Regulation of DNA Methylation Patterns by CK2-Mediated Phosphorylation of Dnmt3a

DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns

Functional Connection between Deimination and Deacetylation of Histones▿

Histone methylation plays key roles in regulating chromatin structure and function. The recent identification of enzymes that antagonize or remove histone methylation offers new opportunities to appreciate histone methylation plasticity in the regulation of epigenetic pathways. Peptidylarginine deiminase 4 (PADI4; also known as PAD4) was the first enzyme shown to antagonize histone methylation. PADI4 functions as a histone deiminase converting a methylarginine residue to citrulline at specific sites on the tails of histones H3 and H4. This activity is linked to repression of the estrogen-regulated pS2 promoter. Very little is known as to how PADI4 silences gene expression. We show here that PADI4 associates with the histone deacetylase 1 (HDAC1). Kinetic chromatin immunoprecipitation assays revealed that PADI4 and HDAC1, and the corresponding activities, associate cyclically and coordinately with the pS2 promoter during repression phases. Knockdown of HDAC1 led to decreased H3 citrullination, concomitantly with increased histone arginine methylation. In cells with a reduced HDAC1 and a slightly decreased PADI4 level, these effects were more pronounced. Our data thus suggest that PADI4 and HDAC1 collaborate to generate a repressive chromatin environment on the pS2 promoter. These findings further substantiate the “transcriptional clock” concept, highlighting the dynamic connection between deimination and deacetylation of histones

Functional and Direct Connection between Deimination and Deacetylation of Histone

Histone methylation plays key roles in the regulation of chromatin structure and function. The recent identification of enzymes that antagonize or remove histone methylation offers new opportunities to appreciate histone methylation plasticity in the regulation of epigenetic pathways. Peptidylarginine deiminase 4 (PADI4) was the first enzyme shown to antagonize histone methylation. PADI4 functions as a histone deiminase converting a methylarginine residue to citrulline at specific sites on the tail of histones H3 and H4. This activity is linked to repression of the estrogen-regulated pS2 promoter. Very little is known as to how PADI4 silences gene expression. To investigate the mechanisms by which histone demethylation and in particular PADI4 functions, and as no PADI4 interactors have been described so far, in vitro interaction assays and coimmunoprecipitations were performed. We found that the histone deacetylase HDAC1 interacts with PADI4, both in vitro and in vivo, and associates with PADI4-mediated histone deiminase activity. Chromatin immunoprecipitations in MDA-ER66 cells using antibodies against PADI4, HDAC1, citrulline H3 and acetylated histones show that PADI4 and HDAC1 appear transiently and in a cyclic manner on the estrogen-responsive promoter pS2, in the presence of estradiol. Their presence correlates with the loss of arginine methylation, acquisition of citrulline, histone deacetylation, and disengagement of RNA polymerase II from the pS2 promoter. Furthermore, sequential ChIP further indicated that PADI4 and HDAC1 bind together to the pS2 promoter in the presence of estrogen. These results further substantiate the “transcriptional clock” concept, highlighting the dynamic interplay between deimination and deacetylation of histones.info:eu-repo/semantics/nonPublishe

Citrullination of DNMT3A by PADI4 regulates its stability and controls DNA methylation

DNA methylation is a central epigenetic modification in mammals, with essential roles in development and disease. De novo DNA methyltransferases establish DNA methylation patterns in specific regions within the genome by mechanisms that remain poorly understood. Here we show that protein citrullination by peptidylarginine deiminase 4 (PADI4) affects the function of the DNA methyltransferase DNMT3A. We found that DNMT3A and PADI4 interact, from overexpressed as well as untransfected cells, and associate with each other's enzymatic activity. Both in vitro and in vivo, PADI4 was shown to citrullinate DNMT3A. We identified a sequence upstream of the PWWP domain of DNMT3A as its primary region citrullinated by PADI4. Increasing the PADI4 level caused the DNMT3A protein level to increase as well, provided that the PADI4 was catalytically active, and RNAi targeting PADI4 caused reduced DNMT3A levels. Accordingly, pulse-chase experiments revealed stabilization of the DNMT3A protein by catalytically active PADI4. Citrullination and increased expression of native DNMT3A by PADI4 were confirmed in PADI4-knockout MEFs. Finally, we showed that PADI4 overexpression increases DNA methyltransferase activity in a catalytic-dependent manner and use bisulfite pyrosequencing to demonstrate that PADI4 knockdown causes significant reduction of CpG methylation at the p21 promoter, a known target of DNMT3A and PADI4. Protein citrullination by PADI4 thus emerges as a novel mechanism for controlling a de novo DNA methyltransferase. Our results shed new light on how post-translational modifications might contribute to shaping the genomic CpG methylation landscape. © 2014 The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Dnmt3L is a transcriptional repressor that recruits histone deacetylase

The Dnmt3L protein belongs to the Dnmt3 family of DNA methyltransferases by virtue of its sequence homology in the plant homeodomain (PHD)-like motif. Dnmt3L is essential for the establishment of maternal genomic imprints and, given its lack of key methyltransferase motifs, is more likely to act as a regulator of methylation rather than as an enzyme that methylates DNA. Here, we show that Dnmt3L, like Dnmt3a and Dnmt3b, interacts both in vitro and in vivo with the histone deacetylase HDAC1. Consistent with the binding to a deacetylase, Dnmt3L purifies histone deacetylase activity from nuclear extracts. We find that Dnmt3L can repress transcription and that this repression is dependent on HDAC1 and is relieved by treatment with the HDAC inhibitor trichostatin A. Binding of Dnmt3L to HDAC1 as well as its repressive function require the PHD-like motif. Our results indicate that Dnmt3L plays a role in transcriptional regulation and that recruitment of the HDAC repressive machinery is a shared and conserved feature of the Dnmt3 family. The fact that, despite the absence of a methyltransferase domain, Dnmt3L retains the capacity to contact deacetylase further substantiates the notion that the Dnmts can repress transcription independently of their methylating activities

Comprehensive identification of long noncoding RNAs in colorectal cancer

Colorectal cancer (CRC) is one of the most common cancers in humans and a leading cause of cancer-related deaths worldwide. As in the case of other cancers, CRC heterogeneity leads to a wide range of clinical outcomes and complicates therapy. Over the years, multiple factors have emerged as markers of CRC heterogeneity, improving tumor classification and selection of therapeutic strategies. Understanding the molecular mechanisms underlying this heterogeneity remains a major challenge. A considerable research effort is therefore devoted to identifying additional features of colorectal tumors, in order to better understand CRC etiology and to multiply therapeutic avenues. Recently, long noncoding RNAs (lncRNAs) have emerged as important players in physiological and pathological processes, including CRC. Here we looked for lncRNAs that might contribute to the various colorectal tumor phenotypes. We thus monitored the expression of 4898 lncRNA genes across 566 CRC samples and identified 282 lncRNAs reflecting CRC heterogeneity. We then inferred potential functions of these lncRNAs. Our results highlight lncRNAs that may participate in the major processes altered in distinct CRC cases, such as WNT/β-catenin and TGF-β signaling, immunity, the epithelial-to-mesenchymal transition (EMT), and angiogenesis. For several candidates, we provide experimental evidence supporting our functional predictions that they may be involved in the cell cycle or the EMT. Overall, our work identifies lncRNAs associated with key CRC characteristics and provides insights into their respective functions. Our findings constitute a further step towards understanding the contribution of lncRNAs to CRC heterogeneity. They may open new therapeutic opportunities.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

MicroRNAs regulate KDM5 histone demethylases in breast cancer cells.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Alteration of miRNA levels is common in tumors and contributes to the pathogenesis of human malignancies. In the present study we examined the role played by miR-137 in breast tumorigenesis. We found miR-137 levels to be lower in breast cancer cells than in their non-tumorigenic counterparts and observed reduced proliferation and migration of breast cancer cells overexpressing miR-137. We further identified KDM5B, a histone demethylase known to be involved in breast cancer tumorigenesis, as a target of miR-137. As the involvement of histone demethylases in cancer is still poorly understood and as the role of miRNAs in controlling epigenetic mechanisms in cancer is emerging, we broadened our study to the whole KDM5 histone demethylase family to see if the genes coding for these epigenetic enzymes might be regulated by miRNAs in cancer cells. We discovered that KDM5C is overexpressed in breast cancer cells, providing evidence that miR-138 regulates its expression. We found miR-138 overexpression to affect breast cancer cell proliferation. Altogether, our findings suggest that miRNAs may regulate KDM5 histone demethylase levels in breast cancer and thereby control breast cancer cell proliferation and migration.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Expression of the Ets transcription factor Erm is regulated through a conventional PKC signaling pathway in the Molt4 lymphoblastic cell line.

Erm, a member of the PEA3 group within the Ets family of transcription factors, is expressed in murine and human lymphocytes. Here, we show that in the human Molt4 lymphoblastic cell line, the erm gene expression is regulated by the conventional PKC (cPKC) pathway. To better characterize the molecular mechanism by which cPKC regulates Erm transcription in Molt4 cells, we tested proximal promoter deletions of the human gene, and identified a specific cPKC-regulated region between positions -420 and -115 upstream of the first exon.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

The interplay between the lysine demethylase KDM1A and DNA methyltransferases in cancer cells is cell cycle dependent

International audienceDNA methylation and histone modifications are key epigenetic regulators of gene expression, and tight connections are known between the two. DNA methyltransferases are upregulated in several tumors and aberrant DNA methylation profiles are a cancer hallmark. On the other hand, histone demethylases are upregulated in cancer cells. Previous work on ES cells has shown that the lysine demethylase KDM1A binds to DNMT1, thereby affecting DNA methylation. In cancer cells, the occurrence of this interaction has not been explored. Here we demonstrate in several tumor cell lines an interaction between KDM1A and both DNMT1 and DNMT3B. Intriguingly and in contrast to what is observed in ES cells, KDM1A depletion in cancer cells was foundnot to trigger any reduction in the DNMT1 or DNMT3B protein level or any change in DNA methylation. In the S-phase, furthermore, KDM1A and DNMT1 were found, to colocalize within the heterochromatin. Using P-LISA, we revealed substantially increased binding of KDM1A to DNMT1 during the S-phase. Together, our findings propose a mechanistic link between KDM1A and DNA methyltransferases in cancer cells and suggest that the KDM1A/DNMT1 interaction may play a role during replication. Our work also strengthens the idea that DNMTs can exert functions unrelated to act on DNA methylation

Portraying breast cancers with long noncoding RNAs

Evidence is emerging that long noncoding RNAs (lncRNAs) may play a role in cancer development, but this role is not yet clear. We performed a genome-wide transcriptional survey to explore the lncRNA landscape across 995 breast tissue samples. We identified 215 lncRNAs whose genes are aberrantly expressed in breast tumors, as compared to normal samples. Unsupervised hierarchical clustering of breast tumors on the basis of their lncRNAs revealed four breast cancer subgroups that correlate tightly with PAM50-defined mRNA-based subtypes. Using multivariate analysis, we identified no less than 210 lncRNAs prognostic of clinical outcome. By analyzing the coexpression of lncRNA genes and protein-coding genes, we inferred potential functions of the 215 dysregulated lncRNAs. We then associated subtype-specific lncRNAs with key molecular processes involved in cancer. A correlation was observed, on the one hand, between luminal A–specific lncRNAs and the activation of phosphatidylinositol 3-kinase, fibroblast growth factor, and transforming growth factor–β pathways and, on the other hand, between basal-like–specific lncRNAs and the activation of epidermal growth factor receptor (EGFR)–dependent pathways and of the epithelial-to-mesenchymal transition. Finally, we showed that a specific lncRNA, which we called CYTOR, plays a role in breast cancer. We confirmed its predicted functions, showing that it regulates genes involved in the EGFR/mammalian target of rapamycin pathway and is required for cell proliferation, cell migration, and cytoskeleton organization. Overall, our work provides the most comprehensive analyses for lncRNA in breast cancers. Our findings suggest a wide range of biological functions associated with lncRNAs in breast cancer and provide a foundation for functional investigations that could lead to new therapeutic approaches.SCOPUS: ar.jinfo:eu-repo/semantics/publishe