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.

Sas3 and Ada2(Gcn5)-dependent histone H3 acetylation is required for transcription elongation at the de-repressed FLO1 gene.

The Saccharomyces cerevisiae FLO1 gene encodes a cell wall protein that imparts cell-cell adhesion. FLO1 transcription is regulated via the antagonistic activities of the Tup1-Cyc8 co-repressor and Swi-Snf co-activator complexes. Tup1-Cyc8 represses transcription through the organization of strongly positioned, hypoacetylated nucleosomes across gene promoters. Swi-Snf catalyzes remodeling of these nucleosomes in a mechanism involving histone acetylation that is poorly understood. Here, we show that FLO1 de-repression is accompanied by Swi-Snf recruitment, promoter histone eviction and Sas3 and Ada2(Gcn5)-dependent histone H3K14 acetylation. In the absence of H3K14 acetylation, Swi-Snf recruitment and histone eviction proceed, but transcription is reduced, suggesting these processes, while essential, are not sufficient for de-repression. Further analysis in the absence of H3K14 acetylation reveals RNAP II recruitment at the FLO1 promoter still occurs, but RNAP II is absent from the gene-coding region, demonstrating Sas3 and Ada2-dependent histone H3 acetylation is required for transcription elongation. Analysis of the transcription kinetics at other genes reveals shared mechanisms coupled to a distinct role for histone H3 acetylation, essential at FLO1, downstream of initiation. We propose histone H3 acetylation in the coding region provides rate-limiting control during the transition from initiation to elongation which dictates whether the gene is permissive for transcription.

The yeast Cyc8-Tup1 complex cooperates with Hda1p and Rpd3p histone deacetylases to robustly repress transcription of the subtelomeric FLO1 gene.

We demonstrate that the yeast flocculation gene, FLO1, is representative of a distinct subset of subtelomeric genes that are robustly repressed by the Cyc8-Tup1 complex. We have examined Cyc8-Tup1 localisation, histone acetylation and long-range chromatin remodelling within the extensive FLO1 upstream region. We show that Cyc8-Tup1 is localised in a DNase I hypersensitive site within an ordered array of strongly positioned nucleosomes around -700 base pairs upstream of the transcription start site. In cyc8 deletion mutant strains, Tup1p localisation is absent, with concomitant histone hyperacetylation of adjacent regions at the FLO1 promoter. This is accompanied by extensive histone depletion across the upstream region and gene activation. The yeast histone deacetylases, Hda1p and Rpd3p, occupy the repressed FLO1 promoter region in a Cyc8-Tup1 dependent manner and coordinate histone deacetylation, nucleosome stabilisation and gene repression. Moreover, we show that the ATP-dependent chromatin remodelling complex Swi-Snf occupies the site vacated by Cyc8-Tup1 in a cyc8 mutant. These data suggest that distinctly bound Cyc8-Tup1 cooperates with Hda1p and Rpd3p to establish or maintain an extensive array of strongly positioned, deacetylated nucleosomes over the FLO1 promoter and upstream region which inhibit histone acetylation, block Swi-Snf binding and prevent transcription.

Sequential effects of spadetail, one-eyed pinhead and no tail on midline convergence of nephric primordia during zebrafish embryogenesis.

Midline convergence of organ primordia is an important mechanism that shapes the vertebrate body plan. Here, we focus on the morphogenetic movements of pronephric glomerular primordia (PGP) occurring during zebrafish embryonic kidney development. To characterize the process of PGP midline convergence, we used Wilms' tumour 1a (wt1a) as a marker to label kidney primordia, and performed quantitative analyses of the migration of the bilateral PGP. The PGP initially are approximately 350 µm apart in a wild type embryo at 10h post fertilization (hpf). The inter-PGP distance decreases exponentially between 10 and 48 hpf, while the anterior-posterior (A-P) dimension of each PGP increases linearly between 10 and 12 hpf, then decreases substantially between 12 and 24 hpf. Using mutants in the Nodal receptor cofactor one-eyed pinhead (oep) and the T-box transcription factors spadetail (spt) and no tail (ntl), we were able to define distinctive regulation underlying these sequential phases of PGP midline migration. Zygotic oep mutants (Zoep(-/-)) exhibited defects in midline convergence after 16 hpf. Spt is necessary for PGP convergence from 10 hpf, whereas ntl's effect on convergence does not begin until 24 hpf. Notably, we observed normal cardiac convergence in spt(-/-) and ntl(-/-) embryos implying that these novel roles of spt and ntl in PGP migration cannot be explained simply by generalised effects on midline convergence. These findings demonstrate that quantitative approaches to developmental migration allow the parsing of early patterning events, and in this instance suggest that the zebrafish may offer insights into midline urogenital migration anomalies in humans.

Non-canonical functions of the DNA methylome in gene regulation.

Methylation of the cytosine base in DNA, DNA methylation, is an essential epigenetic mark in mammals that contributes to the regulation of transcription. Several advances have been made in this area in recent years, leading to a leap forward in our understanding of how this pathway contributes to gene regulation during embryonic development, and the functional consequences of its perturbation in human disease. Critical to these advances is a comprehension of the genomic distribution of modified cytosine bases in unprecedented detail, drawing attention to genomic regions beyond gene promoters. In addition, we have a more complete understanding of the multifactorial manner by which DNA methylation influences gene regulation at the molecular level, and which genes rely directly on the DNA methylome for their normal transcriptional regulation. It is becoming apparent that a major role of DNA modification is to act as a relatively stable, and mitotically heritable, template that contributes to the establishment and maintenance of chromatin states. In this regard, interplay is emerging between DNA methylation and the PcG (Polycomb group) proteins, which act as evolutionarily conserved mediators of cell identity. In the present paper we review these aspects of DNA methylation, and discuss how a multifunctional view of DNA modification as an integral part of chromatin organization is influencing our understanding of this epigenetic mark's contribution to transcriptional regulation.

Histone H4K20me3 and HP1α are late heterochromatin markers in development, but present in undifferentiated embryonic stem cells.

We report here that the formation of heterochromatin in cell nuclei during mouse development is characterised by dynamic changes in the epigenetic modifications of histones. Our observations reveal that heterochromatin in mouse preimplantation embryos is in an immature state that lacks the constitutive heterochromatin markers histone H4 trimethyl Lys20 (H4K20me3) and chromobox homolog 5 (HP1α, also known as CBX5). Remarkably, these somatic heterochromatin hallmarks are not detectable--except in mural trophoblast--until mid-gestation, increasing in level during foetal development. Our results support a developmentally regulated connection between HP1α and H4K20me3. Whereas inner cell mass (ICM) and epiblast stain negative for H4K20me3 and HP1α, embryonic stem (ES) cell lines, by contrast, stain positive for these markers, indicating substantial chromatin divergence. We conclude that H4K20me3 and HP1α are late developmental epigenetic markers, and slow maturation of heterochromatin in tissues that develop from ICM is ectopically induced during ES cell derivation. Our findings suggest that H4K20me3 and HP1α are markers for cell type commitment that can be triggered by developmental or cell context, independently of the differentiation process.

Tup1-Ssn6 and Swi-Snf remodelling activities influence long-range chromatin organization upstream of the yeast SUC2 gene.

The traditional model for chromatin remodelling during transcription has focused upon the remodelling of nucleosomes at gene promoters. However, in this study, we have determined that Tup1-Ssn6 and Swi-Snf chromatin remodelling activities extend far upstream of the SUC2 gene promoter into the intergenic region of the Saccharomyces cerevisiae chromosome. We mapped the nucleosomal array over a 7.5 kb region that encompassed the SUC2 gene promoter and upstream region but was devoid of other transcriptionally active genes. Nucleosome positioning over this region was determined under conditions of glucose repression and derepression, and in snf2, ssn6 and snf2 ssn6 mutant strains. A map detailing remodelling events extending as much as 5 kb upstream of the SUC2 gene promoter underlines the roles of the Tup1-Ssn6 and Swi-Snf complexes in respectively organizing and disrupting nucleosome arrays. The gene specificity of these events suggests a role in gene regulation. We propose that long-range chromatin remodelling activities of Swi-Snf and Tup1-Ssn6 may ultimately influence whether the chromosomal state of the SUC2 gene is proficient for transcription. These data raise the possibility that remodelling of extensive chromatin domains may be a general property of the Swi-Snf and Tup1-Ssn6 complexes.

DNA Methylation Directs Polycomb-Dependent 3D Genome Re-organization in Naive Pluripotency.

The DNA hypomethylation that occurs when embryonic stem cells (ESCs) are directed to the ground state of naive pluripotency by culturing in two small molecule inhibitors (2i) results in redistribution of polycomb (H3K27me3) away from its target loci. Here, we demonstrate that 3D genome organization is also altered in 2i, with chromatin decompaction at polycomb target loci and a loss of long-range polycomb interactions. By preventing DNA hypomethylation during the transition to the ground state, we are able to restore to ESC in 2i the H3K27me3 distribution, as well as polycomb-mediated 3D genome organization that is characteristic of primed ESCs grown in serum. However, these cells retain the functional characteristics of 2i ground-state ESCs. Our findings demonstrate the central role of DNA methylation in shaping major aspects of 3D genome organization but caution against assuming causal roles for the epigenome and 3D genome in gene regulation and function in ESCs.

Cardiac Stem Cells in the Postnatal Heart: Lessons from Development.

Heart development in mammals is followed by a postnatal decline in cell proliferation and cell renewal from stem cell populations. A better understanding of the developmental changes in cardiac microenvironments occurring during heart maturation will be informative regarding the loss of adult regenerative potential. We reevaluate the adult heart's mitotic potential and the reported adult cardiac stem cell populations, as these are two topics of ongoing debate. The heart's early capacity for cell proliferation driven by progenitors and reciprocal signalling is demonstrated throughout development. The mature heart architecture and environment may be more restrictive on niches that can host progenitor cells. The engraftment issues observed in cardiac stem cell therapy trials using exogenous stem cells may indicate a lack of supporting stem cell niches, while tissue injury adds to a hostile microenvironment for transplanted cells. Engraftment may be improved by preconditioning the cultured stem cells and modulating the microenvironment to host these cells. These prospective areas of further research would benefit from a better understanding of cardiac progenitor interactions with their microenvironment throughout development and may lead to enhanced cardiac niche support for stem cell therapy engraftment.

DNA methylation as a genomic marker of exposure to chemical and environmental agents.

Recent progress in interpreting comprehensive genetic and epigenetic profiles for human cellular states has contributed new insights into the developmental origins of disease, elucidated novel signalling pathways and enhanced drug discovery programs. A similar comprehensive approach to decoding the epigenetic readouts from chemical challenges in vivo would yield new paradigms for monitoring and assessing environmental exposure in model systems and humans.

Antibody-Based Detection of Global Nuclear DNA Methylation in Cells, Tissue Sections, and Mammalian Embryos.

Immunostaining is widely used in cell biology for the in situ detection of proteins in fixed cells. The method is based on the specificity of antibodies for recognizing and binding to a selected target, combined with immunolabeling techniques for microscopic imaging. Antibodies with high specificities for modified nucleotides have also been widely developed, and among those, antibodies that recognize modified cytosine: 5-methylcytosine (5mC), and more recently, its derivates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). To allow for their detection, primary antibody signals can be amplified using secondary antibodies coupled to fluorophores for immunofluorescence, or other molecules for immunocytochemistry.Immunostaining can be used to gain information on the spatial distribution and levels of DNA methylation states within the nucleus. Although the resolution remains quite low in genomic terms, advanced microscopy techniques and image analysis can obtain detailed spatial information content from immunostained sites. The technique complements genomic approaches that permit the assessment of DNA methylation on specific sequences, but that cannot provide global nuclear spatial context. Immunostaining is an accessible method of great benefit in several cases: when working with limited material (such as embryos or primary cells), to quickly assess at the level of individual cells the effect of siRNA, drugs, or biological processes that promote or inhibit DNA methylation or demethylation, or to study the 3D nuclear organization of regions with high DNA methylation, such as constitutive heterochromatin.Here, we review and outline protocols for the fluorescent and enzymatic immunodetection of DNA methylation in the nuclei of cells, tissue sections, and mammalian embryos.

A determining influence for CpG dinucleotides on nucleosome positioning in vitro.

DNA sequence information that directs the translational positioning of nucleosomes can be attenuated by cytosine methylation when a short run of CpG dinucleotides is located close to the dyad axis of the nucleosome. Here, we show that point mutations introduced to re-pattern methylation at the (CpG)3 element in the chicken betaA-globin promoter sequence themselves strongly influenced nucleosome formation in reconstituted chromatin. The disruptive effect of cytosine methylation on nucleosome formation was found to be determined by the sequence context of CpG dinucleotides, not just their location in the positioning sequence. Additional mutations indicated that methylation can also promote the occupation of certain nucleosome positions. DNase I analysis demonstrated that these genetic and epigenetic modifications altered the structural characteristics of the (CpG)3 element. Our findings support a proposal that the intrinsic structural properties of the DNA at the -1.5 site, as occupied by (CpG)3 in the nucleosome studied, can be decisive for nucleosome formation and stability, and that changes in anisotropic DNA bending or flexibility at this site explain why nucleosome positioning can be exquisitely sensitive to genetic and epigenetic modification of the DNA sequence.

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.

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.

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.

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.