Structural DNMT-nucleosome contacts are related to DNA methylation patterns

Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana using massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified 10-base periodicities in the DNA methylation status of nucleosome-bound DNA and found that nucleosomal DNA was more highly methylated than flanking DNA. These results indicate that nucleosome positioning influences DNA methylation patterning throughout the genome and that DNA methyltransferases preferentially target nucleosome-bound DNA. We also observed similar trends in human nucleosomal DNA, indicating that the relationships between nucleosomes and DNA methyltransferases are conserved. Finally, as has been observed in animals, nucleosomes were highly enriched on exons, and preferentially positioned at intron-exon and exon-intron boundaries. RNA polymerase II (Pol II) was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is also enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition.

Epigenetics in evolution and disease

Nucleosome sliding is a frequent result of energy-dependent nucleosome remodelling in vitro. This review discusses the possible roles for nucleosome sliding in the assembly and maintenance of dynamic chromatin and for the regulation of diverse functions in eukaryotic nuclei.

Effects of DNA Methylation on Progression to Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Biopsies: A Multi-Omics Approach.

DNA (cytosine-5) methylation represents one of the most widely used mechanisms of enduring cellular memory. Stable patterns of DNA methylation are established during development, resulting in creation of persisting cellular phenotypes. There is growing evidence that the nervous system has co-opted a number of cellular mechanisms used during development to subserve the formation of long term memory. In this study, we examined the role DNA (cytosine-5) methyltransferase (DNMT) activity might play in regulating the induction of synaptic plasticity. We found that the DNA within promoters for reelin and brain-derived neurotrophic factor, genes implicated in the induction of synaptic plasticity in the adult hippocampus, exhibited rapid and dramatic changes in cytosine methylation when DNMT activity was inhibited. Moreover, zebularine and 5-aza-2-deoxycytidine, inhibitors of DNMT activity, blocked the induction of long term potentiation at Schaffer collateral synapses. Activation of protein kinase C in the hippocampus decreased reelin promoter methylation and increased DNMT3A gene expression. Interestingly, DNMT activity is required for protein kinase C-induced increases in histone H3 acetylation. Considered together, these results suggest that DNMT activity is dynamically regulated in the adult nervous system and that DNMT may play a role in regulating the induction of synaptic plasticity in the mature CNS.

In the cell, DNA is wrapped on histone octamers, which reduces its accessibility for DNA interacting enzymes. We investigated de novo methylation of nucleosomal DNA in vitro and show that the Dnmt3a and Dnmt1 DNA methyltransferases efficiently methylate nucleosomal DNA without dissociation of the histone octamer from the DNA. In contrast, the prokaryotic SssI DNA methyltransferase and the catalytic domain of Dnmt3a are strongly inhibited by nucleosomes. We also found that full-length Dnmt1 and Dnmt3a bind to nucleosomes much stronger than their isolated catalytic domains, demonstrating that the N-terminal parts of the MTases are required for the interaction with nucleosomes. Variations of the DNA sequence or the histone tails did not significantly influence the methylation activity of Dnmt3a. The observation that mammalian methyltransferases directly modify nucleosomal DNA provides an insight into the mechanisms by which histone tail and DNA methylation patterns can influence each other because the DNA methylation pattern can be established while histones remain associated to the DNA.

Epigenetic regulation of cellular identity and function is at least partly achieved through changes in covalent modifications on DNA and histones. Much progress has been made in recent years to understand how these covalent modifications affect cell identity and function. Despite the advances, whether and how epigenetic factors contribute to memory formation is still poorly understood. In this review, we discuss recent progress in elucidating epigenetic mechanisms of learning and memory, primarily at the DNA level, and look ahead to discuss their potential implications in reward memory and development of drug addiction.

DNA methylation modulates allograft survival and acute rejection after renal transplantation by regulating the mTOR pathway

Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.

Traditionally cancer is viewed as a disease driven by the accumulation of genetic alterations; nowadays this view has been expanded to include epigenetic alterations. Recent developments such as exome-sequencing have enabled researchers to analyze the entire exome of cancer cell lines and tissue samples to determine possible genetic mutations causing tumor onset/progression, leading to improved disease prognosis and treatment therapies. These studies have uncovered that genes encoding components of the epigenetic machinery are frequently mutated in urothelial cell carcinoma (UCC) of the bladder and many other tumor types. The effects of these mutations on the epigenetic landscape, e.g. DNA methylation, histone modification, and nucleosome positioning are as yet unknown. Given that the disruption of normal epigenetic patterns contributes to carcinogenesis, it is critical to study the effect of genetic alterations on the epigenome, since the latter are reversible by pharmacological intervention. In this study, we sequenced the exomes of 15 bladder cancer cell lines and compared them to 17 additional cell lines analyzed by the Cancer Cell Line Encyclopedia (CCLE). We have identified that epigenetic regulators in UCC of the bladder are commonly mutated, confirming other reports pertaining to UCC. The most frequently mutated genes include chromatin regulators such as KDM6A, ARID1A, HDAC4, and CREBBP. In addition to the discovery of genetic modifications in UCC cell lines, we studied the potential impact of these mutations on DNA methylation and nucleosome positioning by performing the Infinium Human Methylation 450K Array and the novel AcceSssIble assay, which is also based on the 450K array; comparing M.SssI CpG methyltransferase treated chromatin samples against controls, leading to the identification of nucleosome occupied CpG sites. To identify possible correlations between chromatin regulator mutations and DNA methylation patterns, we compared UCC cell lines bearing a specific chromatin regulator mutation to cell lines without the mutation and searched for significantly differentially methylated CpG sites. We found that UCC cell lines with mutations in ARID1A and HDAC4 show significant DNA methylation changes compared to cell lines not carrying the mutation. Our results show a possible direct impact of genetic mutations leading to epigenetic changes that may contribute to the tumor phenotype and might lead to novel therapeutic targets in UCC. Further studies will determine the effects of these specific chromatin regulator mutations on their target gene expression, DNA methylation and nucleosome occupancy. Loss and gain of function studies of these chromatin regulators will further our understanding of their roles in UCC tumor progression. The target genes of these studies will be further interrogated with commonly used epigenetic drugs such as 5-Azacitidine and HDAC inhibitors. Citation Format: Christopher E. Duymich, Jessica Charlet, Gangning Liang, Peter A. Jones. Role of chromatin remodeler mutations in urothelial cell carcinoma of the bladder. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A11.

DNA methylation and nucleosome positioning work together to generate chromatin structures that regulate gene expression. Nucleosomes are typically mapped using nuclease digestion but we have developed a method (NOMe-Seq) that uses a GpC methyltransferase (M.CviP1) and next generation sequencing to generate high resolution footprints of nucleosome positioning genome wide using less than 1 million cells while retaining endogenous DNA methylation information from the same strand. We have developed a novel bioinformatics pipeline that shows a striking anti-correlation between nucleosome occupancy and DNA methylation at CTCF regions that is not present at promoters. The extent of nucleosome depletion of promoters is directly correlated with expression level and can accommodate multiple nucleosomes. Genome wide evidence also shows that expressed non-CpG island promoters are nucleosome depleted. Because the method obtains DNA methylation and nucleosome positioning information from the same DNA molecule we can also use this method to track the relationship between nucleosome occupancy and DNA methylation during differentiation and mitosis. These experiments have shown that nucleosome occupancy precedes DNA methylation in the OCT4 promoter which is consistent with the idea that nucleosomal DNA is the preferred substrate for de novo DNA methylation. We have also used this approach to show that nucleosomes shift during mitosis to cover the transcription start sites of genes. This may be a mechanism to bookmark genes for expression in the following G1 phase of the cell cycle.

Genetic Control of Individual Differences in Gene-Specific Methylation in Human Brain

Aberrant DNA methylation is commonly observed in cancer and is characterized by genome-wide hypomethylation and gene-specific hypermethylation, which is thought to contribute to genomic instability and tumor suppressor gene silencing, respectively. The DNA methyltransferases (DNMTs) are responsible for the establishment (DNMT3A, DNMT3B, DNMT3L) and maintenance (DNMT1) of DNA methylation patterns genome-wide. The mechanism by which DNA methylation patterns are altered in cancer is not well understood. Genome-wide unique and overlapping target sites for each of the DNMTs are also unknown both in normal and cancer states. Identification of DNMT target loci is essential in order to better understand how aberrant DNA methylation occurs in cancer. To study this process, DNMT mRNA levels were depleted both individually and in a combinatorial fashion via RNAi-based techniques in embryonic carcinoma cells. Following reduction in DNMT expression, the resulting DNA and RNA was analyzed for genome-wide DNA methylation and gene expression patterns, respectively. An affinity purification method, Methyl-Binding Domain (MBD)-seq, was used to capture and enrich methylated regions of the genome by utilizing the methyl-binding domain of MBD2b. The enriched methylated DNA was then used to construct an Illumina sequencing library. Global gene expression patterns were analyzed by microarray for each RNA sample. Next generation sequencing and microarray data were analyzed using several available algorithms (e.g. MACS) and high-throughput data software packages (e.g. Partek Genomics Suite) in order to construct and evaluate the DNA methylation profiles and subsequent gene expression changes that result from DNMT depletion. Our data show that distinct changes in DNA methylation profiles occur among the various DNMT knockdown samples that permit us to identify unique and cooperative target loci for each DNMT. Further elucidation of DNMT target sites holds great promise for enhancing our understanding of mechanisms that control aberrant DNA methylation that is observed in cancer as well as provide insight and rationale for targeting specific DNMTs in cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5009. doi:1538-7445.AM2012-5009

To understand the role of CCAAT-binding factor (CBF) in transcription in the context of chromatin-assembled DNA, we used regularly spaced nucleosomal DNA using topoisomerase IIalpha (topo IIalpha) and alpha2(1) collagen promoter templates, which were subsequently reconstituted in an in vitro transcription reaction. Binding of CBF to the nucleosomal wild-type topo IIalpha promoter containing four CBF-binding sites disrupted the regular nucleosomal structure not only in the promoter region containing the CBF-binding sites but also in the downstream region over the transcription start site. In contrast, no nucleosome disruption was observed in a mutant topo IIalpha promoter containing mutations in all CBF-binding sites. Interestingly, CBF also activated transcription from nucleosomal wild-type topo IIalpha promoter. In this experiment, a promoter containing one wild-type CBF-binding site was activated very weakly, whereas the promoter containing mutations in all sites was not activated by CBF. A truncated CBF that lacked the glutamine-rich domains did not activate transcription from nucleosomal wild-type topo IIalpha promoter but disrupted the nucleosomal structure about as much as did the binding of full-length CBF. Two nucleosomal mouse alpha2(1) collagen promoter DNAs, one containing a single and the other containing four CBF- binding sites, were also reconstituted in an in vitro transcription reaction. None of the nucleosomal collagen promoters was activated by CBF. However, both of these collagen promoters were activated by CBF when the transcription reaction was performed using naked DNA templates. Binding of CBF to the nucleosomal collagen promoter containing four binding sites disrupted the nucleosomal structure, similarly as observed in the topo IIalpha promoter. Altogether this study indicates that CBF-mediated nucleosomal disruption occurred independently of transcription activation. It also suggests that specific promoter structure may play a role in the CBF-mediated transcription activation of nucleosomal topo IIalpha promoter template.

DNMT3 proteins are de novo DNA methyltransferases that are responsible for the establishment of DNA methylation patterns in mammalian genomes. Here, we have determined the crystal structures of the ATRX-DNMT3-DNMT3L (ADD) domain of DNMT3A in an unliganded form and in a complex with the amino-terminal tail of histone H3. Combined with the results of biochemical analysis, the complex structure indicates that DNMT3A recognizes the unmethylated state of lysine 4 in histone H3. This finding indicates that the recruitment of DNMT3A onto chromatin, and thereby de novo DNA methylation, is mediated by recognition of the histone modification state by its ADD domain. Furthermore, our biochemical and nuclear magnetic resonance data show mutually exclusive binding of the ADD domain of DNMT3A and the chromodomain of heterochromatin protein 1alpha to the H3 tail. These results indicate that de novo DNA methylation by DNMT3A requires the alteration of chromatin structure.