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  • Human T-lymphotropic virus type 1 transcription and chromatin-remodeling complexes. 20164218

    Human T-lymphotropic virus type 1 (HTLV-1) encodes the viral protein Tax, which is believed to act as a viral transactivator through its interactions with a variety of transcription factors, including CREB and NF-kappaB. As is the case for all retroviruses, the provirus is inserted into the host DNA, where nucleosomes are deposited to ensure efficient packaging. Nucleosomes act as roadblocks in transcription, making it difficult for RNA polymerase II (Pol II) to proceed toward the 3' end of the genome. Because of this, a variety of chromatin remodelers can act to modify nucleosomes, allowing for efficient transcription. While a number of covalent modifications are known to occur on histone tails in HTLV-1 infection (i.e., histone acetyltransferases [HATs], histone deacetylases [HDACs], and histone methyltransferases [HMTs]), evidence points to the use of chromatin remodelers that use energy from ATP hydrolysis to remodel nucleosomes. Here we confirm that BRG1, which is the core subunit of eight chromatin-remodeling complexes, is essential not only for Tax transactivation but also for viral replication. This is especially evident when wild-type infectious clones of HTLV-1 are used. BRG1 associates with Tax at the HTLV-1 long terminal repeat (LTR), and coexpression of BRG1 and Tax results in increased rates of transcription. The interaction of BRG1 with Tax additionally recruits the basal transcriptional machinery and removes some of the core histones from the nucleosome at the start site (Nuc 1). When using the BRG1-deficient cell lines SW13, C33A, and TSUPR1, we observed little viral transcription and no viral replication. Importantly, while these three cell lines do not express detectable levels of BRG1, much of the SWI/SNF complex remains assembled in the cells. Knockdown of BRG1 and associated SWI/SNF subunits suggests that the BRG1-utilizing SWI/SNF complex PBAF is responsible for HTLV-1 nucleosome remodeling. Finally, HTLV-1 infection of cell lines with a knockdown in BRG1 or the PBAF complex results in a significant reduction in viral production. Overall, we concluded that BRG1 is required for Tax transactivation and HTLV-1 viral production and that the PBAF complex appears to be responsible for nucleosome remodeling.
    Document Type:
    Reference
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    Multiple
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    Multiple

  • p53 binding to nucleosomal DNA depends on the rotational positioning of DNA response element. 19887449

    The sequence-specific binding to DNA is crucial for the p53 tumor suppressor function. To investigate the constraints imposed on p53-DNA recognition by nucleosomal organization, we studied binding of the p53 DNA binding domain (p53DBD) and full-length wild-type p53 protein to a single p53 response element (p53RE) placed near the nucleosomal dyad in six rotational settings. We demonstrate that the strongest p53 binding occurs when the p53RE in the nucleosome is bent in the same direction as observed for the p53-DNA complexes in solution and in co-crystals. The p53RE becomes inaccessible, however, if its orientation in the core particle is changed by approximately 180 degrees. Our observations indicate that the orientation of the binding sites on a nucleosome may play a significant role in the initial p53-DNA recognition and subsequent cofactor recruitment.
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    Reference
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  • MRG15 activates the cdc2 promoter via histone acetylation in human cells. 21324423

    Chromatin remodeling is required for transcriptional activation and repression. MRG15 (MORF4L1), a chromatin modulator, is a highly conserved protein and is present in complexes containing histone acetyltransferases (HATs) as well as histone deacetylases (HDACs). Loss of expression of MRG15 in mice and Drosophila results in embryonic lethality and fibroblast and neural stem/progenitor cells cultured from Mrg15 null mouse embryos exhibit marked proliferative defects when compared with wild type cells. To determine the role of MRG15 in cell cycle progression we performed chromatin immunoprecipitation with an antibody to MRG15 on normal human fibroblasts as they entered the cell cycle from a quiescent state, and analyzed various cell cycle gene promoters. The results demonstrated a 3-fold increase in MRG15 occupancy at the cdc2 promoter during S phase of the cell cycle and a concomitant increase in acetylated histone H4. H4 lysine 12 was acetylated at 24 h post-serum stimulation while there was no change in acetylation of lysine 16. HDAC1 and 2 were decreased at this promoter during cell cycle progression. Over-expression of MRG15 in HeLa cells activated a cdc2 promoter-reporter construct in a dose-dependent manner, whereas knockdown of MRG15 resulted in decreased promoter activity. In order to implicate HAT activity, we treated cells with the HAT inhibitor anacardic acid and determined that HAT inhibition results in loss of expression of cdc2 mRNA. Further, chromatin immunoprecipitation with Tip60 localizes the protein to the same 110bp stretch of the cdc2 promoter pulled down by MRG15. Additionally, we determined that cotransfection of MRG15 with the known associated HAT Tip60 had a cooperative effect in activating the cdc2 promoter. These results suggest that MRG15 is acting in a HAT complex involving Tip60 to modify chromatin via acetylation of histone H4 at the cdc2 promoter to activate transcription.
    Document Type:
    Reference
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    Multiple
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  • Mechanism for fetal hemoglobin induction by histone deacetylase inhibitors involves gamma-globin activation by CREB1 and ATF-2. 16896160

    The histone deacetylase inhibitors (HDA-CIs) butyrate and trichostatin A activate gamma-globin expression via a p38 mitogen-activating protein kinase (MAPK)-dependent mechanism. We hypothesized that down-stream effectors of p38 MAPK, namely activating transcription factor-2 (ATF-2) and cyclic AMP response element (CRE) binding protein (CREB), are intimately involved in fetal hemoglobin induction by these agents. In this study, we observed increased ATF-2 and CREB1 phosphorylation mediated by the HDACIs in K562 cells, in conjunction with histone H4 hyperacetylation. Moreover, enhanced DNA-protein interactions occurred in the CRE in the (G)gamma-globin promoter (G-CRE) in vitro after drug treatments; subsequent chromatin immunoprecipitation assay confirmed ATF-2 and CREB1 binding to the G-CRE in vivo. Enforced expression of ATF-2 and CREB produced (G)gamma-promoter trans-activation which was abolished by a 2-base pair mutation in the putative G-CRE. The data presented herein demonstrate that gamma-gene induction by butyrate and trichostatin A involves ATF-2 and CREB1 activation via p38 MAPK signaling.
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    Reference
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    Multiple
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    Multiple

  • Chromatin loading of E2F-MLL complex by cancer-associated coregulator ANCCA via reading a specific histone mark. 20855524

    Histone modifications are regarded as the carrier of epigenetic memory through cell divisions. How the marks facilitate cell cycle-dependent gene expression is poorly understood. The evolutionarily conserved AAA ATPase ANCCA (AAA nuclear coregulator cancer-associated protein)/ATAD2 was identified as a direct target of oncogene AIB1/ACTR/SRC-3 and a transcriptional coregulator for estrogen and androgen receptors and is strongly implicated in tumorigenesis. We report here that ANCCA directly interacts with E2F1 to E2F3 and that its N terminus interacts with both the N and C termini of E2F1. ANCCA preferentially associates via its bromodomain with H3 acetylated at lysine 14 (H3K14ac) and is required for key cell cycle gene expression and cancer cell proliferation. ANCCA associates with chromosomes at late mitosis, and its occupancy at E2F targets peaks at the G(1)-to-S transition. Strikingly, ANCCA is required for recruitment of specific E2Fs to their targets and chromatin assembly of the host cell factor 1 (HCF-1)-MLL histone methyltransferase complex. ANCCA depletion results in a marked decrease of the gene activation-linked H3K4me3 mark. Bromodomain mutations disable ANCCA function as an E2F coactivator and its ability to promote cancer cell proliferation, while ANCCA overexpression in tumors correlates with tumor growth. Together, these results suggest that ANCCA acts as a pioneer factor in E2F-dependent gene activation and that a novel mechanism involving ANCCA bromodomain may contribute to cancer cell proliferation.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
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    Multiple

  • The FUN30 chromatin remodeler, Fft3, protects centromeric and subtelomeric domains from euchromatin formation. 21437270

    The chromosomes of eukaryotes are organized into structurally and functionally discrete domains. This implies the presence of insulator elements that separate adjacent domains, allowing them to maintain different chromatin structures. We show that the Fun30 chromatin remodeler, Fft3, is essential for maintaining a proper chromatin structure at centromeres and subtelomeres. Fft3 is localized to insulator elements and inhibits euchromatin assembly in silent chromatin domains. In its absence, euchromatic histone modifications and histone variants invade centromeres and subtelomeres, causing a mis-regulation of gene expression and severe chromosome segregation defects. Our data strongly suggest that Fft3 controls the identity of chromatin domains by protecting these regions from euchromatin assembly.
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    Reference
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    Multiple
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    Multiple

  • The histone variant macroH2A suppresses melanoma progression through regulation of CDK8. 21179167

    Cancer is a disease consisting of both genetic and epigenetic changes. Although increasing evidence demonstrates that tumour progression entails chromatin-mediated changes such as DNA methylation, the role of histone variants in cancer initiation and progression currently remains unclear. Histone variants replace conventional histones within the nucleosome and confer unique biological functions to chromatin. Here we report that the histone variant macroH2A (mH2A) suppresses tumour progression of malignant melanoma. Loss of mH2A isoforms, histone variants generally associated with condensed chromatin and fine-tuning of developmental gene expression programs, is positively correlated with increasing malignant phenotype of melanoma cells in culture and human tissue samples. Knockdown of mH2A isoforms in melanoma cells of low malignancy results in significantly increased proliferation and migration in vitro and growth and metastasis in vivo. Restored expression of mH2A isoforms rescues these malignant phenotypes in vitro and in vivo. We demonstrate that the tumour-promoting function of mH2A loss is mediated, at least in part, through direct transcriptional upregulation of CDK8. Suppression of CDK8, a colorectal cancer oncogene, inhibits proliferation of melanoma cells, and knockdown of CDK8 in cells depleted of mH2A suppresses the proliferative advantage induced by mH2A loss. Moreover, a significant inverse correlation between mH2A and CDK8 expression levels exists in melanoma patient samples. Taken together, our results demonstrate that mH2A is a critical component of chromatin that suppresses the development of malignant melanoma, a highly intractable cutaneous neoplasm.
    Document Type:
    Reference
    Product Catalog Number:
    05-858
    Product Catalog Name:
    Anti-Histone H4 Antibody, pan, clone 62-141-13, rabbit monoclonal

  • N-terminal tail of a viral histone H4 encoded in Cotesia plutellae bracovirus is essential to suppress gene expression of host histone H4. 19196351

    An endoparasitoid wasp, Cotesia plutellae, possesses a symbiotic bracovirus (CpBV), which facilitates parasitism of a specific host, such as larvae of the diamondback moth, Plutella xylostella. A viral histone H4 (CpBV-H4) has been found in the CpBV genome and its gene product plays a role in impairing the host insect cellular immune response. Based on its high similarity to histone H4 of P. xylostella apart from its extended N-terminal tail, it has been suspected to alter host gene expression. Histone subunits were purified from parasitized P. xylostella larvae and found to contain both host and viral H4s, confirming a previous report of a possible epigenetic mode of action. Moreover, this study showed that the host H4 levels in the parasitized larvae clearly decreased during the parasitization period, whereas CpBV-H4 levels maintained a significant level without significant changes. To understand the decrease of host H4 levels, transcription levels of host H4 were monitored by quantitative reverse-transcriptase PCR (RT-PCR) and showed a significant decrease in parasitized P. xylostella larvae, whereas no significant change of the mRNA level was detected in nonparasitized larvae. This transcriptional control of host H4 expression was also observed by inducing transient expression of CpBV-H4 in nonparasitized P. xylostella. Moreover, co-injection of CpBV-H4 and its specific double-stranded RNA recovered the host H4 expression level. To identify a functional domain of CpBV-H4 involved in the transcriptional control, the extended N-terminal tail of CpBV-H4 was removed by preparing a truncated viral H4 construct in an expression vector by deleting the N-terminal tail of 38 amino acid residues and inducing its expression in nonparasitized P. xylostella larvae. The truncated CpBV-H4 clearly lost its inhibitory effects on host H4 transcription. Moreover, the presence of CpBV-H4 affects the spreading of host haemocytes by an epigenetic effect, which is at least partly restored in larvae expressing the truncated version of CpBV-H4. This study suggests that the viral H4 encoded in CpBV can alter host gene expression with its extended N-terminal tail.
    Document Type:
    Reference
    Product Catalog Number:
    05-858
    Product Catalog Name:
    Anti-Histone H4 Antibody, pan, clone 62-141-13, rabbit monoclonal