DNA methylation and DNA methyltransferases

The prevailing views as to the form, function, and regulation of genomic methylation patterns have their origin many years in the past, at a time when the structure of the mammalian genome was only dimly perceived, when the number of protein-encoding mammalian genes was believed to be at least five times greater than the actual number, and when it was not understood that only ~10% of the genome is under selective pressure and likely to have biological function. We use more recent findings from genome biology and whole-genome methylation profiling to provide a reappraisal of the shape of genomic methylation patterns and the nature of the changes that they undergo during gametogenesis and early development. We observe that the sequences that undergo deep changes in methylation status during early development are largely sequences without regulatory function. We also discuss recent findings that begin to explain the remarkable fidelity of maintenance methylation. Rather than a general overview of DNA methylation in mammals (which has been the subject of many reviews), we present a new analysis of the distribution of methylated CpG dinucleotides across the multiple sequence compartments that make up the mammalian genome, and we offer an updated interpretation of the nature of the changes in methylation patterns that occur in germ cells and early embryos. We discuss the cues that might designate specific sequences for demethylation or de novo methylation during development, and we summarize recent findings on mechanisms that maintain methylation patterns in mammalian genomes. We also describe the several human disorders, each very different from the other, that are caused by mutations in DNA methyltransferase genes

Cytosine Methylation: Remaining Faithful

SummaryDNA methyltransferase-1 (DNMT1) has a higher specific activity on hemimethylated DNA than on unmethylated DNA, but this preference is too small to explain the faithful mitotic inheritance of genomic methylation patterns. New genetic studies in plants and mammals have identified a novel factor that increases the fidelity of maintenance methylation

Abnormal X chromosome inactivation and sex-specific gene dysregulation after ablation of FBXL10

BACKGROUND: Almost all CpG-rich promoters in the mammalian genome are bound by the multidomain FBXL10 protein (also known as KDM2B, JHDM1B, CXXC2, and NDY1). FBXL10 is expressed as two isoforms: FBXL10-1, a longer form that contains an N-terminal histone demethylase domain with C-terminal F-box, CXXC, PHD, RING, and leucine-rich repeat domains, and FBXL10-2, a shorter form that initiates at an alternative internal exon and which lacks the histone demethylase domain but retains all other annotated domains. Selective deletion of Fbxl10-1 had been reported to produce a low penetrance and variable phenotype; most of the mutant animals were essentially normal. We constructed mutant mouse strains that were either null for Fbxl10-2 but wild type for Fbxl10-1 or null for both Fbxl10-1 and Fbxl10-2. RESULTS: Deletion of Fbxl10-2 (in a manner that does not perturb expression of Fbxl10-1) produced a phenotype very different from the Fbxl10-1 mutant, with craniofacial abnormalities, neural tube defects, and increased lethality, especially in females. Mutants that lacked both FBXL10-1 and FBXL10-2 showed embryonic lethality and even more extreme sexual dimorphism, with more severe gene dysregulation in mutant female embryos. X-linked genes were most severely dysregulated, and there was marked overexpression of Xist in mutant females although genes that encode factors that bind to Xist RNA were globally downregulated in mutant female as compared to male embryos. CONCLUSIONS: FBXL10 is the first factor shown to be required both for the normal expression and function of the Xist gene and for normal expression of proteins that associate with Xist RNA; it is proposed that FBXL10 coordinates the expression of Xist RNA with proteins that associate with this RNA. The function of FBXL10 is largely independent of the histone demethylase activity of the long form of the protein. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13072-016-0069-1) contains supplementary material, which is available to authorized users

Decatenation checkpoint deficiency in stem and progenitor cells

SummaryThe decatenation checkpoint normally delays entry into mitosis until chromosomes have been disentangled through the action of topoisomerase II. We have found that the decatenation checkpoint is highly inefficient in mouse embryonic stem cells, mouse neural progenitor cells, and human CD34+ hematopoietic progenitor cells. Checkpoint efficiency increased when embryonic stem cells were induced to differentiate, which suggests that the deficiency is a feature of the undifferentiated state. Embryonic stem cells completed cell division in the presence of entangled chromosomes, which resulted in severe aneuploidy in the daughter cells. The decatenation checkpoint deficiency is likely to increase the rates of chromosome aberrations in progenitor cells, stem cells, and cancer stem cells

Reactivation of a silenced H19 gene in human rhabdomyosarcoma by demethylation of DNA but not by histone hyperacetylation

BACKGROUND: The active copy of the imprinted gene H19 is turned off by inappropriate methylation in several pediatric tumors including Wilms' Tumour and embryonal rhabdomyosarcoma. H19 controls in cis the linked Insulin-like Growth Factor 2 (IGF2) gene, encoding an important growth factor. Recent work has suggested that methylation of a gene may lead to deacetylation of its associated histones and that silenced genes can be reactivated by increasing histone acetylation levels. RESULTS: Treatment of a rhabdomyosarcoma cell line which has a silent, methylated H19 gene with histone deacetylase (HDAC) inhibitors under conditions which gave maximal hyperacetylation of histone 4, both globally and at the H19 gene itself could not reactivate H19 or affect the active Insulin-like Growth Factor 2 (IGF2) gene, but caused clear up-regulation of the Tissue-type Plasminogen Activator (TPA) gene, a non-imprinted gene known to respond to changes in histone acetylation. In contrast, mild treatment of the cells with the methylation inhibitor 5-AzaC-2'-deoxycytidine (AzaC) on its own was able to reactivate H19. Combining AzaC treatment with HDAC inhibitors gave a reduced rather than enhanced reactivation. These findings were confirmed in mouse primary liver and kidney explants which maintain normal imprinting, where we also found that the silent Igf2 gene could not be reactivated by HDAC inhibitors. CONCLUSION: These results suggest that DNA methylation rather than histone acetylation is the primary determinant of silencing of H19 in rhabdomyosarcoma

Coordinate regulation of DNA methyltransferase expression during oogenesis

<p>Abstract</p> <p>Background</p> <p>Normal mammalian development requires the action of DNA methyltransferases (DNMTs) for the establishment and maintenance of DNA methylation within repeat elements and imprinted genes. Here we report the expression dynamics of <it>Dnmt3a </it>and <it>Dnmt3b</it>, as well as a regulator of DNA methylation, <it>Dnmt3L</it>, in isolated female germ cells.</p> <p>Results</p> <p>Our results indicate that these enzymes are coordinately regulated and that their expression peaks during the stage of postnatal oocyte development when maternal methylation imprints are established. We find that Dnmt3a, Dnmt3b, Dnmt3L and Dnmt1o transcript accumulation is related to oocyte diameter. Furthermore, DNMT3L deficient 15 dpp oocytes have aberrantly methylated <it>Snrpn</it>, <it>Peg3 </it>and <it>Igf2r </it>DMRs, but normal IAP and LINE-1 methylation levels, thereby highlighting a male germ cell specific role for DNMT3L in the establishment of DNA methylation at repeat elements. Finally, real-time RT-PCR analysis indicates that the depletion of either DNMT3L or DNMT1o in growing oocytes results in the increased expression of the <it>de novo </it>methyltransferase <it>Dnmt3b</it>, suggesting a potential compensation mechanism by this enzyme for the loss of one of the other DNA methyltransferases.</p> <p>Conclusion</p> <p>Together these results provide a better understanding of the developmental regulation of <it>Dnmt3a</it>, <it>Dnmt3b </it>and <it>Dnmt3L </it>at the time of <it>de novo </it>methylation during oogenesis and demonstrate that the involvement of DNMT3L in retrotransposon silencing is restricted to the male germ line. This in turn suggests the existence of other factors in the oocyte that direct DNA methylation to transposons.</p

Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L

<p>Abstract</p> <p>Background</p> <p>Formation of haploid spermatozoa capable of fertilization requires proper programming of epigenetic information. Exactly how DNMT3L (DNA methyltransferase 3-Like), a postulated regulator of DNA methyltransferase activity, contributes to DNA methylation pattern acquisition during gametogenesis remains unclear. Here we report on the role of DNMT3L in male germ cell development.</p> <p>Results</p> <p>A developmental study covering the first 12 days following birth was conducted on a <it>Dnmt3L </it>mutant mouse model; lower germ cell numbers and delayed entry into meiosis were observed in <it>Dnmt3L</it>^-/-males, pointing to a mitotic defect. A temporal expression study showed that expression of <it>Dnmt3L </it>is highest in prenatal gonocytes but is also detected and developmentally regulated during spermatogenesis. Using a restriction enzyme qPCR assay (qAMP), DNA methylation analyses were conducted on postnatal primitive type A spermatogonia lacking DNMT3L. Methylation levels along 61 sites across chromosomes 4 and X decreased significantly by approximately 50% compared to the levels observed in <it>Dnmt3L</it>^+/+germ cells, suggesting that many loci throughout the genome are marked for methylation by DNMT3L. More so, hypomethylation was more pronounced in regions of lower GC content than in regions of higher GC content.</p> <p>Conclusion</p> <p>Taken together, these data suggest that DNMT3L plays a more global role in genomic methylation patterning than previously believed.</p

Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation

Chromosome synapsis during zygotene is a prerequisite for the timely homologous recombinational repair of meiotic DNA double-strand breaks (DSBs). Unrepaired DSBs are thought to trigger apoptosis during midpachytene of male meiosis if synapsis fails. An early pachytene response to asynapsis is meiotic silencing of unsynapsed chromatin (MSUC), which, in normal males, silences the X and Y chromosomes (meiotic sex chromosome inactivation [MSCI]). In this study, we show that MSUC occurs in Spo11-null mouse spermatocytes with extensive asynapsis but lacking meiotic DSBs. In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response. We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs. All four mutants fail to silence the X and Y chromosomes (MSCI failure), which is sufficient to explain the midpachytene apoptosis. Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI

Dynamic instability of genomic methylation patterns in pluripotent stem cells

<p>Abstract</p> <p>Background</p> <p>Genomic methylation patterns are established during gametogenesis, and perpetuated in somatic cells by faithful maintenance methylation. There have been previous indications that genomic methylation patterns may be less stable in embryonic stem (ES) cells than in differentiated somatic cells, but it is not known whether different mechanisms of <it>de novo </it>and maintenance methylation operate in pluripotent stem cells compared with differentiating somatic cells.</p> <p>Results</p> <p>In this paper, we show that ablation of the DNA methyltransferase regulator DNMT3L (DNA methyltransferase 3-like) in mouse ES cells renders them essentially incapable of <it>de novo </it>methylation of newly integrated retroviral DNA. We also show that ES cells lacking DNMT3L lose DNA methylation over time in culture, suggesting that DNA methylation in ES cells is the result of dynamic loss and gain of DNA methylation. We found that wild-type female ES cells lose DNA methylation at a much faster rate than do male ES cells; this defect could not be attributed to sex-specific differences in expression of DNMT3L or of any DNA methyltransferase. We also found that human ES and induced pluripotent stem cell lines showed marked but variable loss of methylation that could not be attributed to sex chromosome constitution or time in culture.</p> <p>Conclusions</p> <p>These data indicate that DNA methylation in pluripotent stem cells is much more dynamic and error-prone than is maintenance methylation in differentiated cells. DNA methylation requires DNMT3L in stem cells, but DNMT3L is not expressed in differentiating somatic cells. Error-prone maintenance methylation will introduce unpredictable phenotypic variation into clonal populations of pluripotent stem cells, and this variation is likely to be much more pronounced in cultured female cells. This epigenetic variability has obvious negative implications for the clinical applications of stem cells.</p