Dataset Information

Dynamic site-specific recruitment of RBP2 by pocket protein p130 modulates H3K4 methylation on E2F-responsive promoters.

ABSTRACT: The Histone 3 lysine 4 methylation (H3K4me3) mark closely correlates with active transcription. E2F-responsive promoters display dynamic changes in H3K4 methylation during the course of cell cycle progression. However, how and when these marks are reset, is not known. Here we show that the retinoblastoma binding protein RBP2/KDM5A, capable of removing tri-methylation marks on H3K4, associates with the E2F4 transcription factor via the pocket protein-p130-in a cell-cycle-stage specific manner. The association of RBP2 with p130 is LxCxE motif dependent. RNAi experiments reveal that p130 recruits RBP2 to E2F-responsive promoters in early G1 phase to bring about H3K4 demethylation and gene repression. A point mutation in LxCxE motif of RBP2 renders it incapable of p130-interaction and hence, repression of E2F-regulated gene promoters. We also examine how RBP2 may be recruited to non-E2F responsive promoters. Our studies provide insight into how the chromatin landscape needs to be adjusted rapidly and periodically during cell-cycle progression, concomitantly with temporal transcription, to bring about expression/repression of specific gene sets.

SUBMITTER: Zargar ZU

PROVIDER: S-EPMC5758877 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Dynamic site-specific recruitment of RBP2 by pocket protein p130 modulates H3K4 methylation on E2F-responsive promoters.

Zargar Zaffer Ullah ZU Kimidi Mallikharjuna Rao MR Tyagi Shweta S

Nucleic acids research 20180101 1

The Histone 3 lysine 4 methylation (H3K4me3) mark closely correlates with active transcription. E2F-responsive promoters display dynamic changes in H3K4 methylation during the course of cell cycle progression. However, how and when these marks are reset, is not known. Here we show that the retinoblastoma binding protein RBP2/KDM5A, capable of removing tri-methylation marks on H3K4, associates with the E2F4 transcription factor via the pocket protein-p130-in a cell-cycle-stage specific manner. Th ...[more]

PMID: 29059406

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Project description:BackgroundPax6 is a key regulator of the entire cascade of ocular lens formation through specific binding to promoters and enhancers of batteries of target genes. The promoters and enhancers communicate with each other through DNA looping mediated by multiple protein-DNA and protein-protein interactions and are marked by specific combinations of histone posttranslational modifications (PTMs). Enhancers are distinguished from bulk chromatin by specific modifications of core histone H3, including H3K4me1 and H3K27ac, while promoters show increased H3K4me3 PTM. Previous studies have shown the presence of Pax6 in as much as 1/8 of lens-specific enhancers but a much smaller fraction of tissue-specific promoters. Although Pax6 is known to interact with EP300/p300 histone acetyltransferase responsible for generation of H3K27ac, a potential link between Pax6 and histone H3K4 methylation remains to be established.ResultsHere we show that Pax6 co-purifies with H3K4 methyltransferase activity in lens cell nuclear extracts. Proteomic studies show that Pax6 immunoprecipitates with Set1a, Mll1, and Mll2 enzymes, and their associated proteins, i.e., Wdr5, Rbbp5, Ash2l, and Dpy30. ChIP-seq studies using chromatin prepared from mouse lens and cultured lens cells demonstrate that Pax6-bound regions are mostly enriched with H3K4me2 and H3K4me1 in enhancers and promoters, though H3K4me3 marks only Pax6-containing promoters. The shRNA-mediated knockdown of Pax6 revealed down-regulation of a set of direct target genes, including Cap2, Farp1, Pax6, Plekha1, Prox1, Tshz2, and Zfp536. Pax6 knockdown was accompanied by reduced H3K4me1 at enhancers and H3K4me3 at promoters, with little or no changes of the H3K4me2 modifications. These changes were prominent in Plekha1, a gene regulated by Pax6 in both lens and retinal pigmented epithelium.ConclusionsOur study supports a general model of Pax6-mediated recruitment of histone methyltransferases Mll1 and Mll2 to lens chromatin, especially at distal enhancers. Genome-wide data in lens show that Pax6 binding correlates with H3K4me2, consistent with the idea that H3K4me2 PTMs correlate with the binding of transcription factors. Importantly, partial reduction of Pax6 induces prominent changes in local H3K4me1 and H3K4me3 modification. Together, these data open the field to mechanistic studies of Pax6, Mll1, Mll2, and H3K4me1/2/3 dynamics at distal enhancers and promoters of developmentally controlled genes.

Project description:Hepatitis C Virus (HCV) infection is associated with chronically evolving disease and development of hepatocellular carcinoma (HCC), albeit the mechanism of HCC induction by HCV is still controversial. The nucleocapsid (core) protein of HCV has been shown to be directly implicated in cellular transformation and immortalization, enhancing the effect of oncogenes and decreasing the one of tumor suppressor genes, as RB1 and its protein product pRB. With the aim of identifying novel molecular mechanisms of hepatocyte transformation by HCV, we examined the effect of HCV core protein on the expression of the whole Retinoblastoma (RB) family of tumor and growth suppressor factors, i.e. pRb, p107 and pRb2/p130.We used a model system consisting of the HuH-7, HCV-free, human hepatocellular carcinoma cell line and of the HuH-7-CORE cells derived from the former and constitutively expressing the HCV core protein. We determined pRb, p107 and pRb2/p130 protein and mRNA amount of the respective genes RB1, RBL1 and RBL2, RBL2 promoter activity and methylation as well as DNA methyltransferase 1 (DNMT1) and 3b (DNMT3b) expression level. The effect of pRb2/p130 over-expression on the HCV core-expressing HuH-7-CORE cells was also evaluated.We found that the HCV core protein expression down-regulated pRb2/p130 protein and mRNA levels in HuH-7-CORE cells by inducing promoter hyper-methylation with the concomitant up-regulation of DNMT1 and DNMT3b expression. When pRb2/p130 expression was artificially re-established in HuH-7-CORE cells, cell cycle analysis outlined an accumulation in the G0/G1 phase, as expected.HCV core appears indeed able to significantly down-regulate the expression and the function of two out of three RB family tumor and growth suppressor factors, i.e. pRb and pRb2/p130. The functional consequences at the level of cell cycle regulation, and possibly of more complex cell homeostatic processes, may represent a plausible molecular mechanism involved in liver transformation by HCV.

Dataset Information

Dynamic site-specific recruitment of RBP2 by pocket protein p130 modulates H3K4 methylation on E2F-responsive promoters.

Publications

Dynamic site-specific recruitment of RBP2 by pocket protein p130 modulates H3K4 methylation on E2F-responsive promoters.

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