb bodies overlapping with CP190. By using fluorescence in situ hybridization (FISH) probes to identify long-range interaction (kissing) of the Hox gene clusters Antennapedia complex (ANT-C) and Bithorax complex (BX-C), we found the frequency of interaction to be very low. However, these rare kissing events were associated with insulator speckles at a significantly shorter distance and an increased speckle number. This suggests that insulator speckles are associated with long-distance interaction. © 2016. Published by The Company of Biologists Ltd.

  1. The Role of Chromatin in Adenoviral Vector Function

    Directory of Open Access Journals (Sweden)

    Carmen M. Wong

    2013-06-01

    Full Text Available Vectors based on adenovirus (Ad are one of the most commonly utilized platforms for gene delivery to cells in molecular biology studies and in gene therapy applications. Ad is also the most popular vector system in human clinical gene therapy trials, largely due to its advantageous characteristics such as high cloning capacity (up to 36 kb, ability to infect a wide variety of cell types and tissues, and relative safety due to it remaining episomal in transduced cells. The latest generation of Ad vectors, helper‑dependent Ad (hdAd, which are devoid of all viral protein coding sequences, can mediate high-level expression of a transgene for years in a variety of species ranging from rodents to non-human primates. Given the importance of histones and chromatin in modulating gene expression within the host cell, it is not surprising that Ad, a nuclear virus, also utilizes these proteins to protect the genome and modulate virus- or vector‑encoded genes. In this review, we will discuss our current understanding of the contribution of chromatin to Ad vector function.

  2. 4DGenome: a comprehensive database of chromatin interactions.

    Science.gov (United States)

    Teng, Li; He, Bing; Wang, Jiahui; Tan, Kai

    2015-08-01

    The 3D structure of the genome plays a critical role in regulating gene expression. Recent progress in mapping technologies for chromatin interactions has led to a rapid increase in this kind of interaction data. This trend will continue as research in this burgeoning field intensifies. We describe the 4DGenome database that stores chromatin interaction data compiled through comprehensive literature curation. The database currently covers both low- and high-throughput assays, including 3C, 4C-Seq, 5C, Hi-C, ChIA-PET and Capture-C. To complement the set of interactions detected by experimental assays, we also include interactions predicted by a recently developed computational method with demonstrated high accuracy. The database currently contains ∼8 million records, covering 102 cell/tissue types in five organisms. Records in the database are described using a standardized file format, facilitating data exchange. The vast major of the interactions were assigned a confidence score. Using the web interface, users can query and download database records via a number of annotation dimensions. Query results can be visualized along with other genomics datasets via links to the UCSC genome browser. We anticipate that 4DGenome will be a valuable resource for investigating the spatial structure-and-function relationship of genomes. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  3. Insulator speckles associated with long-distance chromatin contacts

    Science.gov (United States)

    Buxa, Melanie K.; Slotman, Johan A.; van Royen, Martin E.; Paul, Maarten W.; Houtsmuller, Adriaan B.

    2016-01-01

    ABSTRACT Nuclear foci of chromatin binding factors are, in many cases, discussed as sites of long-range chromatin interaction in the three-dimensional nuclear space. Insulator binding proteins have been shown to aggregate into insulator bodies, which are large structures not involved in insulation; however, the more diffusely distributed insulator speckles have not been analysed in this respect. Furthermore, insulator binding proteins have been shown to drive binding sites for Polycomb group proteins into Polycomb bodies. Here we find that insulator speckles, marked by the insulator binding protein dCTCF, and Polycomb bodies show differential association with the insulator protein CP190. They differ in number and three-dimensional location with only 26% of the Polycomb bodies overlapping with CP190. By using fluorescence in situ hybridization (FISH) probes to identify long-range interaction (kissing) of the Hox gene clusters Antennapedia complex (ANT-C) and Bithorax complex (BX-C), we found the frequency of interaction to be very low. However, these rare kissing events were associated with insulator speckles at a significantly shorter distance and an increased speckle number. This suggests that insulator speckles are associated with long-distance interaction. PMID:27464669

  4. Chromatin Structure and Radiation-Induced Intrachromosome Exchange

    Science.gov (United States)

    Mangala; Zhang, Ye; Hada, Megumi; Cucinotta, Francis A.; Wu, Honglu

    2011-01-01

    We have recently investigated the location of breaks involved in intrachromosomal type exchange events, using the multicolor banding in situ hybridization (mBAND) technique for human chromosome 3. In human epithelial cells exposed to both low- and high-LET radiations in vitro, intrachromosome exchanges were found to occur preferentially between a break in the 3p21 and one in the 3q11. Exchanges were also observed between a break in 3p21 and one in 3q26, but few exchanges were observed between breaks in 3q11 and 3q26, even though the two regions were on the same arm of the chromosome. To explore the relationships between intrachromosome exchanges and chromatin structure, we used probes that hybridize the three regions of 3p21, 3q11 and 3q26, and measured the distance between two of the three regions in interphase cells. We further analyzed fragile sites on the chromosome that have been identified in various types of cancers. Our results demonstrated that the distribution of breaks involved in radiation-induced intrachromosome aberrations depends upon both the location of fragile sites and the folding of chromatins

  5. Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin

    Directory of Open Access Journals (Sweden)

    Mora Xavier

    2007-05-01

    Full Text Available Abstract Background Histone H1 is involved in the formation and maintenance of chromatin higher order structure. H1 has multiple isoforms; the subtypes differ in timing of expression, extent of phosphorylation and turnover rate. In vertebrates, the amino acid substitution rates differ among subtypes by almost one order of magnitude, suggesting that each subtype might have acquired a unique function. We have devised a competitive assay to estimate the relative binding affinities of histone H1 mammalian somatic subtypes H1a-e and H1° for long chromatin fragments (30–35 nucleosomes in physiological salt (0.14 M NaCl at constant stoichiometry. Results The H1 complement of native chromatin was perturbed by adding an additional amount of one of the subtypes. A certain amount of SAR (scaffold-associated region DNA was present in the mixture to avoid precipitation of chromatin by excess H1. SAR DNA also provided a set of reference relative affinities, which were needed to estimate the relative affinities of the subtypes for chromatin from the distribution of the subtypes between the SAR and the chromatin. The amounts of chromatin, SAR and additional H1 were adjusted so as to keep the stoichiometry of perturbed chromatin similar to that of native chromatin. H1 molecules freely exchanged between the chromatin and SAR binding sites. In conditions of free exchange, H1a was the subtype of lowest affinity, H1b and H1c had intermediate affinities and H1d, H1e and H1° the highest affinities. Subtype affinities for chromatin differed by up to 19-fold. The relative affinities of the subtypes for chromatin were equivalent to those estimated for a SAR DNA fragment and a pUC19 fragment of similar length. Avian H5 had an affinity ~12-fold higher than H1e for both DNA and chromatin. Conclusion H1 subtypes freely exchange in vitro between chromatin binding sites in physiological salt (0.14 M NaCl. The large differences in relative affinity of the H1 subtypes for

  6. The ''self-stirred'' genome: Bulk and surface dynamics of the chromatin globule

    Science.gov (United States)

    Zidovska, Alexandra

    Chromatin structure and dynamics control all aspects of DNA biology yet are poorly understood. In interphase, time between two cell divisions, chromatin fills the cell nucleus in its minimally condensed polymeric state. Chromatin serves as substrate to a number of biological processes, e.g. gene expression and DNA replication, which require it to become locally restructured. These are energy-consuming processes giving rise to non-equilibrium dynamics. Chromatin dynamics has been traditionally studied by imaging of fluorescently labeled nuclear proteins and single DNA-sites, thus focusing only on a small number of tracer particles. Recently, we developed an approach, displacement correlation spectroscopy (DCS) based on time-resolved image correlation analysis, to map chromatin dynamics simultaneously across the whole nucleus in cultured human cells. DCS revealed that chromatin movement was coherent across large regions (4-5 μm) for several seconds. Regions of coherent motion extended beyond the boundaries of single-chromosome territories, suggesting elastic coupling of motion over length scales much larger than those of genes. These large-scale, coupled motions were ATP-dependent and unidirectional for several seconds. Following these observations, we developed a hydrodynamic theory of active chromatin dynamics, using the two-fluid model and describing the content of cell nucleus as a chromatin solution, which is subject to both passive thermal fluctuations and active (ATP-consuming) scalar and vector events. In this work we continue in our efforts to elucidate the mechanism and function of the chromatin dynamics in interphase. We investigate the chromatin interactions with the nuclear envelope and compare the surface dynamics of the chromatin globule with its bulk dynamics.

  7. Super-resolution imaging reveals distinct chromatin folding for different epigenetic states.

    Science.gov (United States)

    Boettiger, Alistair N; Bintu, Bogdan; Moffitt, Jeffrey R; Wang, Siyuan; Beliveau, Brian J; Fudenberg, Geoffrey; Imakaev, Maxim; Mirny, Leonid A; Wu, Chao-ting; Zhuang, Xiaowei

    2016-01-21

    Metazoan genomes are spatially organized at multiple scales, from packaging of DNA around individual nucleosomes to segregation of whole chromosomes into distinct territories. At the intermediate scale of kilobases to megabases, which encompasses the sizes of genes, gene clusters and regulatory domains, the three-dimensional (3D) organization of DNA is implicated in multiple gene regulatory mechanisms, but understanding this organization remains a challenge. At this scale, the genome is partitioned into domains of different epigenetic states that are essential for regulating gene expression. Here we investigate the 3D organization of chromatin in different epigenetic states using super-resolution imaging. We classified genomic domains in Drosophila cells into transcriptionally active, inactive or Polycomb-repressed states, and observed distinct chromatin organizations for each state. All three types of chromatin domains exhibit power-law scaling between their physical sizes in 3D and their domain lengths, but each type has a distinct scaling exponent. Polycomb-repressed domains show the densest packing and most intriguing chromatin folding behaviour, in which chromatin packing density increases with domain length. Distinct from the self-similar organization displayed by transcriptionally active and inactive chromatin, the Polycomb-repressed domains are characterized by a high degree of chromatin intermixing within the domain. Moreover, compared to inactive domains, Polycomb-repressed domains spatially exclude neighbouring active chromatin to a much stronger degree. Computational modelling and knockdown experiments suggest that reversible chromatin interactions mediated by Polycomb-group proteins play an important role in these unique packaging properties of the repressed chromatin. Taken together, our super-resolution images reveal distinct chromatin packaging for different epigenetic states at the kilobase-to-megabase scale, a length scale that is directly

  8. Non-farnesylated B-type lamin can tether chromatin inside the nucleus and its chromatin interaction requires the Ig-fold region.

    Science.gov (United States)

    Uchino, Ryo; Sugiyama, Shin; Katagiri, Motoi; Chuman, Yoshiro; Furukawa, Kazuhiro

    2017-02-01

    Lamins are thought to direct heterochromatin to the nuclear lamina (NL); however, this function of lamin has not been clearly demonstrated in vivo. To address this, we analyzed polytene chromosome morphology when artificial lamin variants were expressed in Drosophila endoreplicating cells. We found that the CaaX-motif-deleted B-type lamin Dm0, but not A-type lamin C, was able to form a nuclear envelope-independent layer that was closely associated with chromatin. Other nuclear envelope proteins were not detected in this "ectopic lamina," and the associated chromatin showed a repressive histone modification maker but not a permissive histone modification marker nor RNA polymerase II proteins. Furthermore, deletion of the C-terminal lamin-Ig-fold domain prevents chromatin association with this ectopic lamina. Thus, non-farnesylated B-type lamin Dm0 can form an ectopic lamina and induce changes to chromatin structure and status inside the interphase nucleus.

  9. Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Joseph Landry

    2008-10-01

    Full Text Available We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor, the largest subunit of NURF (Nucleosome Remodeling Factor in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf(-/- embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf(-/- embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

  10. The dynamics of individual nucleosomes controls the chromatin condensation pathway: direct atomic force microscopy visualization of variant chromatin.

    Science.gov (United States)

    Montel, Fabien; Menoni, Hervé; Castelnovo, Martin; Bednar, Jan; Dimitrov, Stefan; Angelov, Dimitar; Faivre-Moskalenko, Cendrine

    2009-07-22

    Chromatin organization and dynamics is studied at scales ranging from single nucleosome to nucleosomal array by using a unique combination of biochemical assays, single molecule imaging technique, and numerical modeling. We show that a subtle modification in the nucleosome structure induced by the histone variant H2A.Bbd drastically modifies the higher order organization of the nucleosomal arrays. Importantly, as directly visualized by atomic force microscopy, conventional H2A nucleosomal arrays exhibit specific local organization, in contrast to H2A.Bbd arrays, which show "beads on a string" structure. The combination of systematic image analysis and theoretical modeling allows a quantitative description relating the observed gross structural changes of the arrays to their local organization. Our results suggest strongly that higher-order organization of H1-free nucleosomal arrays is determined mainly by the fluctuation properties of individual nucleosomes. Moreover, numerical simulations suggest the existence of attractive interactions between nucleosomes to provide the degree of compaction observed for conventional chromatin fibers.

  11. The Sea Urchin sns5 Chromatin Insulator Shapes the Chromatin Architecture of a Lentivirus Vector Integrated in the Mammalian Genome.

    Science.gov (United States)

    Baiamonte, Elena; Spinelli, Giovanni; Maggio, Aurelio; Acuto, Santina; Cavalieri, Vincenzo

    2016-10-01

    Lentivirus vectors are presently the favorite vehicles for therapeutic gene transfer in hematopoietic cells. Nonetheless, these vectors integrate randomly throughout the genome, exhibiting variegation of transgene expression due to the spreading of heterochromatin into the vector sequences. Moreover, the cis-regulatory elements harbored by the vector could disturb the proper transcription of resident genes neighboring the integration site. The incorporation of chromatin insulators in flanking position to the transferred unit can alleviate both the above-mentioned dangerous effects, due to the insulator-specific barrier and enhancer-blocking activities. In this study, we report the valuable properties of the sea urchin-derived sns5 insulator in improving the expression efficiency of a lentivirus vector integrated in the mammalian erythroid genome. We show that these results neither reflect an intrinsic sns5 enhancer activity nor rely on the recruitment of the erythroid-specific GATA-1 factor to sns5. Furthermore, by using the Chromosome Conformation Capture technology, we report that a single copy of the sns5-insulated vector is specifically organized into an independent chromatin loop at the provirus locus. Our results not only provide new clues concerning the molecular mechanism of sns5 function in the erythroid genome but also reassure the use of sns5 to improve the performance of gene therapy vectors.

  12. Introducing enteral feeding induces intestinal subclinical inflammation and respective chromatin changes in preterm pigs

    DEFF Research Database (Denmark)

    Willems, Rhea; Krych, Lukasz; Rybicki, Verena

    2015-01-01

    AIM: To analyze how enteral food introduction affects intestinal gene regulation and chromatin structure in preterm pigs. MATERIALS & METHODS: Preterm pigs were fed parenteral nutrition plus/minus slowly increasing volumes of enteral nutrition. Intestinal gene-expression and chromatin structure w...

  13. Assembly of Two Transgenes in an Artificial Chromatin Domain Gives Highly Coordinated Expression in Tobacco

    NARCIS (Netherlands)

    Mlynárová, Ludmila; Loonen, Annelies; Mietkiewska, Elzbieta; Jansen, Ritsert C.; Nap, Jan-Peter

    The chromatin loop model predicts that genes within the same chromatin domain exhibit coordinated regulation. We here present the first direct experimental support for this model in plants. Two reporter genes, the E. coli β-glucuronidase gene and the firefly luciferase gene, driven by different

  14. Modern human sperm freezing: Effect on DNA, chromatin and acrosome integrity

    Directory of Open Access Journals (Sweden)

    Tahereh Rahiminia

    2017-08-01

    Conclusion: Sperm in Vapour was healthier in terms of DNA, chromatin and acrosome integrity. In contrast of higher motility and normal morphology; DNA, chromatin and acrosome integrity were decreased in Vit. However, these findings were more acceptable in SSV or Vapour.

  15. A Model of Repetitive-DNA-Organized Chromatin Network of Interphase Chromosomes

    Directory of Open Access Journals (Sweden)

    Shao-Jun Tang

    2012-03-01

    Full Text Available During interphase, chromosomes are relatively de-condensed in the nuclear space. Interphase chromosomes are known to occupy nuclear space in a non-random manner (chromosome territory; however, their internal structures are poorly defined. In particular, little is understood about the molecular mechanisms that govern the internal organization of interphase chromosomes. The author recently proposed that pairing (or interaction of repetitive DNA-containing chromatin regions is a critical driving force that specifies the higher-order organization of eukaryotic chromosomes. Guided by this theoretical framework and published experimental data on the structure of interphase chromosomes and the spatial distribution of repetitive DNA in interphase nuclei, I postulate here a molecular structure of chromatin organization in interphase chromosomes. According to this model, an interphase chromosome is a chromatin mesh (or lattice that is formed by repeat pairing (RP. The mesh consists of two types of structural components: chromosome nodes and loose chromatin fibers. Chromosome nodes are DNA repeat assemblies (RAs that are formed via RP, while loose fibers include chromatin loops that radiate from the nodes. Different loops crosslink by RPs and form a large integrated chromatin network. I suggest that the organization of the chromatin network of a given interphase chromosome is intrinsically specified by the distribution of repetitive DNA elements on the linear chromatin. The stability of the organization is governed by the collection of RA-formed nodes, and the dynamics of the organization is driven by the assembling and disassembling of the nodes.

  16. Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping.

    Science.gov (United States)

    Risca, Viviana I; Denny, Sarah K; Straight, Aaron F; Greenleaf, William J

    2017-01-12

    Chromatin structure at the length scale encompassing local nucleosome-nucleosome interactions is thought to play a crucial role in regulating transcription and access to DNA. However, this secondary structure of chromatin remains poorly understood compared with the primary structure of single nucleosomes or the tertiary structure of long-range looping interactions. Here we report the first genome-wide map of chromatin conformation in human cells at the 1-3 nucleosome (50-500 bp) scale, obtained using ionizing radiation-induced spatially correlated cleavage of DNA with sequencing (RICC-seq) to identify DNA-DNA contacts that are spatially proximal. Unbiased analysis of RICC-seq signal reveals regional enrichment of DNA fragments characteristic of alternating rather than adjacent nucleosome interactions in tri-nucleosome units, particularly in H3K9me3-marked heterochromatin. We infer differences in the likelihood of nucleosome-nucleosome contacts among open chromatin, H3K27me3-marked, and H3K9me3-marked repressed chromatin regions. After calibrating RICC-seq signal to three-dimensional distances, we show that compact two-start helical fibre structures with stacked alternating nucleosomes are consistent with RICC-seq fragmentation patterns from H3K9me3-marked chromatin, while non-compact structures and solenoid structures are consistent with open chromatin. Our data support a model of chromatin architecture in intact interphase nuclei consistent with variable longitudinal compaction of two-start helical fibres.

  17. Multiple interactions between regulatory regions are required to stabilize an active chromatin hub.

    NARCIS (Netherlands)

    G.P. Patrinos (George); M. de Krom (Mariken); E. de Boer (Ernie); A. Langeveld (An); A.M.A. Imam (Ali); J. Strouboulis (John); W.L. de Laat (Wouter); F.G. Grosveld (Frank)

    2004-01-01

    textabstractThe human beta-globin locus control region (LCR) is required for the maintenance of an open chromatin configuration of the locus. It interacts with the genes and the hypersensitive regions flanking the locus to form an active chromatin hub (ACH) transcribing the genes. Proper

  18. Temporal profiling of the chromatin proteome reveals system-wide responses to replication inhibition

    DEFF Research Database (Denmark)

    Khoudoli, Guennadi A; Gillespie, Peter J; Stewart, Graeme

    2008-01-01

    Although the replication, expression, and maintenance of DNA are well-studied processes, the way that they are coordinated is poorly understood. Here, we report an analysis of the changing association of proteins with chromatin (the chromatin proteome) during progression through interphase of the...

  19. Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

    Science.gov (United States)

    Kurat, Christoph F; Yeeles, Joseph T P; Patel, Harshil; Early, Anne; Diffley, John F X

    2017-01-05

    The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Chd1 remodelers maintain open chromatin and regulate the epigenetics of differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Persson, Jenna [Department of Biosciences and Nutrition, Center for Biosciences, Karolinska Institutet (Sweden); Ekwall, Karl, E-mail: karl.ekwall@ki.se [Department of Biosciences and Nutrition, Center for Biosciences, Karolinska Institutet (Sweden); School of Life Sciences, University College Sodertorn, NOVUM, Huddinge (Sweden)

    2010-05-01

    Eukaryotic DNA is packaged around octamers of histone proteins into nucleosomes, the basic unit of chromatin. In addition to enabling meters of DNA to fit within the confines of a nucleus, the structure of chromatin has functional implications for cell identity. Covalent chemical modifications to the DNA and to histones, histone variants, ATP-dependent chromatin remodelers, small noncoding RNAs and the level of chromatin compaction all contribute to chromosomal structure and to the activity or silencing of genes. These chromatin-level alterations are defined as epigenetic when they are heritable from mother to daughter cell. The great diversity of epigenomes that can arise from a single genome permits a single, totipotent cell to generate the hundreds of distinct cell types found in humans. Two recent studies in mouse and in fly have highlighted the importance of Chd1 chromatin remodelers for maintaining an open, active chromatin state. Based on evidence from fission yeast as a model system, we speculate that Chd1 remodelers are involved in the disassembly of nucleosomes at promoter regions, thus promoting active transcription and open chromatin. It is likely that these nucleosomes are specifically marked for disassembly by the histone variant H2A.Z.

  1. 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...

  2. Hi-C Chromatin Interaction Networks Predict Co-expression in the Mouse Cortex

    Science.gov (United States)

    Hulsman, Marc; Lelieveldt, Boudewijn P. F.; de Ridder, Jeroen; Reinders, Marcel

    2015-01-01

    The three dimensional conformation of the genome in the cell nucleus influences important biological processes such as gene expression regulation. Recent studies have shown a strong correlation between chromatin interactions and gene co-expression. However, predicting gene co-expression from frequent long-range chromatin interactions remains challenging. We address this by characterizing the topology of the cortical chromatin interaction network using scale-aware topological measures. We demonstrate that based on these characterizations it is possible to accurately predict spatial co-expression between genes in the mouse cortex. Consistent with previous findings, we find that the chromatin interaction profile of a gene-pair is a good predictor of their spatial co-expression. However, the accuracy of the prediction can be substantially improved when chromatin interactions are described using scale-aware topological measures of the multi-resolution chromatin interaction network. We conclude that, for co-expression prediction, it is necessary to take into account different levels of chromatin interactions ranging from direct interaction between genes (i.e. small-scale) to chromatin compartment interactions (i.e. large-scale). PMID:25965262

  3. The condensed chromatin fiber: an allosteric chemo-mechanical machine for signal transduction and genome processing

    Science.gov (United States)

    Lesne, Annick; Bécavin, Christophe; Victor, Jean–Marc

    2012-02-01

    Allostery is a key concept of molecular biology which refers to the control of an enzyme activity by an effector molecule binding the enzyme at another site rather than the active site (allos = other in Greek). We revisit here allostery in the context of chromatin and argue that allosteric principles underlie and explain the functional architecture required for spacetime coordination of gene expression at all scales from DNA to the whole chromosome. We further suggest that this functional architecture is provided by the chromatin fiber itself. The structural, mechanical and topological features of the chromatin fiber endow chromosomes with a tunable signal transduction from specific (or nonspecific) effectors to specific (or nonspecific) active sites. Mechanical constraints can travel along the fiber all the better since the fiber is more compact and regular, which speaks in favor of the actual existence of the (so-called 30 nm) chromatin fiber. Chromatin fiber allostery reconciles both the physical and biochemical approaches of chromatin. We illustrate this view with two supporting specific examples. Moreover, from a methodological point of view, we suggest that the notion of chromatin fiber allostery is particularly relevant for systemic approaches. Finally we discuss the evolutionary power of allostery in the context of chromatin and its relation to modularity.

  4. Inferring the physical properties of yeast chromatin through Bayesian analysis of whole nucleus simulations.

    Science.gov (United States)

    Arbona, Jean-Michel; Herbert, Sébastien; Fabre, Emmanuelle; Zimmer, Christophe

    2017-05-03

    The structure and mechanical properties of chromatin impact DNA functions and nuclear architecture but remain poorly understood. In budding yeast, a simple polymer model with minimal sequence-specific constraints and a small number of structural parameters can explain diverse experimental data on nuclear architecture. However, how assumed chromatin properties affect model predictions was not previously systematically investigated. We used hundreds of dynamic chromosome simulations and Bayesian inference to determine chromatin properties consistent with an extensive dataset that includes hundreds of measurements from imaging in fixed and live cells and two Hi-C studies. We place new constraints on average chromatin fiber properties, narrowing down the chromatin compaction to ~53-65 bp/nm and persistence length to ~52-85 nm. These constraints argue against a 20-30 nm fiber as the exclusive chromatin structure in the genome. Our best model provides a much better match to experimental measurements of nuclear architecture and also recapitulates chromatin dynamics measured on multiple loci over long timescales. This work substantially improves our understanding of yeast chromatin mechanics and chromosome architecture and provides a new analytic framework to infer chromosome properties in other organisms.

  5. A Testis-Specific Chaperone and the Chromatin Remodeler ISWI Mediate Repackaging of the Paternal Genome

    NARCIS (Netherlands)

    C.M. Doyen (Cécile); G.E. Chalkley (Gillian); O. Voets (Olaf); K. Bezstarosti (Karel); J.A.A. Demmers (Jeroen); Y.M. Moshkin (Yuri); C.P. Verrijzer (Peter)

    2015-01-01

    textabstractDuring spermatogenesis, the paternal genome is repackaged into a non-nucleosomal, highly compacted chromatin structure. Bioinformatic analysis revealed that Drosophila sperm chromatin proteins are characterized by a motif related to the high-mobility group (HMG) box, which we termed

  6. Prediction of highly expressed genes in microbes based on chromatin accessibility

    DEFF Research Database (Denmark)

    Willenbrock, Hanni; Ussery, David

    2007-01-01

    BACKGROUND: It is well known that gene expression is dependent on chromatin structure in eukaryotes and it is likely that chromatin can play a role in bacterial gene expression as well. Here, we use a nucleosomal position preference measure of anisotropic DNA flexibility to predict highly expressed...

  7. Reading the maps: Organization and function of chromatin types in Drosophila

    NARCIS (Netherlands)

    Braunschweig, U.

    2010-01-01

    The work presented in this thesis shows that the Drosophila genome is organized in chromatin domains with many implications for gene regulation, nuclear organization, and evolution. Furthermore it provides examples of how maps of chromatin protein binding, combined with computational approaches, can

  8. High-throughput assessment of context-dependent effects of chromatin proteins

    NARCIS (Netherlands)

    Brueckner, L. (Laura); Van Arensbergen, J. (Joris); Akhtar, W. (Waseem); L. Pagie (Ludo); B. van Steensel (Bas)

    2016-01-01

    textabstractBackground: Chromatin proteins control gene activity in a concerted manner. We developed a high-throughput assay to study the effects of the local chromatin environment on the regulatory activity of a protein of interest. The assay combines a previously reported multiplexing strategy

  9. ZMYM3 regulates BRCA1 localization at damaged chromatin to promote DNA repair.

    Science.gov (United States)

    Leung, Justin W C; Makharashvili, Nodar; Agarwal, Poonam; Chiu, Li-Ya; Pourpre, Renaud; Cammarata, Michael B; Cannon, Joe R; Sherker, Alana; Durocher, Daniel; Brodbelt, Jennifer S; Paull, Tanya T; Miller, Kyle M

    2017-02-01

    Chromatin connects DNA damage response factors to sites of damaged DNA to promote the signaling and repair of DNA lesions. The histone H2A variants H2AX, H2AZ, and macroH2A represent key chromatin constituents that facilitate DNA repair. Through proteomic screening of these variants, we identified ZMYM3 (zinc finger, myeloproliferative, and mental retardation-type 3) as a chromatin-interacting protein that promotes DNA repair by homologous recombination (HR). ZMYM3 is recruited to DNA double-strand breaks through bivalent interactions with both histone and DNA components of the nucleosome. We show that ZMYM3 links the HR factor BRCA1 to damaged chromatin through specific interactions with components of the BRCA1-A subcomplex, including ABRA1 and RAP80. By regulating ABRA1 recruitment to damaged chromatin, ZMYM3 facilitates the fine-tuning of BRCA1 interactions with DNA damage sites and chromatin. Consistent with a role in regulating BRCA1 function, ZMYM3 deficiency results in impaired HR repair and genome instability. Thus, our work identifies a critical chromatin-binding DNA damage response factor, ZMYM3, which modulates BRCA1 functions within chromatin to ensure the maintenance of genome integrity. © 2017 Leung et al.; Published by Cold Spring Harbor Laboratory Press.

  10. Two new insulator proteins, Pita and ZIPIC, target CP190 to chromatin.

    Science.gov (United States)

    Maksimenko, Oksana; Bartkuhn, Marek; Stakhov, Viacheslav; Herold, Martin; Zolotarev, Nickolay; Jox, Theresa; Buxa, Melanie K; Kirsch, Ramona; Bonchuk, Artem; Fedotova, Anna; Kyrchanova, Olga; Renkawitz, Rainer; Georgiev, Pavel

    2015-01-01

    Insulators are multiprotein-DNA complexes that regulate the nuclear architecture. The Drosophila CP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. We have identified two DNA-binding zinc-finger proteins, Pita, and a new factor named ZIPIC, that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCP insulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins. © 2015 Maksimenko et al.; Published by Cold Spring Harbor Laboratory Press.

  11. Two new insulator proteins, Pita and ZIPIC, target CP190 to chromatin

    Science.gov (United States)

    Maksimenko, Oksana; Bartkuhn, Marek; Stakhov, Viacheslav; Herold, Martin; Zolotarev, Nickolay; Jox, Theresa; Buxa, Melanie K.; Kirsch, Ramona; Bonchuk, Artem; Fedotova, Anna; Kyrchanova, Olga

    2015-01-01

    Insulators are multiprotein–DNA complexes that regulate the nuclear architecture. The Drosophila CP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. We have identified two DNA-binding zinc-finger proteins, Pita, and a new factor named ZIPIC, that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCP insulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins. PMID:25342723

  12. Global alterations of the transcriptional landscape during yeast growth and development in the absence of Ume6-dependent chromatin modification.

    Science.gov (United States)

    Lardenois, Aurélie; Becker, Emmanuelle; Walther, Thomas; Law, Michael J; Xie, Bingning; Demougin, Philippe; Strich, Randy; Primig, Michael

    2015-10-01

    Chromatin modification enzymes are important regulators of gene expression and some are evolutionarily conserved from yeast to human. Saccharomyces cerevisiae is a major model organism for genome-wide studies that aim at the identification of target genes under the control of conserved epigenetic regulators. Ume6 interacts with the upstream repressor site 1 (URS1) and represses transcription by recruiting both the conserved histone deacetylase Rpd3 (through the co-repressor Sin3) and the chromatin-remodeling factor Isw2. Cells lacking Ume6 are defective in growth, stress response, and meiotic development. RNA profiling studies and in vivo protein-DNA binding assays identified mRNAs or transcript isoforms that are directly repressed by Ume6 in mitosis. However, a comprehensive understanding of the transcriptional alterations, which underlie the complex ume6Δ mutant phenotype during fermentation, respiration, or sporulation, is lacking. We report the protein-coding transcriptome of a diploid MAT a/α wild-type and ume6/ume6 mutant strains cultured in rich media with glucose or acetate as a carbon source, or sporulation-inducing medium. We distinguished direct from indirect effects on mRNA levels by combining GeneChip data with URS1 motif predictions and published high-throughput in vivo Ume6-DNA binding data. To gain insight into the molecular interactions between successive waves of Ume6-dependent meiotic genes, we integrated expression data with information on protein networks. Our work identifies novel Ume6 repressed genes during growth and development and reveals a strong effect of the carbon source on the derepression pattern of transcripts in growing and developmentally arrested ume6/ume6 mutant cells. Since yeast is a useful model organism for chromatin-mediated effects on gene expression, our results provide a rich source for further genetic and molecular biological work on the regulation of cell growth and cell differentiation in eukaryotes.

  13. The JAZF1-SUZ12 fusion protein disrupts PRC2 complexes and impairs chromatin repression during human endometrial stromal tumorogenesis.

    Science.gov (United States)

    Ma, Xianyong; Wang, Jinglan; Wang, Jianhui; Ma, Charles X; Gao, Xiaobin; Patriub, Vytas; Sklar, Jeffrey L

    2017-01-17

    The Polycomb repressive complex 2 (PRC2), which contains three core proteins EZH2, EED and SUZ12, controls chromatin compaction and transcription repression through trimethylation of lysine 27 on histone 3. The (7;17)(p15;q21) chromosomal translocation present in most cases of endometrial stromal sarcomas (ESSs) results in the in-frame fusion of the JAZF1 and SUZ12 genes. We have investigated whether and how the fusion protein JAZF1-SUZ12 functionally alters PRC2. We found that the fusion protein exists at high levels in ESS containing the t(7;17). Co-transient transfection assay indicated JAZF1-SUZ12 destabilized PRC2 components EZH2 and EED, resulting in decreased histone methyl transferase (HMT) activity, which was confirmed by in vitro studies using reconstituted PRC2 and nucleosome array substrates. We also demonstrated the PRC2 containing the fusion protein decreased the binding affinity to target chromatin loci. In addition, we found that trimethylation of H3K27 was decreased in ESS samples with the t(7;17), but there was no detectable change in H3K9 in these tissues. Moreover, re-expression of SUZ12 in Suz12 (-/-) ES cells rescued the neuronal differentiation while the fusion protein failed to restore this function and enhanced cell proliferation. In summary, our studies reveal that JAZF1-SUZ12 fusion protein disrupts the PRC2 complex, abolishes HMT activity and subsequently activates chromatin/genes normally repressed by PRC2. Such dyesfunction of PRC2 inhibits normal neural differentiation of ES cell and increases cell proliferation. Related changes induced by the JAZF-SUZ12 protein in endometrial stromal cells may explain the oncogenic effect of the t(7;17) in ESS.

  14. Condensin-mediated remodeling of the mitotic chromatin landscape in fission yeast.

    Science.gov (United States)

    Kakui, Yasutaka; Rabinowitz, Adam; Barry, David J; Uhlmann, Frank

    2017-10-01

    The eukaryotic genome consists of DNA molecules far longer than the cells that contain them. They reach their greatest compaction during chromosome condensation in mitosis. This process is aided by condensin, a structural maintenance of chromosomes (SMC) family member. The spatial organization of mitotic chromosomes and how condensin shapes chromatin architecture are not yet fully understood. Here we use chromosome conformation capture (Hi-C) to study mitotic chromosome condensation in the fission yeast Schizosaccharomyces pombe. This showed that the interphase landscape characterized by small chromatin domains is replaced by fewer but larger domains in mitosis. Condensin achieves this by setting up longer-range, intrachromosomal DNA interactions, which compact and individualize chromosomes. At the same time, local chromatin contacts are constrained by condensin, with profound implications for local chromatin function during mitosis. Our results highlight condensin as a major determinant that changes the chromatin landscape as cells prepare their genomes for cell division.

  15. Identification and characterization of nonhistone chromatin proteins: human positive coactivator 4 as a candidate.

    Science.gov (United States)

    Kumari, Sujata; Das, Chandrima; Sikder, Sweta; Kumar, Manoj; Bachu, Mahesh; Ranga, Udaykumar; Kundu, Tapas K

    2015-01-01

    The highly dynamic nucleoprotein structure of eukaryotic genome is organized in an ordered fashion, the unit of which is the nucleosome. The nucleosome is composed of core histones and DNA of variable size wrapped around it. Apart from the histone proteins, several nonhistone proteins also interact with the complex consisting of the DNA, the core and linker histones conferring highly regulated fluidity on the chromatin and permitting fine tuning of its functions. The nonhistone proteins are multifunctional and accentuate diverse cellular outcomes. In spite of the technical challenges, the architectural role of the nonhistone proteins altering the topology of the chromatin has been studied extensively. To appreciate the significance of the chromatin for genome function, it is essential to examine the role of the nonhistone proteins in different physiological conditions. Here, taking the example of a highly abundant chromatin protein, PC4 (Positive coactivator 4), we describe strategies for the identification of the chromatin-associated proteins and their structural and functional characterization.

  16. Chromatin Configuration Determines Cell Responses to Hormone Stimuli | Center for Cancer Research

    Science.gov (United States)

    Ever since selective gene expression was established as the central driver of cell behavior, researchers have been working to understand the forces that control gene transcription. Aberrant gene expression can cause or promote many diseases, including cancer, and alterations in gene expression are the goal of many therapeutic agents. Recent work has focused on the potential role of chromatin structure as a contributor to gene regulation. Chromatin can exist in a tightly packed/inaccessible or loose/accessible configuration depending on the interactions between DNA and its associated proteins. Patterns of chromatin structure can differ between cell types and can also change within cells in response to certain signals. Cancer researchers are particularly interested in the role of chromatin in gene regulation because many of the genomic regions found to be associated with cancer risk are in open chromatin structures.

  17. Epigenetic crosstalk: Pharmacological inhibition of HDACs can rescue defective synaptic morphology and neurotransmission phenotypes associated with loss of the chromatin reader Kismet.

    Science.gov (United States)

    Latcheva, Nina K; Viveiros, Jennifer M; Waddell, Edward A; Nguyen, Phuong T T; Liebl, Faith L W; Marenda, Daniel R

    2018-03-01

    We are beginning to appreciate the complex mechanisms by which epigenetic proteins control chromatin dynamics to tightly regulate normal development. However, the interaction between these proteins, particularly in the context of neuronal function, remains poorly understood. Here, we demonstrate that the activity of histone deacetylases (HDACs) opposes that of a chromatin remodeling enzyme at the Drosophila neuromuscular junction (NMJ). Pharmacological inhibition of HDAC function reverses loss of function phenotypes associated with Kismet, a chromodomain helicase DNA-binding (CHD) protein. Inhibition of HDACs suppresses motor deficits, overgrowth of the NMJ, and defective neurotransmission associated with loss of Kismet. We hypothesize that Kismet and HDACs may converge on a similar set of target genes in the nervous system. Our results provide further understanding into the complex interactions between epigenetic protein function in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Low Dose Radiation-Induced Genome and Epigenome Instability Symposium and Epigenetic Mechanisms, DNA Repair, and Chromatin Symposium at the EMS 2008 Annual Meeting - October 2008

    Energy Technology Data Exchange (ETDEWEB)

    Morgan, William F; Kovalchuk, Olga; Dolinoy, Dana C; Dubrova, Yuri E; Coleman, Matthew A; Schär, Primo; Pogribny, Igor; Hendzel, Michael

    2010-02-19

    The Low Dose Radiation Symposium thoughtfully addressed ionizing radiation non-mutational but transmissable alterations in surviving cells. Deregulation of epigenetic processes has been strongly implicated in carcinogenesis, and there is increasing realization that a significant fraction of non-targeted and adaptive mechanisms in response to ionizing radiation are likely to be epigenetic in nature. Much remains to be learned about how chromatin and epigenetic regulators affect responses to low doses of radiation, and how low dose radiation impacts other epigenetic processes. The Epigenetic Mechanisms Symposium focused on on epigenetic mechanisms and their interplay with DNA repair and chromatin changes. Addressing the fact that the most well understood mediators of epigenetic regulation are histone modifications and DNA methylation. Low levels of radiation can lead to changes in the methylation status of certain gene promoters and the expression of DNA methyltransferases, However, epigenetic regulation can also involve changes in higher order chromosome structure.

  19. Time-resolved spectroscopy and fluorescence resonance energy transfer in the study of excimer laser damage of chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Radu, L. [Department of Molecular Genetics and Radiobiology, Babes National Institute, Bucharest (Romania)], E-mail: lilianajradu@yahoo.fr; Mihailescu, I. [Department of Lasers, Laser, Plasma and Radiation Physics Institute, Bucharest (Romania); Radu, S. [Department of Computer Science, Polytechnics University, Bucharest (Romania); Gazdaru, D. [Department of Biophysics, Bucharest University (Romania)

    2007-09-21

    The analysis of chromatin damage produced by a 248 nm excimer laser radiation, for doses of 0.3-3 MJ/m{sup 2} was carried out by time-resolved spectroscopy and fluorescence resonance energy transfer (FRET). The chromatin was extracted from a normal and a tumoral tissue of Wistar rats. The decrease with laser dose of the relative contribution of the excited state lifetimes of ethidium bromide (EtBr) bounded to chromatin constitutes an evidence of the reduction of chromatin deoxyribonucleic acid (DNA) double-strand structure. FRET was performed from dansyl chloride to acridine orange, both coupled to chromatin. The increase of the average distance between these ligands, under the action of laser radiation, reflects a loosening of the chromatin structure. The radiosensitivity of tumor tissue chromatin is higher than that of a normal tissue. The determination of the chromatin structure modification in an excimer laser field can be of interest in laser therapy.

  20. Snake-like chromatin in conjunctival cells of a population aged 30-60 years from Copenhagen City

    DEFF Research Database (Denmark)

    Bjerrum, Kirsten Birgitte

    1998-01-01

    ophthalmology, keratoconjunctivitis sicca, Sjögrens Syndrome, epidemiology, imprint biopsy, snake-like chromatin......ophthalmology, keratoconjunctivitis sicca, Sjögrens Syndrome, epidemiology, imprint biopsy, snake-like chromatin...

  1. C/EBP maintains chromatin accessibility in liver and facilitates glucocorticoid receptor recruitment to steroid response elements

    DEFF Research Database (Denmark)

    Grøntved, Lars; John, Sam; Baek, Songjoon

    2013-01-01

    Mechanisms regulating transcription factor interaction with chromatin in intact mammalian tissues are poorly understood. Exploiting an adrenalectomized mouse model with depleted endogenous glucocorticoids, we monitor changes of the chromatin landscape in intact liver tissue following glucocortico...

  2. Cytology of DNA Replication Reveals Dynamic Plasticity of Large-Scale Chromatin Fibers.

    Science.gov (United States)

    Deng, Xiang; Zhironkina, Oxana A; Cherepanynets, Varvara D; Strelkova, Olga S; Kireev, Igor I; Belmont, Andrew S

    2016-09-26

    In higher eukaryotic interphase nuclei, the 100- to >1,000-fold linear compaction of chromatin is difficult to reconcile with its function as a template for transcription, replication, and repair. It is challenging to imagine how DNA and RNA polymerases with their associated molecular machinery would move along the DNA template without transient decondensation of observed large-scale chromatin "chromonema" fibers [1]. Transcription or "replication factory" models [2], in which polymerases remain fixed while DNA is reeled through, are similarly difficult to conceptualize without transient decondensation of these chromonema fibers. Here, we show how a dynamic plasticity of chromatin folding within large-scale chromatin fibers allows DNA replication to take place without significant changes in the global large-scale chromatin compaction or shape of these large-scale chromatin fibers. Time-lapse imaging of lac-operator-tagged chromosome regions shows no major change in the overall compaction of these chromosome regions during their DNA replication. Improved pulse-chase labeling of endogenous interphase chromosomes yields a model in which the global compaction and shape of large-Mbp chromatin domains remains largely invariant during DNA replication, with DNA within these domains undergoing significant movements and redistribution as they move into and then out of adjacent replication foci. In contrast to hierarchical folding models, this dynamic plasticity of large-scale chromatin organization explains how localized changes in DNA topology allow DNA replication to take place without an accompanying global unfolding of large-scale chromatin fibers while suggesting a possible mechanism for maintaining epigenetic programming of large-scale chromatin domains throughout DNA replication. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Interplay between chromatin state, regulator binding, and regulatory motifs in six human cell types

    Science.gov (United States)

    Ernst, Jason; Kellis, Manolis

    2013-01-01

    The regions bound by sequence-specific transcription factors can be highly variable across different cell types despite the static nature of the underlying genome sequence. This has been partly attributed to changes in chromatin accessibility, but a systematic picture has been hindered by the lack of large-scale data sets. Here, we use 456 binding experiments for 119 regulators and 84 chromatin maps generated by the ENCODE in six human cell types, and relate those to a global map of regulatory motif instances for these factors. We find specific and robust chromatin state preferences for each regulator beyond the previously reported open-chromatin association, suggesting a much richer chromatin landscape beyond simple accessibility. The preferentially bound chromatin states of regulators were enriched for sequence motifs of regulators relative to all states, suggesting that these preferences are at least partly encoded by the genomic sequence. Relative to all regions bound by a regulator, however, regulatory motifs were surprisingly depleted in the regulator's preferentially bound states, suggesting additional non-sequence-specific binding beyond the level predicted by the regulatory motifs. Such permissive binding was largely restricted to open-chromatin regions showing histone modification marks characteristic of active enhancer and promoter regions, whereas open-chromatin regions lacking such marks did not show permissive binding. Lastly, the vast majority of cobinding of regulator pairs is predicted by the chromatin state preferences of individual regulators. Overall, our results suggest a joint role of sequence motifs and specific chromatin states beyond mere accessibility in mediating regulator binding dynamics across different cell types. PMID:23595227

  4. Chromatin decondensation is accompanied by a transient increase in transcriptional output.

    Science.gov (United States)

    Vaňková Hausnerová, Viola; Lanctôt, Christian

    2017-01-01

    The levels of chromatin condensation usually correlate inversely with the levels of transcription. The mechanistic links between chromatin condensation and RNA polymerase II activity remain to be elucidated. In the present work, we sought to experimentally determine whether manipulation of chromatin condensation levels can have a direct effect on transcriptional activity. We generated a U-2-OS cell line in which the nascent transcription of a reporter gene could be imaged alongside chromatin compaction levels in living cells. The transcripts were tagged at their 5' end with PP7 stem loops, which can be detected upon expression of a PP7 capsid protein fused to green fluorescent protein. Cycles of global chromatin hypercondensation and decondensation were performed by perfusing culture media of different osmolarities during imaging. We used the fluorescence recovery after photobleaching technique to analyse the transcriptional dynamics in both conditions. Surprisingly, we found that, despite a drop in signal intensity, nascent transcription appeared to continue at the same rate in hypercondensed chromatin. Furthermore, quantification of transcriptional profiles revealed that chromatin decondensation was accompanied by a brief and transient spike in transcriptional output. We propose a model whereby the initiation of transcription is not impaired in condensed chromatin, but inefficient elongation in these conditions leads to the accumulation of RNA polymerase II at the transcription site. Upon chromatin decondensation, release of the RNA polymerase II halt triggers a wave of transcription, which we detect as a transient spike in activity. The results presented here shed light on the activity of RNA polymerase II during chromatin condensation and decondensation. As such, they point to a new level of transcriptional regulation. © 2016 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

  5. Regulation of Chromatin Assembly and Cell Transformation by Formaldehyde Exposure in Human Cells.

    Science.gov (United States)

    Chen, Danqi; Fang, Lei; Mei, Shenglin; Li, Hongjie; Xu, Xia; Des Marais, Thomas L; Lu, Kun; Liu, X Shirley; Jin, Chunyuan

    2017-09-21

    Formaldehyde (FA) is an environmental and occupational chemical carcinogen. Recent studies have shown that exogenous FA causes only a modest increase in DNA adduct formation compared with the amount of adducts formed by endogenous FA, raising the possibility that epigenetic mechanisms may contribute to FA-mediated carcinogenicity. We investigated the effects of FA exposure on histone modifications and chromatin assembly. We also examined the role of defective chromatin assembly in FA-mediated transcription and cell transformation. Cellular fractionation and Western blot analysis were used to measure the levels of histone modifications in human bronchial epithelial BEAS-2B cells and human nasal RPMI2650 cells in the presence of FA. Chromatin immunoprecipitation (ChIP) and micrococcal nuclease (MNase) digest assays were performed to examine the changes in chromatin assembly and accessibility after FA exposure. RNA sequencing (RNA-seq) and real-time polymerase chain reaction (PCR) were used to examine transcriptional dysregulation. Finally, anchorage-independent cell growth ability was tested by soft agar assay following FA exposure. Exposure to FA dramatically decreased the acetylation of the N-terminal tails of cytosolic histones. These modifications are important for histone nuclear import and subsequent chromatin assembly. Histone proteins were depleted in both the chromatin fraction and at most of the genomic loci tested following FA exposure, suggesting that FA compromises chromatin assembly. Moreover, FA increased chromatin accessibility and altered the expression of hundreds of cancer-related genes. Knockdown of the histone H3.3 gene (an H3 variant), which mimics inhibition of chromatin assembly, facilitated FA-mediated anchorage-independent cell growth. We propose that the inhibition of chromatin assembly represents a novel mechanism of cell transformation induced by the environmental and occupational chemical carcinogen FA. https://doi.org/10.1289/EHP1275.

  6. Epigenetic chromatin modifications in barley after mutagenic treatment.

    Science.gov (United States)

    Braszewska-Zalewska, Agnieszka; Tylikowska, Marta; Kwasniewska, Jolanta; Szymanowska-Pulka, Joanna

    2014-11-01

    In addition to their normal developmental processes, plants have evolved complex genetic and epigenetic regulatory mechanisms to cope with various environmental stresses. It has been shown that both DNA methylation and histone modifications are involved in DNA damage response to various types of stresses. In this study, we focused on the involvement of two mutagenic agents, chemical (maleic acid hydrazide; MH) and physical (gamma rays), on the global epigenetic modifications of chromatin in barley. Our results indicate that both mutagens strongly influence the level of histone methylation and acetylation. Moreover, we found that gamma irradiation, in contrast to MH, has a more robust influence on the DNA methylation level. This is the first study that brings together mutagenic treatment along with its impact at the level of epigenetic modifications examined using the immunohistochemical method.

  7. Formation of mammalian erythrocytes: chromatin condensation and enucleation.

    Science.gov (United States)

    Ji, Peng; Murata-Hori, Maki; Lodish, Harvey F

    2011-07-01

    In all vertebrates, the cell nucleus becomes highly condensed and transcriptionally inactive during the final stages of red cell biogenesis. Enucleation, the process by which the nucleus is extruded by budding off from the erythroblast, is unique to mammals. Enucleation has critical physiological and evolutionary significance in that it allows an elevation of hemoglobin levels in the blood and also gives red cells their flexible biconcave shape. Recent experiments reveal that enucleation involves multiple molecular and cellular pathways that include histone deacetylation, actin polymerization, cytokinesis, cell-matrix interactions, specific microRNAs and vesicle trafficking; many evolutionarily conserved proteins and genes have been recruited to participate in this uniquely mammalian process. In this review, we discuss recent advances in mammalian erythroblast chromatin condensation and enucleation, and conclude with our perspectives on future studies. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Transcriptional and chromatin regulation during fasting – The genomic era

    Science.gov (United States)

    Goldstein, Ido; Hager, Gordon L.

    2015-01-01

    An elaborate metabolic response to fasting is orchestrated by the liver and is heavily reliant upon transcriptional regulation. In response to hormones (glucagon, glucocorticoids) many transcription factors (TFs) are activated and regulate various genes involved in metabolic pathways aimed at restoring homeostasis: gluconeogenesis, fatty acid oxidation, ketogenesis and amino acid shuttling. We summarize the recent discoveries regarding fasting-related TFs with an emphasis on genome-wide binding patterns. Collectively, the summarized findings reveal a large degree of co-operation between TFs during fasting which occurs at motif-rich DNA sites bound by a combination of TFs. These new findings implicate transcriptional and chromatin regulation as major determinants of the response to fasting and unravels the complex, multi-TF nature of this response. PMID:26520657

  9. Live visualization of chromatin dynamics with fluorescent TALEs.

    Science.gov (United States)

    Miyanari, Yusuke; Ziegler-Birling, Céline; Torres-Padilla, Maria-Elena

    2013-11-01

    The spatiotemporal organization of genomes in the nucleus is an emerging key player to regulate genome function. Live imaging of nuclear organization dynamics would be a breakthrough toward uncovering the functional relevance and mechanisms regulating genome architecture. Here, we used transcription activator-like effector (TALE) technology to visualize endogenous repetitive genomic sequences. We established TALE-mediated genome visualization (TGV) to label genomic sequences and follow nuclear positioning and chromatin dynamics in cultured mouse cells and in the living organism. TGV is highly specific, thus allowing differential labeling of parental chromosomes by distinguishing between single-nucleotide polymorphisms (SNPs). Our findings provide a framework to address the function of genome architecture through visualization of nuclear dynamics in vivo.

  10. Chromatin Dynamics in Vivo: A Game of Musical Chairs

    Directory of Open Access Journals (Sweden)

    Daniël P. Melters

    2015-08-01

    Full Text Available Histones are a major component of chromatin, the nucleoprotein complex fundamental to regulating transcription, facilitating cell division, and maintaining genome integrity in almost all eukaryotes. In addition to canonical, replication-dependent histones, replication-independent histone variants exist in most eukaryotes. In recent years, steady progress has been made in understanding how histone variants assemble, their involvement in development, mitosis, transcription, and genome repair. In this review, we will focus on the localization of the major histone variants H3.3, CENP-A, H2A.Z, and macroH2A, as well as how these variants have evolved, their structural differences, and their functional significance in vivo.

  11. Human Transcriptome and Chromatin Modifications: An ENCODE Perspective

    Directory of Open Access Journals (Sweden)

    Li Shen

    2013-06-01

    Full Text Available A decade-long project, led by several international research groups, called the Encyclopedia of DNA Elements (ENCODE, recently released an unprecedented amount of data. The ambitious project covers transcriptome, cistrome, epigenome, and interactome data from more than 1,600 sets of experiments in human. To make use of this valuable resource, it is important to understand the information it represents and the techniques that were used to generate these data. In this review, we introduce the data that ENCODE generated, summarize the observations from the data analysis, and revisit a computational approach that ENCODE used to predict gene expression, with a focus on the human transcriptome and its association with chromatin modifications.

  12. Chromatin Constrains the Initiation and Elongation of DNA Replication.

    Science.gov (United States)

    Devbhandari, Sujan; Jiang, Jieqing; Kumar, Charanya; Whitehouse, Iestyn; Remus, Dirk

    2017-01-05

    Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Causes and consequences of chromatin variation between inbred mice.

    Directory of Open Access Journals (Sweden)

    Mona Hosseini

    2013-06-01

    Full Text Available Variation at regulatory elements, identified through hypersensitivity to digestion by DNase I, is believed to contribute to variation in complex traits, but the extent and consequences of this variation are poorly characterized. Analysis of terminally differentiated erythroblasts in eight inbred strains of mice identified reproducible variation at approximately 6% of DNase I hypersensitive sites (DHS. Only 30% of such variable DHS contain a sequence variant predictive of site variation. Nevertheless, sequence variants within variable DHS are more likely to be associated with complex traits than those in non-variant DHS, and variants associated with complex traits preferentially occur in variable DHS. Changes at a small proportion (less than 10% of variable DHS are associated with changes in nearby transcriptional activity. Our results show that whilst DNA sequence variation is not the major determinant of variation in open chromatin, where such variants exist they are likely to be causal for complex traits.

  14. Snake-like chromatin in conjunctival cells of normal elderly persons and of patients with primary Sjögren's syndrome and other connective tissue diseases

    DEFF Research Database (Denmark)

    Bjerrum, Kirsten Birgitte

    1995-01-01

    Ophthalmology, snake-like chromatin, cytoplasm ratio, keratoconjunctivitis sicca, nucleus, goblet cell......Ophthalmology, snake-like chromatin, cytoplasm ratio, keratoconjunctivitis sicca, nucleus, goblet cell...

  15. Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes

    Science.gov (United States)

    Sanborn, Adrian; Rao, Suhas; Huang, Su-Chen; Durand, Neva; Huntley, Miriam; Jewett, Andrew; Bochkov, Ivan; Chinnappan, Dharmaraj; Cutkosky, Ashok; Li, Jian; Geeting, Kristopher; McKenna, Doug; Stamenova, Elena; Gnirke, Andreas; Melnikov, Alexandre; Lander, Eric; Aiden, Erez

    Our recent kilobase-resolution genome-wide maps of DNA self-contacts demonstrated that mammalian genomes are organized into domains and loops demarcated by the DNA-binding protein CTCF. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that domains are inconsistent with equilibrium and fractal models. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during condensation leads to formation of an anisotropic ``tension globule.'' In the other, CTCF and cohesin act together to extrude unknotted loops. Both models are consistent with the observed domains and loops. However, the extrusion model explains a far wider array of observations, such as why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The extrusion model predicts in silico the experimental maps using only CTCF-binding sites. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.

  16. Functional genomics indicates yeast requires Golgi/ER transport, chromatin remodeling, and DNA repair for low dose DMSO tolerance

    Directory of Open Access Journals (Sweden)

    Brandon David Gaytán

    2013-08-01

    Full Text Available Dimethyl sulfoxide (DMSO is frequently utilized as a solvent in toxicological and pharmaceutical investigations. It is therefore important to establish the cellular and molecular targets of DMSO in order to differentiate its intrinsic effects from those elicited by a compound of interest. We performed a genome-wide functional screen in Saccharomyces cerevisiae to identify deletion mutants exhibiting sensitivity to 1% DMSO, a concentration standard to yeast chemical profiling studies. We report that mutants defective in Golgi/ER transport are sensitive to DMSO, including those lacking components of the conserved oligomeric Golgi (COG complex. Moreover, strains deleted for members of the SWR1 histone exchange complex are hypersensitive to DMSO, with additional chromatin remodeling mutants displaying a range of growth defects. We also identify DNA repair genes important for DMSO tolerance. Finally, we demonstrate that overexpression of histone H2A.Z, which replaces chromatin-associated histone H2A in a SWR1-catalyzed reaction, confers resistance to DMSO. Many yeast genes described in this study have homologs in more complex organisms, and the data provided is applicable to future investigations into the cellular and molecular mechanisms of DMSO toxicity.

  17. ATM/Wip1 activities at chromatin control Plk1 re-activation to determine G2 checkpoint duration.

    Science.gov (United States)

    Jaiswal, Himjyot; Benada, Jan; Müllers, Erik; Akopyan, Karen; Burdova, Kamila; Koolmeister, Tobias; Helleday, Thomas; Medema, René H; Macurek, Libor; Lindqvist, Arne

    2017-07-14

    After DNA damage, the cell cycle is arrested to avoid propagation of mutations. Arrest in G2 phase is initiated by ATM-/ATR-dependent signaling that inhibits mitosis-promoting kinases such as Plk1. At the same time, Plk1 can counteract ATR-dependent signaling and is required for eventual resumption of the cell cycle. However, what determines when Plk1 activity can resume remains unclear. Here, we use FRET-based reporters to show that a global spread of ATM activity on chromatin and phosphorylation of ATM targets including KAP1 control Plk1 re-activation. These phosphorylations are rapidly counteracted by the chromatin-bound phosphatase Wip1, allowing cell cycle restart despite persistent ATM activity present at DNA lesions. Combining experimental data and mathematical modeling, we propose a model for how the minimal duration of cell cycle arrest is controlled. Our model shows how cell cycle restart can occur before completion of DNA repair and suggests a mechanism for checkpoint adaptation in human cells. © 2017 The Authors.

  18. The chromatin of Candida albicans pericentromeric repeats bears features of both euchromatin and heterochromatin

    Directory of Open Access Journals (Sweden)

    Veronica eFreire-Beneitez

    2016-05-01

    Full Text Available Centromeres, sites of kinetochore assembly, are important for chromosome stability and integrity. Most eukaryotes have regional centromeres epigenetically specified by the presence of the histone H3 variant CENP-A. CENP-A chromatin is often surrounded by pericentromeric regions packaged into transcriptionally silent heterochromatin. Candida albicans, the most common human fungal pathogen, possesses small regional centromeres assembled into CENP-A chromatin. The chromatin state of C. albicans pericentromeric regions is unknown. Here, for the first time, we address this question. We find that C. albicans pericentromeres are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Pericentromeric chromatin is associated with nucleosomes that are highly acetylated, as found in euchromatic regions of the genome, and hypomethylated on H3K4, as found in heterochromatin. This intermediate chromatin state is inhibitory to transcription and partially represses expression of proximal genes and inserted marker genes. Our analysis identifies a new chromatin state associated with pericentromeric regions.

  19. Mitotic post-translational modifications of histones promote chromatin compaction in vitro.

    Science.gov (United States)

    Zhiteneva, Alisa; Bonfiglio, Juan Jose; Makarov, Alexandr; Colby, Thomas; Vagnarelli, Paola; Schirmer, Eric C; Matic, Ivan; Earnshaw, William C

    2017-09-01

    How eukaryotic chromosomes are compacted during mitosis has been a leading question in cell biology since the nineteenth century. Non-histone proteins such as condensin complexes contribute to chromosome shaping, but appear not to be necessary for mitotic chromatin compaction. Histone modifications are known to affect chromatin structure. As histones undergo major changes in their post-translational modifications during mitotic entry, we speculated that the spectrum of cell-cycle-specific histone modifications might contribute to chromosome compaction during mitosis. To test this hypothesis, we isolated core histones from interphase and mitotic cells and reconstituted chromatin with them. We used mass spectrometry to show that key post-translational modifications remained intact during our isolation procedure. Light, atomic force and transmission electron microscopy analysis showed that chromatin assembled from mitotic histones has a much greater tendency to aggregate than chromatin assembled from interphase histones, even under low magnesium conditions where interphase chromatin remains as separate beads-on-a-string structures. These observations are consistent with the hypothesis that mitotic chromosome formation is a two-stage process with changes in the spectrum of histone post-translational modifications driving mitotic chromatin compaction, while the action of non-histone proteins such as condensin may then shape the condensed chromosomes into their classic mitotic morphology. © 2017 The Authors.

  20. Characterization of chick liver chromatin and analysis of its in vitro transcription products.

    Science.gov (United States)

    Dierks-Ventling, C; Stalder, J; Gautschi, J

    1978-07-01

    Carefully controlled preparation of chromatin from purified chick liver nuclei yielded over 50% native chromatin as shown by the analysis of the nucleosome pattern after micrococcal nuclease digestion. The size of DNA in this chromatin as analyzed on alkaline sucrose gradients varied from 10S to 19S, the majority being 14S. All endogenous RNA polymerases were represented in the chromatin preparation although to different extents: RNA polymerase I was the most and RNA polymerase II the least abundant. Initiation studies showed that endogenous RNA polymerase II was capable of initiating RNA chains during 5 min. Saturation of chromatin with purified homologous RNA polymerase II increased initiation to 10 min. The addition of heparin caused the RNA transcribed to be larger in size and of increased yield. Chromatin transcription with added purified RNA polymerase II in the presence of heparin produced RNA as large as 32S. A chromatin preparation of this kind would therefore be suitable to transcribe any eukaryotic gene invitro provided additional homologous RNA polymerase II is used.Images

  1. Non coding RNA: sequence-specific guide for chromatin modification and DNA damage signaling

    Directory of Open Access Journals (Sweden)

    Sofia eFrancia

    2015-11-01

    Full Text Available Chromatin conformation shapes the environment in which our genome is transcribed into RNA. Transcription is a source of DNA damage, thus it often occurs concomitantly to DNA damage signaling. Growing amounts of evidence suggest that different types of RNAs can, independently from their protein-coding properties, directly affect chromatin conformation, transcription and splicing, as well as promote the activation of the DNA damage response (DDR and DNA repair. Therefore, transcription paradoxically functions to both threaten and safeguard genome integrity. On the other hand, DNA damage signaling is known to modulate chromatin to suppress transcription of the surrounding genetic unit. It is thus intriguing to understand how transcription can modulate DDR signaling while, in turn, DDR signaling represses transcription of chromatin around the DNA lesion. An unexpected player in this field is the RNA interference (RNAi machinery, which play roles in transcription, splicing and chromatin modulation in several organisms. Non-coding RNAs (ncRNAs and several protein factors involved in the RNAi pathway are well known master regulators of chromatin while only recent reports suggest that ncRNAs are involved in DDR signaling and homology-mediated DNA repair. Here, we discuss the experimental evidence supporting the idea that ncRNAs act at the genomic loci from which they are transcribed to modulate chromatin, DDR signaling and DNA repair.

  2. Heterogeneous pattern of DNA methylation in developmentally important genes correlates with its chromatin conformation.

    Science.gov (United States)

    Sinha, Puja; Singh, Kiran; Sachan, Manisha

    2017-01-11

    DNA methylation is a major epigenetic modification, playing a crucial role in the development and differentiation of higher organisms. DNA methylation is also known to regulate transcription by gene repression. Various developmental genes such as c-mos, HoxB5, Sox11, and Sry show tissue-specific gene expression that was shown to be regulated by promoter DNA methylation. The aim of the present study is to investigate the establishment of chromatin marks (active or repressive) in relation to heterogeneous methylation in the promoter regions of these developmentally important genes. Chromatin-immunoprecipitation (ChIP) assays were performed to immuno-precipitate chromatin by antibodies against both active (H3K4me3) and repressive (H3K9me3) chromatin regions. The analysis of ChIP results showed that both the percentage input and fold enrichment of activated chromatin was higher in tissues expressing the respective genes as compared to the tissues not expressing the same set of genes. This was true for all the genes selected for the study (c-mos, HoxB5, Sox11, and Sry). These findings illustrate that inconsistent DNA methylation patterns (sporadic, mosaic and heterogeneous) may also influence gene regulation, thereby resulting in the modulation of chromatin conformation. These findings illustrate that various patterns of DNA methylation (asynchronous, mosaic and heterogeneous) correlates with chromatin modification, resulting in the gene regulation.

  3. DNA Methylation Landscape Reflects the Spatial Organization of Chromatin in Different Cells.

    Science.gov (United States)

    Zhang, Ling; Xie, Wen Jun; Liu, Sirui; Meng, Luming; Gu, Chan; Gao, Yi Qin

    2017-10-03

    The relationship between DNA methylation and chromatin structure is still largely unknown. By analyzing a large set of published sequencing data, we observed a long-range power law correlation of DNA methylation with cell class-specific scaling exponents in the range of tens of kilobases. We showed that such cell class-specific scaling exponents are caused by different patchiness of DNA methylation in different cells. By modeling the chromatin structure using high-resolution chromosome conformation capture data and mapping the methylation level onto the modeled structure, we demonstrated that the patchiness of DNA methylation is related to chromatin structure. The scaling exponents of the power law correlation are thus a display of the spatial organization of chromatin. Besides the long-range correlation, we also showed that the local correlation of DNA methylation is associated with nucleosome positioning. The local correlation of partially methylated domains is different from that of nonpartially methylated domains, suggesting that their chromatin structures differ at the scale of several hundred base pairs (covering a few nucleosomes). Our study provides a novel, to our knowledge, view of the spatial organization of chromatin structure from a perspective of DNA methylation, in which both long-range and local correlations of DNA methylation along the genome reflect the spatial organization of chromatin. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  4. Real-Time Tracking of Parental Histones Reveals Their Contribution to Chromatin Integrity Following DNA Damage.

    Science.gov (United States)

    Adam, Salomé; Dabin, Juliette; Chevallier, Odile; Leroy, Olivier; Baldeyron, Céline; Corpet, Armelle; Lomonte, Patrick; Renaud, Olivier; Almouzni, Geneviève; Polo, Sophie E

    2016-10-06

    Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  5. Widespread Chromatin Accessibility at Repetitive Elements Links Stem Cells with Human Cancer.

    Science.gov (United States)

    Gomez, Nicholas C; Hepperla, Austin J; Dumitru, Raluca; Simon, Jeremy M; Fang, Fang; Davis, Ian J

    2016-11-01

    Chromatin regulation is critical for differentiation and disease. However, features linking the chromatin environment of stem cells with disease remain largely unknown. We explored chromatin accessibility in embryonic and multipotent stem cells and unexpectedly identified widespread chromatin accessibility at repetitive elements. Integrating genomic and biochemical approaches, we demonstrate that these sites of increased accessibility are associated with well-positioned nucleosomes marked by distinct histone modifications. Differentiation is accompanied by chromatin remodeling at repetitive elements associated with altered expression of genes in relevant developmental pathways. Remarkably, we found that the chromatin environment of Ewing sarcoma, a mesenchymally derived tumor, is shared with primary mesenchymal stem cells (MSCs). Accessibility at repetitive elements in MSCs offers a permissive environment that is exploited by the critical oncogene responsible for this cancer. Our data demonstrate that stem cells harbor a unique chromatin landscape characterized by accessibility at repetitive elements, a feature associated with differentiation and oncogenesis. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Protooncogene Ski cooperates with the chromatin-remodeling factor Satb2 in specifying callosal neurons.

    Science.gov (United States)

    Baranek, Constanze; Dittrich, Manuela; Parthasarathy, Srinivas; Bonnon, Carine Gaiser; Britanova, Olga; Lanshakov, Dmitriy; Boukhtouche, Fatiha; Sommer, Julia E; Colmenares, Clemencia; Tarabykin, Victor; Atanasoski, Suzana

    2012-02-28

    First insights into the molecular programs orchestrating the progression from neural stem cells to cortical projection neurons are emerging. Loss of the transcriptional regulator Ski has been linked to the human 1p36 deletion syndrome, which includes central nervous system defects. Here, we report critical roles for Ski in the maintenance of the neural stem cell pool and the specification of callosal neurons. Ski-deficient callosal neurons lose their identity and ectopically express the transcription factor Ctip2. The misspecified callosal neurons largely fail to form the corpus callosum and instead redirect their axons toward subcortical targets. We identify the chromatin-remodeling factor Satb2 as a partner of Ski, and show that both proteins are required for transcriptional repression of Ctip2 in callosal neurons. We propose a model in which Satb2 recruits Ski to the Ctip2 locus, and Ski attracts histone deacetylases, thereby enabling the formation of a functional nucleosome remodeling and deacetylase repressor complex. Our findings establish a central role for Ski-Satb2 interactions in regulating transcriptional mechanisms of callosal neuron specification.

  7. Modulation of chromatin remodelling induced by the freshwater cyanotoxin cylindrospermopsin in human intestinal caco-2 cells.

    Directory of Open Access Journals (Sweden)

    Antoine Huguet

    Full Text Available Cylindrospermopsin (CYN is a cyanotoxin that has been recognised as an emerging potential public health risk. Although CYN toxicity has been demonstrated, the mechanisms involved have not been fully characterised. To identify some key pathways related to this toxicity, we studied the transcriptomic profile of human intestinal Caco-2 cells exposed to a sub-toxic concentration of CYN (1.6 µM for 24hrs using a non-targeted approach. CYN was shown to modulate different biological functions which were related to growth arrest (with down-regulation of cdkn1a and uhrf1 genes, and DNA recombination and repair (with up-regulation of aptx and pms2 genes. Our main results reported an increased expression of some histone-modifying enzymes (histone acetyl and methyltransferases MYST1, KAT5 and EHMT2 involved in chromatin remodelling, which is essential for initiating transcription. We also detected greater levels of acetylated histone H2A (Lys5 and dimethylated histone H3 (Lys4, two products of these enzymes. In conclusion, CYN overexpressed proteins involved in DNA damage repair and transcription, including modifications of nucleosomal histones. Our results highlighted some new cell processes induced by CYN.

  8. Features of mammalian microRNA promoters emerge from polymerase II chromatin immunoprecipitation data.

    Directory of Open Access Journals (Sweden)

    David L Corcoran

    Full Text Available MicroRNAs (miRNAs are short, non-coding RNA regulators of protein coding genes. miRNAs play a very important role in diverse biological processes and various diseases. Many algorithms are able to predict miRNA genes and their targets, but their transcription regulation is still under investigation. It is generally believed that intragenic miRNAs (located in introns or exons of protein coding genes are co-transcribed with their host genes and most intergenic miRNAs transcribed from their own RNA polymerase II (Pol II promoter. However, the length of the primary transcripts and promoter organization is currently unknown.We performed Pol II chromatin immunoprecipitation (ChIP-chip using a custom array surrounding regions of known miRNA genes. To identify the true core transcription start sites of the miRNA genes we developed a new tool (CPPP. We showed that miRNA genes can be transcribed from promoters located several kilobases away and that their promoters share the same general features as those of protein coding genes. Finally, we found evidence that as many as 26% of the intragenic miRNAs may be transcribed from their own unique promoters.miRNA promoters have similar features to those of protein coding genes, but miRNA transcript organization is more complex.

  9. Neuronal necrosis is regulated by a conserved chromatin-modifying cascade

    Science.gov (United States)

    Liu, Kai; Ding, Lianggong; Li, Yuhong; Yang, Hui; Zhao, Chunyue; Lei, Ye; Han, Shuting; Tao, Wei; Miao, Dengshun; Steller, Hermann; Welsh, Michael J.; Liu, Lei

    2014-01-01

    Neuronal necrosis induced by calcium overload causes devastating brain dysfunction in diseases such as stroke and brain trauma. It has been considered a stochastic event lacking genetic regulation, and pharmacological means to suppress neuronal necrosis are lacking. Using a Drosophila model of calcium overloading, we found JIL-1/mitogen- and stress-activated protein kinase 1/2 is a regulator of neuronal necrosis through phosphorylation of histone H3 serine 28 (H3S28ph). Further, we identified its downstream events including displacement of polycomb repressive complex 1 (PRC1) and activation of Trithorax (Trx). To test the role of JIL-1/PRC1/Trx cascade in mammals, we studied the necrosis induced by glutamate in rat cortical neuron cultures and rodent models of brain ischemia and found the cascade is activated in these conditions and inhibition of the cascade suppresses necrosis in vitro and in vivo. Together, our research demonstrates that neuronal necrosis is regulated by a chromatin-modifying cascade, and this discovery may provide potential therapeutic targets and biomarkers for neuronal necrosis. PMID:25201987

  10. Histone H3 lysine-to-methionine mutants as a paradigm to study chromatin signaling.

    Science.gov (United States)

    Herz, Hans-Martin; Morgan, Marc; Gao, Xin; Jackson, Jessica; Rickels, Ryan; Swanson, Selene K; Florens, Laurence; Washburn, Michael P; Eissenberg, Joel C; Shilatifard, Ali

    2014-08-29

    Histone H3 lysine(27)-to-methionine (H3K27M) gain-of-function mutations occur in highly aggressive pediatric gliomas. We established a Drosophila animal model for the pathogenic histone H3K27M mutation and show that its overexpression resembles polycomb repressive complex 2 (PRC2) loss-of-function phenotypes, causing derepression of PRC2 target genes and developmental perturbations. Similarly, an H3K9M mutant depletes H3K9 methylation levels and suppresses position-effect variegation in various Drosophila tissues. The histone H3K9 demethylase KDM3B/JHDM2 associates with H3K9M-containing nucleosomes, and its misregulation in Drosophila results in changes of H3K9 methylation levels and heterochromatic silencing defects. We have established histone lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation pathways that also function as biochemical reagents for capturing site-specific histone-modifying enzymes, thus providing molecular insight into chromatin signaling pathways. Copyright © 2014, American Association for the Advancement of Science.

  11. Dynamic Rewiring of Promoter-Anchored Chromatin Loops during Adipocyte Differentiation.

    Science.gov (United States)

    Siersbæk, Rasmus; Madsen, Jesper Grud Skat; Javierre, Biola Maria; Nielsen, Ronni; Bagge, Emilie Kristine; Cairns, Jonathan; Wingett, Steven William; Traynor, Sofie; Spivakov, Mikhail; Fraser, Peter; Mandrup, Susanne

    2017-05-04

    Interactions between transcriptional promoters and their distal regulatory elements play an important role in transcriptional regulation; however, the extent to which these interactions are subject to rapid modulations in response to signals is unknown. Here, we use promoter capture Hi-C to demonstrate a rapid reorganization of promoter-anchored chromatin loops within 4 hr after inducing differentiation of 3T3-L1 preadipocytes. The establishment of new promoter-enhancer loops is tightly coupled to activation of poised (histone H3 lysine 4 mono- and dimethylated) enhancers, as evidenced by the acquisition of histone H3 lysine 27 acetylation and the binding of MED1, SMC1, and P300 proteins to these regions, as well as to activation of target genes. Intriguingly, formation of loops connecting activated enhancers and promoters is also associated with extensive recruitment of corepressors such as NCoR and HDACs, indicating that this class of coregulators may play a previously unrecognized role during enhancer activation. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. [Loosening of condensed chromatin in human blood lymphocytes exposed to irradiation with a low-energy He-Ne laser].

    Science.gov (United States)

    Manteĭfel', V M; Karu, T I

    2009-01-01

    It was shown that, 1 h after irradiation of human blood lymphocytes with a He-Ne laser at 56 J/m2 (5.6 W/m2, 10 s), the relative optical density of condensed chromatin masses observed in ultrathin sections was decreased (p irradiation also results in dispersion of condensed chromatin clumps in the nucleoplasm and enhancement of their angularity, i.e., in extension of the clump surface. These shifts, correlating with the activation of transcription, may be due to decompaction of the chromatin fibers not only on the periphery of chromatin clusters in the center of the nucleus, but also within the masses of condensed chromatin.

  13. A new and improved algorithm for the quantification of chromatin condensation from microscopic data shows decreased chromatin condensation in regenerating axolotl limb cells.

    Directory of Open Access Journals (Sweden)

    Julian Sosnik

    Full Text Available The nuclear landscape plays an important role in the regulation of tissue and positional specific genes in embryonic and developing cells. Changes in this landscape can be dynamic, and are associated with the differentiation of cells during embryogenesis, and the de-differentiation of cells during induced pluripotent stem cell (iPSC formation and in many cancers. However, tools to quantitatively characterize these changes are limited, especially in the in vivo context, where numerous tissue types are present and cells are arranged in multiple layers. Previous tools have been optimized for the monolayer nature of cultured cells. Therefore, we present a new algorithm to quantify the condensation of chromatin in two in vivo systems. We first developed this algorithm to quantify changes in chromatin compaction and validated it in differentiating spermatids in zebrafish testes. Our algorithm successfully detected the typical increase in chromatin compaction as these cells differentiate. We then employed the algorithm to quantify the changes that occur in amphibian limb cells as they participate in a regenerative response. We observed that the chromatin in the limb cells de-compacts as they contribute to the regenerating organ. We present this new tool as an open sourced software that can be readily accessed and optimized to quantify chromatin compaction in complex multi-layered samples.

  14. A new and improved algorithm for the quantification of chromatin condensation from microscopic data shows decreased chromatin condensation in regenerating axolotl limb cells.

    Science.gov (United States)

    Sosnik, Julian; Vieira, Warren A; Webster, Kaitlyn A; Siegfried, Kellee R; McCusker, Catherine D

    2017-01-01

    The nuclear landscape plays an important role in the regulation of tissue and positional specific genes in embryonic and developing cells. Changes in this landscape can be dynamic, and are associated with the differentiation of cells during embryogenesis, and the de-differentiation of cells during induced pluripotent stem cell (iPSC) formation and in many cancers. However, tools to quantitatively characterize these changes are limited, especially in the in vivo context, where numerous tissue types are present and cells are arranged in multiple layers. Previous tools have been optimized for the monolayer nature of cultured cells. Therefore, we present a new algorithm to quantify the condensation of chromatin in two in vivo systems. We first developed this algorithm to quantify changes in chromatin compaction and validated it in differentiating spermatids in zebrafish testes. Our algorithm successfully detected the typical increase in chromatin compaction as these cells differentiate. We then employed the algorithm to quantify the changes that occur in amphibian limb cells as they participate in a regenerative response. We observed that the chromatin in the limb cells de-compacts as they contribute to the regenerating organ. We present this new tool as an open sourced software that can be readily accessed and optimized to quantify chromatin compaction in complex multi-layered samples.

  15. Repressed template activity of chromatin of pea roots treated by aluminium

    OpenAIRE

    Hideaki, MATSUMOTO; Seiichiro, Morimura; Institute for Agricultural and Biological Sciences, Okayama University; Institute for Agricultural and Biological Sciences, Okayama University(Present)Nippon Shinyaku Co. Ltd.

    1980-01-01

    To determine the mechanism of aluminium toxicity in the pea root, we investigated both the in vivo and in vitro effects of aluminium on chromatin. The ratio of histone or non-histone proteins to DNA in chromatin was slightly increased by aluminium treatment. However, chromatin with an rf of 0.086 (rf: molar ratio of bound aluminium to DNA-phosphate) prepared from aluminium treated roots had template activity for RNA synthesis that was half of the control. The template activity of the chromati...

  16. Data on force-dependent structural changes of chromatin fibers measured with magnetic tweezers

    Directory of Open Access Journals (Sweden)

    Fan-Tso Chien

    2014-12-01

    Full Text Available The compaction of chromatin fibers regulates the accessibility of embedded DNA, highly associated with transcriptional activities [1]. Single molecule force spectroscopy has revealed the great details of the structural changes of chromatin fibers in the presence of external exerted force [2–7]. However, most of the studies focus on a specific force regime [2,3,8,9]. The data here show force-extension (FE traces of chromatin fibers as measured with magnetic tweezers, covering the force regime from 0 pN to 27 pN. Those traces provide information for further studies at varied force regimes.

  17. Identification of a crenarchaeal orthologue of Elf1: implications for chromatin and transcription in Archaea

    Directory of Open Access Journals (Sweden)

    Kelly Steven

    2009-07-01

    Full Text Available Abstract The transcription machineries of Archaea and eukaryotes are similar in many aspects, but little is understood about archaeal chromatin and its role in transcription. Here, we describe the identification in hyperthermophilic Crenarchaeota and a Korarchaeon of an orthologue of the eukaryotic transcription elongation factor Elf1, which has been shown to function in chromatin structure maintenance of actively transcribed templates. Our discovery has implications for the relationship of chromatin and transcription in Archaea and the evolution of these processes in eukaryotes. Reviewers This article was reviewed by Chris P. Ponting and Eugene V. Koonin.

  18. Identification of a crenarchaeal orthologue of Elf1: implications for chromatin and transcription in Archaea

    Science.gov (United States)

    Daniels, Jan-Peter; Kelly, Steven; Wickstead, Bill; Gull, Keith

    2009-01-01

    The transcription machineries of Archaea and eukaryotes are similar in many aspects, but little is understood about archaeal chromatin and its role in transcription. Here, we describe the identification in hyperthermophilic Crenarchaeota and a Korarchaeon of an orthologue of the eukaryotic transcription elongation factor Elf1, which has been shown to function in chromatin structure maintenance of actively transcribed templates. Our discovery has implications for the relationship of chromatin and transcription in Archaea and the evolution of these processes in eukaryotes. This article was reviewed by Chris P. Ponting and Eugene V. Koonin. PMID:19640276

  19. Human CDT1 associates with CDC7 and recruits CDC45 to chromatin during S phase

    DEFF Research Database (Denmark)

    Ballabeni, Andrea; Zamponi, Raffaela; Caprara, Greta

    2009-01-01

    pre-Initiation Complexes, which are a prerequisite for DNA replication. The CDT1 protein is required for the formation of the pre-Replicative Complexes. Here we show that human CDT1 associates with the CDC7 kinase and recruits CDC45 to chromatin. Moreover, we show that the amount of CDT1 bound...... to chromatin is regulated by CDC7. We propose a model in which chromatin bound CDT1 is first stabilized and subsequently displaced by CDC7 activity, thereby ensuring the timely execution of DNA replication....

  20. Brd2 gene disruption causes "metabolically healthy" obesity: epigenetic and chromatin-based mechanisms that uncouple obesity from type 2 diabetes.

    Science.gov (United States)

    W