Lsh is essential for maintaining global DNA methylation levels in amphibia and fish and interacts directly with Dnmt1

Eukaryotic genomes are methylated at cytosine bases in the context of CpG dinucleotides, a pattern which is maintained through cell division by the DNA methyltransferase Dnmt1. Dramatic methylation losses are observed in plant and mouse cells lacking Lsh (lymphoid specific helicase), predominantly at repetitive sequences and gene promoters. However, the mechanism by which Lsh contributes to the maintenance of DNA methylation is unknown. Here we show that DNA methylation is lost in Lsh depleted frog and fish embryos, both of which exhibit developmental delay. Additionally, we show that both Lsh and Dnmt1 are associated with chromatin and that Lsh knockdown leads to a decreased Dnmt1-chromatin association. Coimmunoprecipitation experiments reveal that Lsh and Dnmt1 are found in the same protein complex, and pulldowns show this interaction is direct. Our data indicate that Lsh is usually diffuse in the nucleus but can be recruited to heterochromatin in a HP1α-dependent manner. These data together (a) show that the role of Lsh in DNA methylation is conserved in plants, amphibian, fish, and mice and (b) support a model in which Lsh contributes to Dnmt1 binding to chromatin, explaining how its loss can potentially lead to perturbations in DNA methylation maintenance

Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques

The epigenetic modification of 5-hydroxymethylcytosine (5hmC) is receiving great attention due to its potential role in DNA methylation reprogramming and as a cell state identifier. Given this interest, it is important to identify reliable and cost-effective methods for the enrichment of 5hmC marked DNA for downstream analysis. We tested three commonly used affinity-based enrichment techniques; (i) antibody, (ii) chemical capture and (iii) protein affinity enrichment and assessed their ability to accurately and reproducibly report 5hmC profiles in mouse tissues containing high (brain) and lower (liver) levels of 5hmC. The protein-affinity technique is a poor reporter of 5hmC profiles, delivering 5hmC patterns that are incompatible with other methods. Both antibody and chemical capture-based techniques generate highly similar genome-wide patterns for 5hmC, which are independently validated by standard quantitative PCR (qPCR) and glucosyl-sensitive restriction enzyme digestion (gRES-qPCR). Both antibody and chemical capture generated profiles reproducibly link to unique chromatin modification profiles associated with 5hmC. However, there appears to be a slight bias of the antibody to bind to regions of DNA rich in simple repeats. Ultimately, the increased specificity observed with chemical capture-based approaches makes this an attractive method for the analysis of locus-specific or genome-wide patterns of 5hm

Lsh regulates LTR retrotransposon repression independently of Dnmt3b function

BACKGROUND: DNA methylation contributes to genomic integrity by suppressing repeat-associated transposition. In addition to the canonical DNA methyltransferases, several auxiliary chromatin factors are required to maintain DNA methylation at intergenic and satellite repeats. The interaction between Lsh, a chromatin helicase, and the de novo methyltransferase Dnmt3b facilitates deposition of DNA methylation at stem cell genes, which are hypomethylated in Lsh(−/−) embryos. We wished to determine if a similar targeting mechanism operates to maintain DNA methylation at repetitive sequences. RESULTS: We mapped genome-wide DNA methylation patterns in Lsh(−/−) and Dnmt3b(−/−) somatic cells. DNA methylation is predominantly lost from specific genomic repeats in Lsh(−/−) cells: LTR -retrotransposons, LINE-1 repeats and mouse satellites. RNA-seq experiments demonstrate that specific IAP LTRs and satellites, but not LINE-1 elements, are aberrantly transcribed in Lsh(−/−) cells. LTR hypomethylation in Dnmt3b(−/−) cells is moderate, whereas IAP, LINE-1 and satellite elements are hypomethylated but silent. Repressed LINE-1 elements in Lsh(−/−) cells gain H3K4me3, but H3K9me3 levels are unaltered, indicating that DNA hypomethylation alone is not permissive for their transcriptional activation. Mis-expressed IAPs and satellites lose H3K9me3 and gain H3K4me3 in Lsh(−/−) cells. CONCLUSIONS: Our study emphasizes that regulation of repetitive elements by Lsh and DNA methylation is selective and context dependent. Silencing of repeats in somatic cells appears not to be critically dependent on Dnmt3b function. We propose a model where Lsh is specifically required at a precise developmental window to target de novo methylation to repeat sequences, which is subsequently maintained by Dnmt1 to enforce selective repeat silencing

Redistribution of H3K27me3 upon DNA hypomethylation results in de-repression of Polycomb target genes

BACKGROUND: DNA methylation and the Polycomb repression system are epigenetic mechanisms that play important roles in maintaining transcriptional repression. Recent evidence suggests that DNA methylation can attenuate the binding of Polycomb protein components to chromatin and thus plays a role in determining their genomic targeting. However, whether this role of DNA methylation is important in the context of transcriptional regulation is unclear. RESULTS: By genome-wide mapping of the Polycomb Repressive Complex 2-signature histone mark, H3K27me3, in severely DNA hypomethylated mouse somatic cells, we show that hypomethylation leads to widespread H3K27me3 redistribution, in a manner that reflects the local DNA methylation status in wild-type cells. Unexpectedly, we observe striking loss of H3K27me3 and Polycomb Repressive Complex 2 from Polycomb target gene promoters in DNA hypomethylated cells, including Hox gene clusters. Importantly, we show that many of these genes become ectopically expressed in DNA hypomethylated cells, consistent with loss of Polycomb-mediated repression. CONCLUSIONS: An intact DNA methylome is required for appropriate Polycomb-mediated gene repression by constraining Polycomb Repressive Complex 2 targeting. These observations identify a previously unappreciated role for DNA methylation in gene regulation and therefore influence our understanding of how this epigenetic mechanism contributes to normal development and disease

Defending the genome from the enemy within:mechanisms of retrotransposon suppression in the mouse germline

The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline

Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques

The epigenetic modification of 5-hydroxymethylcytosine (5hmC) is receiving great attention due to its potential role in DNA methylation reprogramming and as a cell state identifier. Given this interest, it is important to identify reliable and cost-effective methods for the enrichment of 5hmC marked DNA for downstream analysis. We tested three commonly used affinity-based enrichment techniques; (i) antibody, (ii) chemical capture and (iii) protein affinity enrichment and assessed their ability to accurately and reproducibly report 5hmC profiles in mouse tissues containing high (brain) and lower (liver) levels of 5hmC. The protein-affinity technique is a poor reporter of 5hmC profiles, delivering 5hmC patterns that are incompatible with other methods. Both antibody and chemical capture-based techniques generate highly similar genome-wide patterns for 5hmC, which are independently validated by standard quantitative PCR (qPCR) and glucosyl-sensitive restriction enzyme digestion (gRES-qPCR). Both antibody and chemical capture generated profiles reproducibly link to unique chromatin modification profiles associated with 5hmC. However, there appears to be a slight bias of the antibody to bind to regions of DNA rich in simple repeats. Ultimately, the increased specificity observed with chemical capture-based approaches makes this an attractive method for the analysis of locus-specific or genome-wide patterns of 5hmC

Apoptosis and DNA methylation

Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG

Rapid reprogramming of epigenetic and transcriptional profiles in mammalian culture systems

BackgroundThe DNA methylation profile of mammalian cell lines differs from the primary tissue from which they were derived, exhibiting increasing divergence from the in vivo methylation profile with extended time in culture. Few studies have directly examined the initial epigenetic and transcriptional consequences of adaptation of primary mammalian cells to culture, and the potential mechanisms through which this epigenetic dysregulation occurs is unknown.ResultsWe demonstrate that adaptation of mouse embryonic fibroblast, MEFS, to cell culture results in a rapid reprogramming of epigenetic and transcriptional states. We observed global 5-hydroxymethylcytosine (5hmC) erasure within three days of culture initiation. Loss of genic 5hmC was independent of global 5-methylcytosine (5mC) levels and could be partially rescued by addition of Vitamin C. Significantly, 5hmC loss was not linked to concomitant changes in transcription. Discrete promoter-specific gains of 5mC were also observed within seven days of culture initiation. Against this background of global 5hmC loss we identified a handful of developmentally important genes that maintained their 5hmC profile in culture, including the imprinted loci Gnas and H19. Similar outcomes were identified in the adaption of CD4+ T-cells to culture.ConclusionsWe report a dramatic and novel consequence of adaptation of mammalian cells to culture in which global loss of 5hmC occurs; suggesting rapid concomitant loss of methylcytosine dioxygenase activity. The observed epigenetic and transcriptional re-programming occurs much earlier than previously assumed, and has significant implications for the use of cell lines as faithful mimics of in vivo epigenetic and physiological processes.We thank Professors Adrian Bird and Nicholas Hastie for their comments on our manuscript. JT and RO are funded by IMI-MARCAR (under grant agreement number (115001) (MARCAR project)). Work in RM's lab is supported by the MRC, IMI-MARCAR and the BBSRC. This work in RM's lab was also initially funded by the Breakthrough Breast Cancer charity. Work in MB's lab was supported by Linkoping University strategic research funding and the Ake Wibergs fund (3772738). Work in SP's lab is supported by the BBSRC.</p