Modulation of Dnmt3b function in vitro by interactions with Dnmt3L, Dnmt3a and Dnmt3b splice variants

DNA methylation, an essential regulator of transcription and chromatin structure, is established and maintained by the coordinated action of three DNA methyltransferases: DNMT1, DNMT3A and DNMT3B, and the inactive accessory factor DNMT3L. Disruptions in DNMT3B function are linked to carcinogenesis and genetic disease. DNMT3B is also highly alternatively spliced in a tissue- and disease-specific manner. The impact of intra-DNMT3 interactions and alternative splicing on the function of DNMT3 family members remains unclear. In the present work, we focused on DNMT3B. Using a panel of in vitro assays, we examined the consequences of DNMT3B splicing and mutations on its ability to bind DNA, interact with itself and other DNMT3's, and methylate DNA. Our results show that, while the C-terminal catalytic domain is critical for most DNMT3B functions, parts of the N-terminal region, including the PWWP domain, are also important. Alternative splicing and domain deletions also influence DNMT3B’s cellular localization. Furthermore, our data reveal the existence of extensive DNMT3B self-interactions that differentially impact on its activity. Finally, we show that catalytically inactive isoforms of DNMT3B are capable of modulating the activity of DNMT3A–DNMT3L complexes. Our studies therefore suggest that seemingly ‘inactive’ DNMT3B isoforms may influence genomic methylation patterns in vivo

Statistical Inference of In Vivo Properties of Human DNA Methyltransferases from Double-Stranded Methylation Patterns

DNA methyltransferases establish methylation patterns in cells and transmit these patterns over cell generations, thereby influencing each cell's epigenetic states. Three primary DNA methyltransferases have been identified in mammals: DNMT1, DNMT3A and DNMT3B. Extensive in vitro studies have investigated key properties of these enzymes, namely their substrate specificity and processivity. Here we study these properties in vivo, by applying novel statistical analysis methods to double-stranded DNA methylation patterns collected using hairpin-bisulfite PCR. Our analysis fits a novel Hidden Markov Model (HMM) to the observed data, allowing for potential bisulfite conversion errors, and yields statistical estimates of parameters that quantify enzyme processivity and substrate specificity. We apply this model to methylation patterns established in vivo at three loci in humans: two densely methylated inactive X (Xi)-linked loci ( and ), and an autosomal locus (), where methylation densities are tissue-specific but moderate. We find strong evidence for a high level of processivity of DNMT1 at and , with the mean association tract length being a few hundred base pairs. Regardless of tissue types, methylation patterns at are dominated by DNMT1 maintenance events, similar to the two Xi-linked loci, but are insufficiently informative regarding processivity to draw any conclusions about processivity at that locus. At all three loci we find that DNMT1 shows a strong preference for adding methyl groups to hemi-methylated CpG sites over unmethylated sites. The data at all three loci also suggest low (possibly 0) association of the de novo methyltransferases, the DNMT3s, and are consequently uninformative about processivity or preference of these enzymes. We also extend our HMM to reanalyze published data on mouse DNMT1 activities in vitro. The results suggest shorter association tracts (and hence weaker processivity), and much longer non-association tracts than human DNMT1 in vivo

Transcriptional provirus silencing as a crosstalk of de novo DNA methylation and epigenomic features at the integration site

The autonomous transcription of integrated retroviruses strongly depends on genetic and epigenetic effects of the chromatin at the site of integration. These effects are mostly suppressive and proviral activity can be finally silenced by mechanisms, such as DNA methylation and histone modifications. To address the role of the integration site at the whole-genome-scale, we performed clonal analysis of provirus silencing with an avian leucosis/sarcoma virus-based reporter vector and correlated the transcriptional silencing with the epigenomic landscape of respective integrations. We demonstrate efficient provirus silencing in human HCT116 cell line, which is strongly but not absolutely dependent on the de novo DNA methyltransferase activity, particularly of Dnmt3b. Proviruses integrated close to the transcription start sites of active genes into the regions enriched in H3K4 trimethylation display long-term stability of expression and are resistant to the transcriptional silencing after over-expression of Dnmt3a or Dnmt3b. In contrast, proviruses in the intergenic regions tend to spontaneous transcriptional silencing even in Dnmt3a−/− Dnmt3b−/− cells. The silencing of proviruses within genes is accompanied with DNA methylation of long terminal repeats, whereas silencing in intergenic regions is DNA methylation-independent. These findings indicate that the epigenomic features of integration sites are crucial for their permissivity to the proviral expression

In Vivo Control of CpG and Non-CpG DNA Methylation by DNA Methyltransferases

The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position–, cell type–, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs

Regulatory feedback from nascent RNA to chromatin and transcription

Transcription and chromatin function are regulated by proteins that bind to DNA, nucleosomes or RNA polymerase II, with specific non-coding RNAs (ncRNAs) functioning to modulate their recruitment or activity. Unlike ncRNAs, nascent pre-mRNA was considered to be primarily a passive player in these processes. In this Opinion article, we describe recently identified interactions between nascent pre-mRNAs and regulatory proteins, highlight commonalities between the functions of nascent pre-mRNA and nascent ncRNA, and propose that both types of RNA have an active role in transcription and chromatin regulation

The complexity of epigenetic diseases

Over the past 30 years, a plethora of pathogenic mutations affecting enhancer regions and epigenetic regulators have been identified. Coupled with more recent genome‐wide association studies (GWAS) and epigenome‐wide association studies (EWAS) implicating major roles for regulatory mutations in disease, it is clear that epigenetic mechanisms represent important biomarkers for disease development and perhaps even therapeutic targets. Here, we discuss the diversity of disease‐causing mutations in enhancers and epigenetic regulators, with a particular focus on cancer. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

Cooperative DNA Binding and Protein/DNA Fiber Formation Increases the Activity of the Dnmt3a DNA Methyltransferase

The Dnmt3a DNA methyltransferase has been shown to bind cooperatively to DNA and to form large multimeric protein/DNA fibers. However, it has also been reported to methylate DNA in a processive manner, a property that is incompatible with protein/DNA fiber formation. We show here that the DNA methylation rate of Dnmt3a increases more than linearly with increasing enzyme concentration on a long DNA substrate, but not on a short 30-mer oligonucleotide substrate. We also show that addition of a catalytically inactive Dnmt3a mutant, which carries an amino acid exchange in the catalytic center, increases the DNA methylation rate by wild type Dnmt3a on the long substrate but not on the short one. In agreement with this finding, preincubation experiments indicate that stable protein/DNA fibers are formed on the long, but not on the short substrate. In addition, methylation experiments with substrates containing one or two CpG sites did not provide evidence for a processive mechanism over a wide range of enzyme concentrations. These data clearly indicate that Dnmt3a binds to DNA in a cooperative reaction and that the formation of stable protein/DNA fibers increases the DNA methylation rate. Fiber formation occurs at low μm concentrations of Dnmt3a, which are in the range of Dnmt3a concentrations in the nucleus of embryonic stem cells. Understanding the mechanism of Dnmt3a is of vital importance because Dnmt3a is a hotspot of somatic cancer mutations one of which has been implicated in changing Dnmt3a processivity