Search

DNA methyltransferase (cytosine-5) 1 (Dnmt1) is the principal enzyme responsible for maintenance of CpG methylation and is essential for the regulation of gene expression, silencing of parasitic DNA elements, genomic imprinting and embryogenesis. Dnmt1 is needed in S phase to methylate newly replicated CpGs occurring opposite methylated ones on the mother strand of the DNA, which is essential for the epigenetic inheritance of methylation patterns in the genome. Despite an intrinsic affinity of Dnmt1 for such hemi-methylated DNA, the molecular mechanisms that ensure the correct loading of Dnmt1 onto newly replicated DNA in vivo are not understood. The Np95 (also known as Uhrf1 and ICBP90) protein binds methylated CpG through its SET and RING finger-associated (SRA) domain. Here we show that localization of mouse Np95 to replicating heterochromatin is dependent on the presence of hemi-methylated DNA. Np95 forms complexes with Dnmt1 and mediates the loading of Dnmt1 to replicating heterochromatic regions. By using Np95-deficient embryonic stem cells and embryos, we show that Np95 is essential in vivo to maintain global and local DNA methylation and to repress transcription of retrotransposons and imprinted genes. The link between hemi-methylated DNA, Np95 and Dnmt1 thus establishes key steps of the mechanism for epigenetic inheritance of DNA methylation.

DNA methyltransferase 1 (DNMT1) plays an important role in the inheritance of genomic DNA methylation, which is coupled to the DNA replication process. Early embryonic lethality in DNMT1-null mutant (Dnmt1(c)) mice indicates that DNA methylation is essential for mammalian development. DNMT1, however, interacts with a number of transcriptional regulators and has a transcriptional repressor activity independent of its catalytic activity. To examine the roles of the catalytic activity of DNMT1 in vivo, we generated a Dnmt1(ps) allele that expresses a point-mutated protein that lacks catalytic activity (DNMT1-C1229S). Dnmt1(ps) mutant mice showed developmental arrest shortly after gastrulation, near-complete loss of DNA methylation, and an altered distribution of repressive chromatin markers in the nuclei; these phenotypes are quite similar to those of the Dnmt1(c) mutant. The mutant DNMT1 protein failed to associate with replication foci in Dnmt1(ps) cells. Reconstitution experiments and replication labeling in Dnmt1-/- Dnmt3a-/- Dnmt3b-/- (i.e., unmethylated) embryonic stem cells revealed that preexisting DNA methylation is a major determinant for the cell cycle-dependent localization of DNMT1. The C-terminal catalytic domain of DNMT1 inhibited its stable association with unmethylated chromatin. Our results reveal essential roles for the DNA methylation mark in mammalian development and in DNMT1 localization.

Background: Environmental and endogenous factors under genetic predisposition are considered to initiate the human intervertebral disc (IVD) degeneration. DNA methylation is an essential mechanism to ensure cell-specific gene expression for normal development and tissue stability. Aberrant epigenetic alterations play a pivotal role in several diseases, including osteoarthritis. However, epigenetic alternations, including DNA methylation, in IVD degeneration have not been evaluated. The purpose of this study was to comprehensively compare the genome-wide DNA methylation profiles of human IVD tissues, specifically nucleus pulpous (NP) tissues, with early and advanced stages of disc degeneration. Methods: Human NP tissues were used in this study. The samples were divided into two groups: early stage degeneration (n = 8, Pfirrmann’s MRI grade: I-III) and advanced stage degeneration (n = 8, grade: IV). Genomic DNA was processed for genome-wide DNA methylation profiling using the Infinium MethylationEPIC BeadChip array. Extraction of raw methylation data, clustering and scatter plot of each group values of each sample were performed using a methylation module in GenomeStudio software. The identification of differentially methylated loci (DMLs) and the Gene Ontology (GO) analysis were performed using R software with the ChAMP package. Results: Unsupervised hierarchical clustering revealed that early and advanced stage degenerated IVD samples segregated into two main clusters by their DNA methylome. A total of 220 DMLs were identified between early and advanced disc degeneration stages. Among these, four loci were hypomethylated and 216 loci were hypermethylated in the advanced disc degeneration stage. The GO enrichment analysis of genes containing DMLs identified two significant GO terms for biological processes, hemophilic cell adhesion and cell-cell adhesion. Conclusions: We conducted a genome-wide DNA methylation profile comparative study and observed significant differences in DNA methylation profiles between early and advanced stages of human IVD degeneration. These results implicate DNA methylation in the process of human IVD degeneration. [ABSTRACT FROM AUTHOR]

Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the contexts of development and disease. DNA methylation by DNA methyltransferases (Dnmts) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell-based and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various Dnmt-mutant mouse embryonic stem (ES) cell lines that included a cell line with a drug-inducible Dnmt3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) was shown to be correlated with redistribution of H3K27me3 induced by DNA hypomethylation: Later replicating PH coincided with H3K27me3-enriched regions. In contrast, this relationship with H3K27me3 was not evident within chromosomal arm regions undergoing either early-to-late (EtoL) or late-to-early (LtoE) switching of replication timing upon loss of the Dnmts. Interestingly, Dnmt-sensitive transcriptional up- and downregulation frequently coincided with earlier and later shifts in replication timing of the chromosomal arm regions, respectively. Our study revealed the previously unrecognized complex and diverse effects of the Dnmts loss on the mammalian DNA replication landscape. [ABSTRACT FROM AUTHOR]