Activation of transcription factor circuity in 2i-induced ground state pluripotency is independent of repressive global epigenetic landscapes.

Mouse embryonic stem cells (mESCs) cultured with MEK/ERK and GSK3ß (2i) inhibitors transition to ground state pluripotency. Gene expression changes, redistribution of histone H3K27me3 profiles and global DNA hypomethylation are hallmarks of 2i exposure, but it is unclear whether epigenetic alterations are required to achieve and maintain ground state or occur as an outcome of 2i signal induced changes. Here we show that ESCs with three epitypes, WT, constitutively methylated, or hypomethylated, all undergo comparable morphological, protein expression and transcriptome changes independently of global alterations of DNA methylation levels or changes in H3K27me3 profiles. Dazl and Fkbp6 expression are induced by 2i in all three epitypes, despite exhibiting hypermethylated promoters in constitutively methylated ESCs. We identify a number of activated gene promoters that undergo 2i dependent loss of H3K27me3 in all three epitypes, however genetic and pharmaceutical inhibition experiments show that H3K27me3 is not required for their silencing in non-2i conditions. By separating and defining their contributions, our data suggest that repressive epigenetic systems play minor roles in mESC self-renewal and naïve ground state establishment by core sets of dominant pluripotency associated transcription factor networks, which operate independently from these epigenetic processes.

SMCHD1 mutations associated with a rare muscular dystrophy can also cause isolated arhinia and Bosma arhinia microphthalmia syndrome.

Arhinia, or absence of the nose, is a rare malformation of unknown etiology that is often accompanied by ocular and reproductive defects. Sequencing of 40 people with arhinia revealed that 84% of probands harbor a missense mutation localized to a constrained region of SMCHD1 encompassing the ATPase domain. SMCHD1 mutations cause facioscapulohumeral muscular dystrophy type 2 (FSHD2) via a trans-acting loss-of-function epigenetic mechanism. We discovered shared mutations and comparable DNA hypomethylation patterning between these distinct disorders. CRISPR/Cas9-mediated alteration of smchd1 in zebrafish yielded arhinia-relevant phenotypes. Transcriptome and protein analyses in arhinia probands and controls showed no differences in SMCHD1 mRNA or protein abundance but revealed regulatory changes in genes and pathways associated with craniofacial patterning. Mutations in SMCHD1 thus contribute to distinct phenotypic spectra, from craniofacial malformation and reproductive disorders to muscular dystrophy, which we speculate to be consistent with oligogenic mechanisms resulting in pleiotropic outcomes.

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.

Rapid reprogramming of epigenetic and transcriptional profiles in mammalian culture systems.

BACKGROUND: The DNA methylation profiles of mammalian cell lines differ from those of the primary tissues 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. RESULTS: We demonstrate that adaptation of mouse embryonic fibroblasts 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. CONCLUSIONS: We 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.

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.

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.

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.

Senescent cells harbour features of the cancer epigenome.

Altered DNA methylation and associated destabilization of genome integrity and function is a hallmark of cancer. Replicative senescence is a tumour suppressor process that imposes a limit on the proliferative potential of normal cells that all cancer cells must bypass. Here we show by whole-genome single-nucleotide bisulfite sequencing that replicative senescent human cells exhibit widespread DNA hypomethylation and focal hypermethylation. Hypomethylation occurs preferentially at gene-poor, late-replicating, lamin-associated domains and is linked to mislocalization of the maintenance DNA methyltransferase (DNMT1) in cells approaching senescence. Low-level gains of methylation are enriched in CpG islands, including at genes whose methylation and silencing is thought to promote cancer. Gains and losses of methylation in replicative senescence are thus qualitatively similar to those in cancer, and this 'reprogrammed' methylation landscape is largely retained when cells bypass senescence. Consequently, the DNA methylome of senescent cells might promote malignancy, if these cells escape the proliferative barrier.

Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer.

LINE-1 retrotransposons are abundant repetitive elements of viral origin, which in normal cells are kept quiescent through epigenetic mechanisms. Activation of LINE-1 occurs frequently in cancer and can enable LINE-1 mobilization but also has retrotransposition-independent consequences. We previously reported that in cancer, aberrantly active LINE-1 promoters can drive transcription of flanking unique sequences giving rise to LINE-1 chimeric transcripts (LCTs). Here, we show that one such LCT, LCT13, is a large transcript (>300 kb) running antisense to the metastasis-suppressor gene TFPI-2. We have modelled antisense RNA expression at TFPI-2 in transgenic mouse embryonic stem (ES) cells and demonstrate that antisense RNA induces silencing and deposition of repressive histone modifications implying a causal link. Consistent with this, LCT13 expression in breast and colon cancer cell lines is associated with silencing and repressive chromatin at TFPI-2. Furthermore, we detected LCT13 transcripts in 56% of colorectal tumours exhibiting reduced TFPI-2 expression. Our findings implicate activation of LINE-1 elements in subsequent epigenetic remodelling of surrounding genes, thus hinting a novel retrotransposition-independent role for LINE-1 elements in malignancy.

The CXXC-TET bridge--mind the methylation gap!

CG-rich DNA "reader" proteins that bind non-methylated CpG sequences have emerged as critical factors to the process of cell differentiation and development. In a recent paper in Nature, Ko et al. show that the CXXC domain protein, IDAX, plays a crucial role as a CG-rich DNA-binding factor in the regulation of Ten-Eleven-Translocation 2 (TET2) protein function.

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.

Promoter DNA methylation couples genome-defence mechanisms to epigenetic reprogramming in the mouse germline.

Mouse primordial germ cells (PGCs) erase global DNA methylation (5mC) as part of the comprehensive epigenetic reprogramming that occurs during PGC development. 5mC plays an important role in maintaining stable gene silencing and repression of transposable elements (TE) but it is not clear how the extensive loss of DNA methylation impacts on gene expression and TE repression in developing PGCs. Using a novel epigenetic disruption and recovery screen and genetic analyses, we identified a core set of germline-specific genes that are dependent exclusively on promoter DNA methylation for initiation and maintenance of developmental silencing. These gene promoters appear to possess a specialised chromatin environment that does not acquire any of the repressive H3K27me3, H3K9me2, H3K9me3 or H4K20me3 histone modifications when silenced by DNA methylation. Intriguingly, this methylation-dependent subset is highly enriched in genes with roles in suppressing TE activity in germ cells. We show that the mechanism for developmental regulation of the germline genome-defence genes involves DNMT3B-dependent de novo DNA methylation. These genes are then activated by lineage-specific promoter demethylation during distinct global epigenetic reprogramming events in migratory (~E8.5) and post-migratory (E10.5-11.5) PGCs. We propose that genes involved in genome defence are developmentally regulated primarily by promoter DNA methylation as a sensory mechanism that is coupled to the potential for TE activation during global 5mC erasure, thereby acting as a failsafe to ensure TE suppression and maintain genomic integrity in the germline.

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.

MBD4 and MLH1 are required for apoptotic induction in xDNMT1-depleted embryos.

Loss of the of the maintenance methyltransferase xDNMT1 during Xenopus development results in premature transcription and activation of a p53-dependent apoptotic program that accounts for embryo lethality. Here, we show that activation of the apoptotic response is signalled through the methyl-CpG binding protein xMBD4 and the mismatch repair pathway protein xMLH1. Depletion of xMBD4 or xMLH1 increases the survival rate of xDNMT1-depleted embryos, whereas overexpression of these proteins in embryos induces programmed cell death at the onset of gastrulation. MBD4 interacts directly with both DNMT1 and MLH1, leading to recruitment of the latter to heterochromatic sites that are coincident with DNMT1 localisation. Time-lapse microscopy of micro-irradiated mammalian cells shows that MLH1/MBD4 (like DNMT1) can accumulate at DNA damage sites. We propose that xMBD4/xMLH1 participates in a novel G2 checkpoint that is responsive to xDNMT1p levels in developing embryos and cells.

The interaction of xKaiso with xTcf3: a revised model for integration of epigenetic and Wnt signalling pathways.

We demonstrate that a direct interaction between the methyl-CpG-dependent transcription repressor Kaiso and xTcf3, a transducer of the Wnt signalling pathway, results in their mutual disengagement from their respective DNA-binding sites. Thus, the transcription functions of xTcf3 can be inhibited by overexpression of Kaiso in cell lines and Xenopus embryos. The interaction of Kaiso with xTcf3 is highly conserved and is dependent on its zinc-finger domains (ZF1-3) and the corresponding HMG DNA-binding domain of TCF3/4 factors. Our data rule out a model suggesting that xKaiso is a direct repressor of Wnt signalling target genes in early Xenopus development via binding to promoter-proximal CTGCNA sequences as part of a xTcf3 repressor complex. Instead, we propose that mutual inhibition by Kaiso/TCF3 of their DNA-binding functions may be important in developmental or cancer contexts and acts as a regulatory node that integrates epigenetic and Wnt signalling pathways.

The non-methylated DNA-binding function of Kaiso is not required in early Xenopus laevis development.

Mammalian forms of the transcription repressor, Kaiso, can reportedly bind methylated DNA and non-methylated CTGCNA motifs. Here we compare the DNA-binding properties of Kaiso from frog, fish and chicken and demonstrate that only the methyl-CpG-binding function of Kaiso is evolutionarily conserved. We present several independent experimental lines of evidence that the phenotypic abnormalities associated with xKaiso-depleted Xenopus laevis embryos are independent of the putative CTGCNA-dependent DNA-binding function of xKaiso. Our analysis suggests that xKaiso does not play a role in the regulation of either xWnt11 or Siamois, key signalling molecules in the Wnt pathway during X. laevis gastrulation. The major phenotypic defects associated with xKaiso depletion are premature transcription activation before the mid-blastula transition and concomitant activation of a p53-dependent cell-death pathway.

xDnmt1 regulates transcriptional silencing in pre-MBT Xenopus embryos independently of its catalytic function.

We previously reported that the maintenance cytosine methyltransferase xDnmt1 is essential for gene silencing in early Xenopus laevis embryos. In the present study, we show that silencing is independent of its catalytic function and that xDnmt1 possesses an intrinsic transcription repression function. We show that reduction of xDnmt1p by morpholino (xDMO) injection prematurely activates gene expression without global changes in DNA methylation before the mid-blastula transition (MBT). Repression of xDnmt1p target genes can be reimposed in xDMO morphants with an mRNA encoding a catalytically inactive form of human DNMT1. Moreover, target gene promoter analysis indicates that silencing is not reliant on dynamic changes in DNA methylation. We demonstrate that xDnmt1 can suppress transcription activator function and can be specifically localised to non-methylated target promoters. These data imply that xDnmt1 has a major silencer role in early Xenopus development before the MBT as a direct transcription repressor protein.

Epigenetic silencing in embryogenesis.

Transcription profiling of early embryos emphasizes that differential gene expression is a fundamental control mechanism of development. Precise regulatory mechanisms operate on a background of epigenetic changes in chromatin composition, modification, and architecture that are integral for the development of a pluripotent embryo into an adult.

Kaiso is a genome-wide repressor of transcription that is essential for amphibian development.

DNA methylation in animals is thought to repress transcription via methyl-CpG specific binding proteins, which recruit enzymatic machinery promoting the formation of inactive chromatin at targeted loci. Loss of DNA methylation can result in the activation of normally silent genes during mouse and amphibian development. Paradoxically, global changes in gene expression have not been observed in mice that are null for the methyl-CpG specific repressors MeCP2, MBD1 or MBD2. Here, we demonstrate that xKaiso, a novel methyl-CpG specific repressor protein, is required to maintain transcription silencing during early Xenopus laevis development. In the absence of xKaiso function, premature zygotic gene expression occurs before the mid-blastula transition (MBT). Subsequent phenotypes (developmental arrest and apoptosis) strongly resemble those observed for hypomethylated embryos. Injection of wild-type human kaiso mRNA can rescue the phenotype and associated gene expression changes of xKaiso-depleted embryos. Our results, including gene expression profiling, are consistent with an essential role for xKaiso as a global repressor of methylated genes during early vertebrate development.