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ABSTRACT: Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage.
Journal of Biological Chemistry 07/2012; 287(35):29801-14. · 4.77 Impact Factor
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ABSTRACT: In an effort to understand the epigenetic regulation of ribosomal RNA gene (rDNA) expression we have previously demonstrated the role of DNA methyltransferases and methyl CpG binding proteins in rRNA synthesis. Here, we studied the role of protein arginine methyltransferase PRMT5 and the two methylated histones H3R8Me2 and H4R3Me2, in rDNA expression in Epstein Barr virus- transformed primary B-cells (LCLs) and in HeLa cells responding to serum-regulated growth. Chromatin immunoprecipitation assay showed that histones H3 and H4 associated with rRNA promoters were differentially methylated at arginine residues 8 and 3, respectively, depending on its transcriptional activity. Association of PRMT5 and methylated H3 with the unmethylated promoters in resting B-cells was significantly reduced in rapidly growing LCLs. Unlike PRMT5 and H3R8Me2, histone H4 associated with both methylated and unmethylated rRNA promoters in resting B-cells was methylated at the R3 residue. However, a dramatic decrease in R3 methylation of H4 recruited to the unmethylated rRNA promoters was observed in LCLs while it remained unaltered in the fraction bound to the methylated promoters. Differential interaction of PRMT5 and methylation of H3 and H4 associated with the rRNA promoters was also observed when serum starved HeLa cells were allowed to grow in serum replenished media. Ectopic expression of PRMT5 suppressed activity of both unmethylated and methylated rRNA promoter in transient transfection assay whereas siRNA mediated knockdown of PRMT5 increased rRNA synthesis in HeLa cells. These data suggest a key role of PRMT5 and the two methylated histones in regulating rRNA promoter activity.
Journal of Cellular Biochemistry 12/2009; 109(3):553-63. · 2.87 Impact Factor
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ABSTRACT: The three basic DNA-binding domain mutations of the microphthalmia-associated transcription factor (Mitf), Mitf(mi/mi), Mitf(or/or), and Mitf(wh/wh) affect osteoclast differentiation with variable penetrance while completely impairing melanocyte development. Mitf(or/or) mice exhibit osteopetrosis that improves with age and their osteoclasts form functional multinuclear osteoclasts, raising the question as to why the Mitf(or/or) mutation results in osteopetrosis. Here we show that Mitf(or/or) osteoclasts express normal levels of acid phosphatase 5 (Acp5) mRNA and significantly lower levels of Cathepsin K (Ctsk) mRNA during receptor activator of nuclear factor kappa B (NFkappaB) ligand (RANKL)-mediated differentiation. Studies using chromatin immunoprecipitation (ChIP) analysis indicate that low levels of Mitf(or/or) protein are recruited to the Ctsk promoter. However, enrichment of Mitf-transcriptional co-activators PU.1 and Brahma-related gene 1 (Brg1) are severely impaired at the Ctsk promoter of Mitf(or/or) osteoclast precursors, indicating that defective recruitment of co-activators by the mutant Mitf(or/or) results in impaired Ctsk expression in osteoclasts. Cathepsin K may thus represent a unique class of Mitf-regulated osteoclast-specific genes that are important for osteoclast function.
Journal of Cellular Physiology 04/2009; 220(1):230-7. · 3.87 Impact Factor
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ABSTRACT: CCAAT/enhancer binding proteindelta (C/EBPdelta) gene transcription is highly induced in G(0) growth arrested mammary epithelial cells and "loss of function" alterations in C/EBPdelta have been reported in human breast cancer. To gain a better understanding of the positive and negative factors that control C/EBPdelta gene expression we investigated the role of transcriptional activators, coactivators, repressors, histone modifications, chromatin remodeling and basal transcriptional machinery components in growing and growth arrested HC11 mouse mammary epithelial cells. Growth arrest treatments result in increased STAT3 activation (pSTAT3) and increased C/EBPdelta expression. Co-immunoprecipitation and chromatin immunoprecipitation (ChIP) assays demonstrated that pSTAT3 and Sp1 interact and bind to the transcriptionally active C/EBPdelta promoter. ChIP assays performed under exponentially growing (C/EBPdelta non-expressing) conditions demonstrated that the C/EBPdelta promoter is preloaded with transcriptional activators (Sp1 and CREB) and transcriptional machinery components (TBP and RNA Pol II). In contrast, under G(0) growth arrest (C/EBPdelta expressing) conditions ChIP analysis detected pSTAT3, Sp1, NCoA/SRC1, CBP/p300, pCREB, TBP, and serine 2 phosphorylated Pol II (pPol II) in association with the C/EBPdelta proximal promoter. C/EBPdelta promoter-associated histone post-translational modification analysis revealed histone H3 and H4 acetylation and methylation patterns consistent with a constitutively "open" chromatin conformation. Chromatin remodeling experiments demonstrated that BRG1, the ATPase component of the SWI/SNF chromatin remodeling complex, is required for C/EBPdelta transcription. Finally, C/EBPdelta expression is repressed in proliferating mammary epithelial cells by c-Myc via a mechanism that involves the binding of c-Myc:Max dimers to C/EBPdelta promoter-bound Miz-1. These results provide a molecular model of C/EBPdelta transcriptional regulation under G(0) growth arrest conditions.
Journal of Cellular Biochemistry 01/2008; 102(5):1256-70. · 2.87 Impact Factor
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ABSTRACT: CCAAT/enhancer binding proteinδ (C/EBPδ) gene transcription is highly induced in G0 growth arrested mammary epithelial cells and “loss of function” alterations in C/EBPδ have been reported in human breast cancer. To gain a better understanding of the positive and negative factors that control C/EBPδ gene expression we investigated the role of transcriptional activators, coactivators, repressors, histone modifications, chromatin remodeling and basal transcriptional machinery components in growing and growth arrested HC11 mouse mammary epithelial cells. Growth arrest treatments result in increased STAT3 activation (pSTAT3) and increased C/EBPδ expression. Co-immunoprecipitation and chromatin immunoprecipitation (ChIP) assays demonstrated that pSTAT3 and Sp1 interact and bind to the transcriptionally active C/EBPδ promoter. ChIP assays performed under exponentially growing (C/EBPδ non-expressing) conditions demonstrated that the C/EBPδ promoter is preloaded with transcriptional activators (Sp1 and CREB) and transcriptional machinery components (TBP and RNA Pol II). In contrast, under G0 growth arrest (C/EBPδ expressing) conditions ChIP analysis detected pSTAT3, Sp1, NCoA/SRC1, CBP/p300, pCREB, TBP, and serine 2 phosphorylated Pol II (pPol II) in association with the C/EBPδ proximal promoter. C/EBPδ promoter-associated histone post-translational modification analysis revealed histone H3 and H4 acetylation and methylation patterns consistent with a constitutively “open” chromatin conformation. Chromatin remodeling experiments demonstrated that BRG1, the ATPase component of the SWI/SNF chromatin remodeling complex, is required for C/EBPδ transcription. Finally, C/EBPδ expression is repressed in proliferating mammary epithelial cells by c-Myc via a mechanism that involves the binding of c-Myc:Max dimers to C/EBPδ promoter-bound Miz-1. These results provide a molecular model of C/EBPδ transcriptional regulation under G0 growth arrest conditions. J. Cell. Biochem. 102: 1256–1270, 2007. © 2007 Wiley-Liss, Inc.
Journal of Cellular Biochemistry 11/2007; 102(5):1256 - 1270. · 2.87 Impact Factor
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ABSTRACT: Transcription factors NFATc1, PU.1, and MITF collaborate to regulate specific genes in response to colony-stimulating factor-1 (CSF-1) and receptor activator of NF-kappaB ligand (RANKL) signaling during osteoclast differentiation. However, molecular details concerning timing and mechanism of specific events remain ill-defined. In bone marrow-derived precursors, CSF-1 alone promoted assembly of MITF-PU.1 complexes at osteoclast target gene promoters like cathepsin K and acid 5 phosphatase without increasing gene expression. The combination of RANKL and CSF-1 concurrently increased the levels of MAPK-phosphorylated forms of MITF, p38 MAPK, and SWI/SNF chromatin-remodeling complexes bound to these target promoters and markedly increased expression of the genes. NFATc1 was subsequently recruited to complexes at the promoters during terminal stages of osteoclast differentiation. Genetic analysis of Mitf and Pu.1 in mouse models supported the critical interaction of these genes in osteoclast differentiation. The results define MITF and PU.1 as nuclear effectors that integrate CSF-1/RANKL signals during osteoclast differentiation to initiate expression of target genes, whereas a complex that includes NFATc1 may act to maintain target gene expression in differentiated cells.
Journal of Biological Chemistry 05/2007; 282(21):15921-9. · 4.77 Impact Factor
