Chromatin In Embryonic Stem Cells Research Articles

From the membrane to the nucleus: mechanical signals and transcription regulation.

Mechanical forces drive and modulate a wide variety of processes in eukaryotic cells including those occurring in the nucleus. Relevantly, forces are fundamental during development since they guide lineage specifications of embryonic stem cells. A sophisticated macromolecular machinery transduces mechanical stimuli received at the cell surface into a biochemical output; a key component in this mechanical communication is the cytoskeleton, a complex network of biofilaments in constant remodeling that links the cell membrane to the nuclear envelope. Recent evidence highlights that forces transmitted through the cytoskeleton directly affect the organization of chromatin and the accessibility of transcription-related molecules to their targets in the DNA. Consequently, mechanical forces can directly modulate transcription and change gene expression programs. Here, we will revise the biophysical toolbox involved in the mechanical communication with the cell nucleus and discuss how mechanical forces impact on the organization of this organelle and more specifically, on transcription. We will also discuss how live-cell fluorescence imaging is producing exquisite information to understand the mechanical response of cells and to quantify the landscape of interactions of transcription factors with chromatin in embryonic stem cells. These studies are building new biophysical insights that could be fundamental to achieve the goal of manipulating forces to guide cell differentiation in culture systems.

Sumoylation and the oncogenic E17K mutation affect AKT1 subcellular distribution and impact on Nanog-binding dynamics to chromatin in embryonic stem cells

AKT/PKB is a kinase involved in the regulation of a plethora of cell processes. Particularly, in embryonic stem cells (ESCs), AKT is crucial for the maintenance of pluripotency. Although the activation of this kinase relies on its recruitment to the cellular membrane and subsequent phosphorylation, multiple other post-translational modifications (PTMs), including SUMOylation, fine-tune its activity and target specificity. Since this PTM can also modify the localization and availability of different proteins, in this work we explored if SUMOylation impacts on the subcellular compartmentalization and distribution of AKT1 in ESCs. We found that this PTM does not affect AKT1 membrane recruitment, but it modifies the AKT1 nucleus/cytoplasm distribution, increasing its nuclear presence. Additionally, within this compartment, we found that AKT1 SUMOylation also impacts on the chromatin-binding dynamics of NANOG, a central pluripotency transcription factor. Remarkably, the oncogenic E17K AKT1 mutant produces major changes in all these parameters increasing the binding of NANOG to its targets, also in a SUMOylation dependent manner. These findings demonstrate that SUMOylation modulates AKT1 subcellular distribution, thus adding an extra layer of regulation of its function, possibly by affecting the specificity and interaction with its downstream targets.

Nascent RNA antagonizes the interaction of a set of regulatory proteins with chromatin

A number of regulatory factors are recruited to chromatin by specialized RNAs. Whether RNA has a more general role in regulating the interaction of proteins with chromatin has not been determined. We used proteomics methods to measure the global impact of nascent RNA on chromatin in embryonic stem cells. Surprisingly, we found that nascent RNA primarily antagonized the interaction of chromatin modifiers and transcriptional regulators with chromatin. Transcriptional inhibition and RNA degradation induced recruitment of a set of transcriptional regulators, chromatin modifiers, nucleosome remodelers, and regulators of higher-order structure. RNA directly bound to factors, including BAF, NuRD, EHMT1, and INO80 and inhibited their interaction with nucleosomes. The transcriptional elongation factor P-TEFb directly bound pre-mRNA, and its recruitment to chromatin upon Pol II inhibition was regulated by the 7SK ribonucleoprotein complex. We postulate that by antagonizing the interaction of regulatory proteins with chromatin, nascent RNA links transcriptional output with chromatin composition.

Bivalent genes that undergo transcriptional switching identify networks of key regulators of embryonic stem cell differentiation

BackgroundBivalent promoters marked with both H3K27me3 and H3K4me3 histone modifications are characteristic of poised promoters in embryonic stem (ES) cells. The model of poised promoters postulates that bivalent chromatin in ES cells is resolved to monovalency upon differntiation. With the availability of single-cell RNA sequencing (scRNA-seq) data, subsequent switches in transcriptional state at bivalent promoters can be studied more closely.ResultsWe develop an approach for capturing genes undergoing transcriptional switching by detecting ‘bimodal’ gene expression patterns from scRNA-seq data. We integrate the identification of bimodal genes in ES cell differentiation with analysis of chromatin state, and identify clear cell-state dependent patterns of bimodal, bivalent genes. We show that binarization of bimodal genes can be used to identify differentially expressed genes from fractional ON/OFF proportions. In time series data from differentiating cells, we build a pseudotime approximation and use a hidden Markov model to infer gene activity switching pseudotimes, which we use to infer a regulatory network. We identify pathways of switching during differentiation, novel details of those pathway, and transcription factor coordination with downstream targets.ConclusionsGenes with expression levels too low to be informative in conventional scRNA analysis can be used to infer transcriptional switching networks that connect transcriptional activity to chromatin state. Since chromatin bivalency is a hallmark of gene promoters poised for activity, this approach provides an alternative that complements conventional scRNA-seq analysis while focusing on genes near the ON/OFF boundary of activity. This offers a novel and productive means of inferring regulatory networks from scRNA-seq data.

BAZ2A safeguards genome architecture of ground-state pluripotent stem cells.

Chromosomes have an intrinsic tendency to segregate into compartments, forming long‐distance contacts between loci of similar chromatin states. How genome compartmentalization is regulated remains elusive. Here, comparison of mouse ground‐state embryonic stem cells (ESCs) characterized by open and active chromatin, and advanced serum ESCs with a more closed and repressed genome, reveals distinct regulation of their genome organization due to differential dependency on BAZ2A/TIP5, a component of the chromatin remodeling complex NoRC. On ESC chromatin, BAZ2A interacts with SNF2H, DNA topoisomerase 2A (TOP2A) and cohesin. BAZ2A associates with chromatin sub‐domains within the active A compartment, which intersect through long‐range contacts. We found that ground‐state chromatin selectively requires BAZ2A to limit the invasion of active domains into repressive compartments. BAZ2A depletion increases chromatin accessibility at B compartments. Furthermore, BAZ2A regulates H3K27me3 genome occupancy in a TOP2A‐dependent manner. Finally, ground‐state ESCs require BAZ2A for growth, differentiation, and correct expression of developmental genes. Our results uncover the propensity of open chromatin domains to invade repressive domains, which is counteracted by chromatin remodeling to establish genome partitioning and preserve cell identity.

Regulation and Roles of the Nucleolus in Embryonic Stem Cells: From Ribosome Biogenesis to Genome Organization.

The nucleolus is the largest compartment of the eukaryotic cell's nucleus. It acts as a ribosome factory, thereby sustaining the translation machinery. The nucleolus is also the subnuclear compartment with the highest transcriptional activity in the cell, where hundreds of ribosomal RNA (rRNA) genes transcribe the overwhelming majority of RNAs. The structure and composition of the nucleolus change according to the developmental state. For instance, in embryonic stem cells (ESCs), rRNA genes display a hyperactive transcriptional state and open chromatin structure compared with differentiated cells. Increasing evidence indicates that the role of the nucleolus and rRNA genes might go beyond the control of ribosome biogenesis. One such role is linked to the genome architecture, since repressive domains are often located close to the nucleolus. This review highlights recent findings describing how the nucleolus is regulated in ESCs and its role in regulating ribosome biogenesis and genome organization for the maintenance of stem cell identity.

Abstract A20: Disruption and restoration of pioneer transcription factor function in bladder cancer

Abstract Urothelial bladder cancer (BLCA) is characterized by the failure to complete epithelial-differentiation. The mechanisms underlying this failure are unknown. To address this knowledge gap, we used unbiased analyzes to identify 1,000 genes significantly suppressed in BLCA vs normal bladder (p<0.001, FDR<0.01) (n=426, TCGA RNA-seq). Remarkably, these genes are located in compacted chromatin in embryonic stem cells (ESC) (decreased H3K4me3, increased CpG methylation), while 1,000 genes upregulated in BLCA vs normal bladder are located in open chromatin at this same ultimate baseline. This striking pattern in unbiased analyses suggested pioneer transcription factor (TF) loss of function in BLCA: pioneer TF access compacted chromatin to initiate the remodeling necessary for gene activation. The pioneer TF hub containing FOXA1, GATA4 is highly expressed in normal bladder, and these pioneer TFs are frequently haploinsufficient in BLCA, 32% and 56%, respectively. We pulled down endogenous FOXA1, the most expressed TF, from UM-UC-6 BLCA cells. Proteomic analyzes by mass spectrometry revealed enrichment for corepressors (DNMT1) over coactivators (ARID1A) in this pioneer TF hub. Genetic analyses showed frequent mutation/deletion of the coactivators: 43% bi-allelic ARID1A inactivation, while corepressors, e.g., CHD4 and DNMT1, were amplified or unaltered. Restoration of ARID1A into UM-UC-6 upregulated epithelial genes, downregulated MYC, upregulated CDKN1B, and terminated proliferation. To translate into therapy, we confirmed using immunoprecipitation LCMS/MS that DNMT1-depletion using noncytotoxic concentrations of the clinical drug decitabine (0.2-0.5 μM) shifted hub composition to coactivators, to thereby induce terminal epithelial-differentiation of BLCA cells. This was also seen in vivo in UM-UC-6 xenograft: very low dose non-cytotoxic decitabine (0.1 mg/kg) combined with an inhibitor of its degradation, THU, decreased tumor size more than standard high-dose cytotoxic gemcitabine/cisplatinum. Thus, BLCA suppresses hundreds of epithelial-differentiation genes via corepressor/coactivator imbalance in the bladder pioneer TF hub. Sensitivity of the pioneer TF hub to corepressor/coactivator stoichiometry, however, also enables pharmacologic reduction of corepressors to compensate for genetic reduction of coactivators, to thereby release BLCA cells to destined terminal epithelial fates. Citation Format: Caroline Schuerger, Xiaorong Gu, Yvonne Parker, Daniel Lindner, Babal Jha. Disruption and restoration of pioneer transcription factor function in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A20.

PHF19 mediated regulation of proliferation and invasiveness in prostate cancer cells.

The Polycomb-like protein PHF19/PCL3 associates with PRC2 and mediates its recruitment to chromatin in embryonic stem cells. PHF19 is also overexpressed in many cancers. However, neither PHF19 targets nor misregulated pathways involving PHF19 are known. Here, we investigate the role of PHF19 in prostate cancer cells. We find that PHF19 interacts with PRC2 and binds to PRC2 targets on chromatin. PHF19 target genes are involved in proliferation, differentiation, angiogenesis, and extracellular matrix organization. Depletion of PHF19 triggers an increase in MTF2/PCL2 chromatin recruitment, with a genome-wide gain in PRC2 occupancy and H3K27me3 deposition. Transcriptome analysis shows that PHF19 loss promotes deregulation of key genes involved in growth, metastasis, invasion, and of factors that stimulate blood vessels formation. Consistent with this, PHF19 silencing reduces cell proliferation, while promotes invasive growth and angiogenesis. Our findings reveal a role for PHF19 in controlling the balance between cell proliferation and invasiveness in prostate cancer.

Kat6b Modulates Oct4 and Nanog Binding to Chromatin in Embryonic Stem Cells and Is Required for Efficient Neural Differentiation

Chromatin remodeling is fundamental for the dynamical changes in transcriptional programs that occur during development and stem cell differentiation. The histone acetyltransferase Kat6b is relevant for neurogenesis in mouse embryos, and mutations of this gene cause intellectual disability in humans. However, the molecular mechanisms involved in Kat6b mutant phenotype and the role of this chromatin modifier in embryonic stem (ES) cells remain elusive. In this work, we show that Kat6b is expressed in ES cells and is repressed during differentiation. Moreover, we found that this gene is regulated by the pluripotency transcription factors Nanog and Oct4. To study the functional relevance of Kat6b in ES cells, we generated a Kat6b knockout ES cell line (K6b−/−) using CRISPR/Cas9. Fluorescence correlation spectroscopy analyses suggest a more compact chromatin organization in K6b−/− cells and impaired interactions of Oct4 and Nanog with chromatin. Remarkably, K6b−/− cells showed a reduced efficiency to differentiate to neural lineage. These results reveal a role of Kat6b as a modulator of chromatin plasticity, its impact on chromatin-transcription factors interactions and its influence on cell fate decisions during neural development.

The CUE1 domain of the SNF2-like chromatin remodeler SMARCAD1 mediates its association with KRAB-associated protein 1 (KAP1) and KAP1 target genes

Chromatin in embryonic stem cells (ESCs) differs markedly from that in somatic cells, with ESCs exhibiting a more open chromatin configuration. Accordingly, ATP-dependent chromatin remodeling complexes are important regulators of ESC homeostasis. Depletion of the remodeler SMARCAD1, an ATPase of the SNF2 family, has been shown to affect stem cell state, but the mechanistic explanation for this effect is unknown. Here, we set out to gain further insights into the function of SMARCAD1 in mouse ESCs. We identified KRAB-associated protein 1 (KAP1) as the stoichiometric binding partner of SMARCAD1 in ESCs. We found that this interaction occurs on chromatin and that SMARCAD1 binds to different classes of KAP1 target genes, including zinc finger protein (ZFP) and imprinted genes. We also found that the RING B-box coiled-coil (RBCC) domain in KAP1 and the proximal coupling of ubiquitin conjugation to ER degradation (CUE) domain in SMARCAD1 mediate their direct interaction. Of note, retention of SMARCAD1 in the nucleus depended on KAP1 in both mouse ESCs and human somatic cells. Mutations in the CUE1 domain of SMARCAD1 perturbed the binding to KAP1 in vitro and in vivo Accordingly, an intact CUE1 domain was required for tethering this remodeler to the nucleus. Moreover, mutation of the CUE1 domain compromised SMARCAD1 binding to KAP1 target genes. Taken together, our results reveal a mechanism that localizes SMARCAD1 to genomic sites through the interaction of SMARCAD1's CUE1 motif with KAP1.

H2A.Z.1 Monoubiquitylation Antagonizes BRD2 to Maintain Poised Chromatin in ESCs.

Histone variant H2A.Z occupies the promoters of active and poised, bivalent genes in embryonic stem cells (ESCs) to regulate developmental programs, yet how it contributes to these contrasting states is poorly understood. Here, we investigate the function of H2A.Z.1 monoubiquitylation (H2A.Z.1ub) by mutation of the PRC1 target residues (H2A.Z.1(K3R3)). We show that H2A.Z.1(K3R3) is properly incorporated at target promoters in murine ESCs (mESCs), but loss of monoubiquitylation leads to de-repression of bivalent genes, loss of Polycomb binding, and faulty lineage commitment. Using quantitative proteomics, we find that tandem bromodomain proteins, including the BET family member BRD2, are enriched in H2A.Z.1 chromatin. We further show that BRD2 is gained at de-repressed promoters in H2A.Z.1(K3R3) mESCs, whereas BRD2 inhibition restores gene silencing at these sites. Together, our study reveals an antagonistic relationship between H2A.Z.1ub and BRD2 to regulate the transcriptional balance at bivalent genes to enable proper execution of developmental programs.

Embryonic stem cell differentiation requires full length Chd1.

The modulation of chromatin dynamics by ATP-dependent chromatin remodeling factors has been recognized as an important mechanism to regulate the balancing of self-renewal and pluripotency in embryonic stem cells (ESCs). Here we have studied the effects of a partial deletion of the gene encoding the chromatin remodeling factor Chd1 that generates an N-terminally truncated version of Chd1 in mouse ESCs in vitro as well as in vivo. We found that a previously uncharacterized serine-rich region (SRR) at the N-terminus is not required for chromatin assembly activity of Chd1 but that it is subject to phosphorylation. Expression of Chd1 lacking this region in ESCs resulted in aberrant differentiation properties of these cells. The self-renewal capacity and ESC chromatin structure, however, were not affected. Notably, we found that newly established ESCs derived from Chd1Δ2/Δ2 mutant mice exhibited similar differentiation defects as in vitro generated mutant ESCs, even though the N-terminal truncation of Chd1 was fully compatible with embryogenesis and post-natal life in the mouse. These results underscore the importance of Chd1 for the regulation of pluripotency in ESCs and provide evidence for a hitherto unrecognized critical role of the phosphorylated N-terminal SRR for full functionality of Chd1.

LncRNA Maturation to Initiate Heterochromatin Formation in the Nucleolus Is Required for Exit from Pluripotency in ESCs

The open chromatin of embryonic stem cells (ESCs) condenses into repressive heterochromatin as cells exit the pluripotent state. How the 3D genome organization is orchestrated and implicated in pluripotency and lineage specification is not understood. Here, we find that maturation of the long noncoding RNA (lncRNA) pRNA is required for establishment of heterochromatin at ribosomal RNA genes, the genetic component of nucleoli, and this process is inactivated in pluripotent ESCs. By using mature pRNA to tether heterochromatin at nucleoli of ESCs, we find that localized heterochromatin condensation of ribosomal RNA genes initiates establishment of highly condensed chromatin structures outside of the nucleolus. Moreover, we reveal that formation of such highly condensed, transcriptionally repressed heterochromatin promotes transcriptional activation of differentiation genes and loss of pluripotency. Our findings unravel the nucleolus as an active regulator of chromatin plasticity and pluripotency and challenge current views on heterochromatin regulation and function in ESCs.

Abstract 3053: Epigenetic control of cell phenotypes in the stem cell compartments of human prostate and prostate cancer: Implications for enhancement of prostate cancer therapies

Abstract There is increasing evidence that the phenotype of heterogeneous prostate tumors can be determined to some extent by reversible (non-mutagenic/epigenetic) genetic mechanisms. In the complex microenvironment of a developing cancer, this is an excellent survival strategy. The target cell (and molecular targets) for primary therapeutic intervention in prostate cancer is frequently determined by hormone responsiveness. We have shown that, in cells fractionated from human cancers (relative to cell lines or multiply passaged xenografts), the human prostate cancer-inducing cell (TIC) is a primitive, more basal-like cell with stem cell characteristics and limited hormone sensitivity. The TICs, and indeed normal tissue stem cells, have proven remarkably resistant to both radiotherapy and chemotherapy, as a result of their unique stem cell-like chromatin configuration, which can be ‘unlocked’ using common epigenetic control disruptors (e.g. HDAC inhibition), resulting in differentiation and the enhancement of therapeutic efficacy. This result prompted us to explore the nature of epigenetic control in multiple cellular fractions of both basal and luminal cells purified from established prostate cell lines, primary tumor cultures, primary xenografts of prostate cancer and small directed biopsies of freshly biopsied prostate tumor tissues. The data indicate a remarkable difference between multiple ‘standard’ cell line models of prostate cancer and cell types from near-patient models. Chromatin status in terms of histone modification, genomic methylation patterns, microRNA expression are all cell-type specific, in contrast to the variable data reported in extracts of unfractionated human tumor biopsies, reflecting tissue heterogeneity. The TIC fraction has retained (or acquired) the bivalent chromatin of embryonic stem cells in a fully undifferentiated state. Dominant epigenetic signals are associated with cellular differentiation and maintenance of the proliferating luminal phenotype, which can mask underlying changes responsible for tumor induction, stem cell maintenance and metastatic capability. Therapeutic targeting of the former simply shrinks tumors without affecting metastatic induction and patient survival. Unlinked clusters of coregulated genes define the differentiation process, and contain novel molecular targets such as stem cell maintenance genes, which could render all cells in a cancer susceptible to current therapies as part of a combination treatment strategy. The lower concentrations of stem cell modifying agents (e.g. retinoic acid and HDAC inhibitors), when used as phenotype modifiers rather than toxins, in combination with more established (cytotoxic) therapies, are more likely to be well tolerated in patients, compared to the toxic clinical doses of current ‘differentiating’ agents. Citation Format: Norman J. Maitland, Davide Pellacani, Jayant K. Rane, Fiona M. Frame, Anne T. Collins. Epigenetic control of cell phenotypes in the stem cell compartments of human prostate and prostate cancer: Implications for enhancement of prostate cancer therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3053. doi:10.1158/1538-7445.AM2014-3053

FIREWACh: high-throughput functional detection of transcriptional regulatory modules in mammalian cells

Promoters and enhancers establish precise gene transcription patterns. The development of functional approaches for their identification in mammalian cells has been complicated by the size of these genomes. Here we report a new method called FIREWACh (Functional Identification of Regulatory Elements Within Accessible Chromatin), a high-throughput functional assay for directly identifying active promoter and enhancer elements. FIREWACh simultaneously assessed over 80,000 DNA fragments derived from “nucleosome-free regions” within embryonic stem cell (ESC) chromatin to identify 6,364 new active regulatory elements. Many FIREWACh DNAs represent newly discovered ESC-specific enhancers and their analyses identified enriched binding site motifs for ESC transcription factors including SOX2, OCT4 (POU5f1), and KLF4. Thus FIREWACh identifies endogenous regulators of gene expression and can be used for the discovery of key cell-specific transcription factors. The application of FIREWACh to additional cultured cell types will facilitate functional annotation of the genome and expand our view of transcriptional network dynamics.

Gene regulation and priming by topoisomerase IIα in embryonic stem cells

Topoisomerases resolve torsional stress, while their function in gene regulation, especially during cellular differentiation, remains unknown. Here we find that the expression of topo II isoforms, topoisomerase IIα and topoisomerase IIβ, is the characteristic of dividing and postmitotic tissues, respectively. In embryonic stem cells, topoisomerase IIα preferentially occupies active gene promoters. Topoisomerase IIα inhibition compromises genomic integrity, which results in epigenetic changes, altered kinetics of RNA Pol II at target promoters and misregulated gene expression. Common targets of topoisomerase IIα and topoisomerase IIβ are housekeeping genes, while unique targets are involved in proliferation/pluripotency and neurogenesis, respectively. Topoisomerase IIα targets exhibiting bivalent chromatin resolve upon differentiation, concomitant with their activation and occupancy by topoisomerase IIβ, features further observed for long genes. These long silent genes display accessible chromatin in embryonic stem cells that relies on topoisomerase IIα activity. These findings suggest that topoisomerase IIα not only contributes to stem-cell transcriptome regulation but also primes developmental genes for subsequent activation upon differentiation.

The Trithorax Group Protein Ash2l Is Essential for Pluripotency and Maintaining Open Chromatin in Embryonic Stem Cells

Embryonic stem (ES) cells exhibit general characteristics of open chromatin, a state that may be necessary for ES cells to efficiently self-renew while remaining poised for differentiation. Histone H3K4 and H3K9 trimethylation associate as a general rule, with open and silenced chromatin, respectively, for ES cell pluripotency maintenance. However, how histone modifications are regulated to maintain open chromatin in ES cells remains largely unknown. Here, we demonstrate that trithorax protein Ash2l, homologue of the Drosophila Ash2 (absent, small, homeotic-2) protein, is a key regulator of open chromatin in ES cells. Consistent with Ash2l being a core subunit of mixed lineage leukemia methyltransferase complex, RNAi knockdown of Ash2l was sufficient to reduce H3K4 methylation levels and drive ES cells to a silenced chromatin state with high H3K9 trimethylation. Genome-wide ChIP-seq analysis indicated that Ash2l is recruited to target loci through two distinct modes and enriched at a family of genes implicated in open chromatin regulation, including chromatin remodeler Cdh7, transcription factor c-Myc, and H3K9 demethylase Kdm4c. Our results underscore the importance of Ash2l in open chromatin regulation and provide insight into how the open chromatin landscape is maintained in ES cells.

Tet Proteins Connect the O-Linked N-acetylglucosamine Transferase Ogt to Chromatin in Embryonic Stem Cells

O-linked N-acetylglucosamine (O-GlcNAc) transferase (Ogt) activity is essential for embryonic stem cell (ESC) viability and mouse development. Ogt is present both in the cytoplasm and the nucleus of different cell types and catalyzes serine and threonine glycosylation. We have characterized the biochemical features of nuclear Ogt and identified the ten-eleven translocation (TET) proteins Tet1 and Tet2 as stable partners of Ogt in the nucleus of ESCs. We show at a genome-wide level that Ogt preferentially associates with Tet1 to genes promoters in close proximity of CpG-rich transcription start sites. These regions are characterized by low levels of DNA modification, suggesting a link between Tet1 and Ogt activities in regulating CpG island methylation. Finally, we show that Tet1 is required for binding of Ogt to chromatin affecting Tet1 activity. Taken together, our data characterize how O-GlcNAcylation is recruited to chromatin and interacts with the activity of 5-methylcytosine hydroxylases.

The Epigenomics of Embryonic Stem Cell Differentiation

Embryonic stem cells (ESCs) possess an open and highly dynamic chromatin landscape, which underlies their plasticity and ultimately maintains ESC pluripotency. The ESC epigenome must not only maintain the transcription of pluripotency-associated genes but must also, through gene priming, facilitate rapid and cell type-specific activation of developmental genes upon lineage commitment. Trans-generational inheritance ensures that the ESC chromatin state is stably transmitted from one generation to the next; yet at the same time, epigenetic marks are highly dynamic, reversible and responsive to extracellular cues. Once committed to differentiation, the ESC epigenome is remodeled and resolves into a more compact chromatin state. A thorough understanding of the role of chromatin modifiers in ESC fate and differentiation will be important if they are to be used for therapeutic purposes.Recent technical advances, particularly in next-generation sequencing technologies, have provided a genome-scale view of epigenetic marks and chromatin modifiers. More affordable and faster sequencing platforms have led to a comprehensive characterization of the ESC epigenome and epigenomes of differentiated cell types. In this review, we summarize and discuss the recent progress that has highlighted the central role of histone modifications, histone variants, DNA methylation and chromatin modifiers in ESC pluripotency and ESC fate. We provide a detailed and comprehensive discussion of genome-wide studies that are pertinent to our understanding of mammalian development.

Influence of Threonine Metabolism on S -Adenosylmethionine and Histone Methylation

Threonine is the only amino acid critically required for the pluripotency of mouse embryonic stem cells (mESCs), but the detailed mechanism remains unclear. We found that threonine and S-adenosylmethionine (SAM) metabolism are coupled in pluripotent stem cells, resulting in regulation of histone methylation. Isotope labeling of mESCs revealed that threonine provides a substantial fraction of both the cellular glycine and the acetyl-coenzyme A (CoA) needed for SAM synthesis. Depletion of threonine from the culture medium or threonine dehydrogenase (Tdh) from mESCs decreased accumulation of SAM and decreased trimethylation of histone H3 lysine 4 (H3K4me3), leading to slowed growth and increased differentiation. Thus, abundance of SAM appears to influence H3K4me3, providing a possible mechanism by which modulation of a metabolic pathway might influence stem cell fate.