Histone Code Modifications on Pluripotential Nuclei of Reprogrammed Somatic Cells

ABSTRACT Following hybridization with embryonic stem (ES) cells, somatic genomes are epigenetically reprogrammed and acquire pluripotency. This results in the transcription of somatic genome-derived tissue-specific genes upon differentiation. During nuclear reprogramming, it is expected that DNA and chromatin modifications, believed to function in cell-type-specific epigenotype memory, should be significantly modified. Indeed, current evidence indicates that acetylation and methylation of histone H3 and H4 amino termini play a major role in the regulation of gene activity through the modulation of chromatin conformation. Here, we show that the reprogrammed somatic genome of ES hybrid cells becomes hyperacetylated at H3 and H4, while lysine 4 (K4) of H3 becomes globally hyper-di- and -tri-methylated. In the Oct4 promoter region, histones H3 and H4 are acetylated and H3-K4 is highly tri-methylated on both the ES and reprogrammed somatic genomes, which correlates with gene activation and DNA demethylation. However, H3-K4 is also di- and tri-methylated in the promoter regions of Neurofilament-M (Nfm), Nfl, and Thy-1, which are all silent in both ES and hybrid cells. Thus, H3-K4 di- and tri-methylation of reprogrammed somatic genomes is independent of gene activity and represents one of the major events that occurs during somatic genome reprogramming towards a transcriptional activation-permissive state.

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MLL3/MLL4 Histone Methyltranferase Activity Dependent Chromatin Organization at Enhancers during Embryonic Stem Cell Differentiation

10.1101/2021.03.17.435905 ◽

2021 ◽

Author(s):

Naoki Kubo ◽

Rong Hu ◽

Zhen Ye ◽

Bing Ren

Keyword(s):

Cell Differentiation ◽

Long Range ◽

Transcriptional Activation ◽

Cellular Differentiation ◽

Es Cells ◽

Gene Activation ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Embryonic Stem Cell Differentiation ◽

Activity Dependent

MLL3 (KMT2C) and MLL4 (KMT2D), the major mono-methyltransferases of histone H3 lysine 4 (H3K4), are required for cellular differentiation and embryonic development in mammals. We previously observed that MLL3/4 promote long-range chromatin interactions at enhancers, however, it is still unclear how their catalytic activities contribute to enhancer-dependent gene activation in mammalian cell differentiation. To address this question, we mapped histone modifications, long-range chromatin contacts as well as gene expression in MLL3/4 catalytically deficient mouse embryonic stem (ES) cells undergoing differentiation toward neural precursor cells. We showed that MLL3/4 activities are responsible for deposition of H3K4me1 modification and formation of long-range enhancer-promoter contacts at a majority of putative enhancers gained during cell differentiation, but are dispensable for most candidate enhancers found in undifferentiated ES cells that persist through differentiation. While transcriptional induction at most genes is unaltered in the MLL3/4 catalytically deficient cells, genes making more contacts with MLL3/4-dependent putative enhancers are disproportionately affected. These results support that MLL3/4 contributes to cellular differentiation through histone-methyltransferase-activity dependent induction of enhancer-promoter contacts and transcriptional activation at a subset of lineage-specific genes.

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The chromosomal protein SMCHD1 regulates DNA methylation and the 2c-like state of embryonic stem cells by antagonizing TET proteins

Science Advances ◽

10.1126/sciadv.abb9149 ◽

2021 ◽

Vol 7 (4) ◽

pp. eabb9149

Author(s):

Zhijun Huang ◽

Jiyoung Yu ◽

Wei Cui ◽

Benjamin K. Johnson ◽

Kyunggon Kim ◽

...

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Homeobox Gene ◽

Dna Demethylation ◽

Chromosomal Protein ◽

Dna Hypomethylation ◽

Tet Proteins ◽

Target Sites ◽

Structural Maintenance Of Chromosomes ◽

Hinge Domain

5-Methylcytosine (5mC) oxidases, the ten-eleven translocation (TET) proteins, initiate DNA demethylation, but it is unclear how 5mC oxidation is regulated. We show that the protein SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) is found in complexes with TET proteins and negatively regulates TET activities. Removal of SMCHD1 from mouse embryonic stem (ES) cells induces DNA hypomethylation, preferentially at SMCHD1 target sites and accumulation of 5-hydroxymethylcytosine (5hmC), along with promoter demethylation and activation of the Dux double-homeobox gene. In the absence of SMCHD1, ES cells acquire a two-cell (2c) embryo–like state characterized by activation of an early embryonic transcriptome that is substantially imposed by Dux. Using Smchd1/Tet1/Tet2/Tet3 quadruple-knockout cells, we show that DNA demethylation, activation of Dux, and other genes upon SMCHD1 loss depend on TET proteins. These data identify SMCHD1 as an antagonist of the 2c-like state of ES cells and of TET-mediated DNA demethylation.

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Gene activation precedes DNA demethylation in response to infection in human dendritic cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814700116 ◽

2019 ◽

Vol 116 (14) ◽

pp. 6938-6943 ◽

Cited By ~ 39

Author(s):

Alain Pacis ◽

Florence Mailhot-Léonard ◽

Ludovic Tailleux ◽

Haley E. Randolph ◽

Vania Yotova ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Dendritic Cells ◽

Transcriptional Activation ◽

Time Course ◽

Gene Activation ◽

Dna Demethylation ◽

Epigenetic Mark ◽

Distal Enhancer ◽

Human Dendritic Cells

DNA methylation is considered to be a relatively stable epigenetic mark. However, a growing body of evidence indicates that DNA methylation levels can change rapidly; for example, in innate immune cells facing an infectious agent. Nevertheless, the causal relationship between changes in DNA methylation and gene expression during infection remains to be elucidated. Here, we generated time-course data on DNA methylation, gene expression, and chromatin accessibility patterns during infection of human dendritic cells withMycobacterium tuberculosis. We found that the immune response to infection is accompanied by active demethylation of thousands of CpG sites overlapping distal enhancer elements. However, virtually all changes in gene expression in response to infection occur before detectable changes in DNA methylation, indicating that the observed losses in methylation are a downstream consequence of transcriptional activation. Footprinting analysis revealed that immune-related transcription factors (TFs), such as NF-κB/Rel, are recruited to enhancer elements before the observed losses in methylation, suggesting that DNA demethylation is mediated by TF binding to cis-acting elements. Collectively, our results show that DNA demethylation plays a limited role to the establishment of the core regulatory program engaged upon infection.

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The Polycomb Group Protein Suz12 Is Required for Embryonic Stem Cell Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.01432-06 ◽

2007 ◽

Vol 27 (10) ◽

pp. 3769-3779 ◽

Cited By ~ 461

Author(s):

Diego Pasini ◽

Adrian P. Bracken ◽

Jacob B. Hansen ◽

Manuela Capillo ◽

Kristian Helin

Keyword(s):

Cell Differentiation ◽

Transcriptional Activation ◽

Es Cells ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Polycomb Group ◽

Embryonic Stem Cell Differentiation ◽

Es Cell ◽

Polycomb Group Protein ◽

Specific Expression

ABSTRACT Polycomb group (PcG) proteins form multiprotein complexes, called Polycomb repressive complexes (PRCs). PRC2 contains the PcG proteins EZH2, SUZ12, and EED and represses transcription through methylation of lysine (K) 27 of histone H3 (H3). Suz12 is essential for PRC2 activity and its inactivation results in early lethality of mouse embryos. Here, we demonstrate that Suz12 −/− mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12 −/− ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation-specific genes. Moreover, Suz12 −/− ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-specific genes. Finally, we demonstrate that the PcGs are actively recruited to several genes during ES cell differentiation, which despite an increase in H3K27me3 levels is not always sufficient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of specific expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different mechanisms to regulate transcription during cellular differentiation.

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Evidence for a Bigenic Chromatin Subdomain in Regulation of the Fetal-to-Adult Hemoglobin Switch

Molecular and Cellular Biology ◽

10.1128/mcb.01735-08 ◽

2008 ◽

Vol 29 (6) ◽

pp. 1635-1648 ◽

Cited By ~ 7

Author(s):

Hugues Beauchemin ◽

Marie Trudel

Keyword(s):

Transcriptional Activation ◽

Gene Activation ◽

Dna Demethylation ◽

Open Chromatin ◽

Chromosome Conformation ◽

Hemoglobin Switching ◽

Switching Patterns ◽

Adult Hemoglobin ◽

Globin Promoter

ABSTRACT During development, human β-globin locus regulation undergoes two critical switches, the embryonic-to-fetal and fetal-to-adult hemoglobin switches. To define the role of the fetal Aγ-globin promoter in switching, human β-globin-YAC transgenic mice were produced with the Aγ-globin promoter replaced by the erythroid porphobilinogen deaminase (PBGD) promoter (PBGDAγ-YAC). Activation of the stage-independent PBGDAγ-globin strikingly stimulated native Gγ-globin expression at the fetal and adult stages, identifying a fetal gene pair or bigenic cooperative mechanism. This impaired fetal silencing severely suppressed both δ- and β-globin expression in PBGDAγ-YAC mice from fetal to neonatal stages and altered kinetics and delayed switching of adult β-globin. This regulation evokes the two human globin switching patterns in the mouse. Both patterns of DNA demethylation and chromatin immunoprecipitation analysis correlated with gene activation and open chromatin. Locus control region (LCR) interactions detected by chromosome conformation capture revealed distinct spatial fetal and adult LCR bigenic subdomains. Since both intact fetal promoters are critical regulators of fetal silencing at the adult stage, we concluded that fetal genes are controlled as a bigenic subdomain rather than a gene-autonomous mechanism. Our study also provides evidence for LCR complex interaction with spatial fetal or adult bigenic functional subdomains as a niche for transcriptional activation and hemoglobin switching.

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Mice develop normally in the absence of Smad4 nucleocytoplasmic shuttling

Biochemical Journal ◽

10.1042/bj20061830 ◽

2007 ◽

Vol 404 (2) ◽

pp. 235-245 ◽

Cited By ~ 12

Author(s):

Christine A. Biondi ◽

Debipriya Das ◽

Michael Howell ◽

Ayesha Islam ◽

Elizabeth K. Bikoff ◽

...

Keyword(s):

Transcriptional Activation ◽

Nuclear Export ◽

Transforming Growth Factor ◽

Es Cells ◽

Nuclear Export Signal ◽

Embryonic Stem ◽

Nucleocytoplasmic Shuttling ◽

Mouse Development ◽

Developmental Defects

Smad4 in partnership with R-Smads (receptor-regulated Smads) activates TGF-β (transforming growth factor-β)-dependent signalling pathways essential for early mouse development. Smad4 null embryos die shortly after implantation due to severe defects in cell proliferation and visceral endoderm differentiation. In the basal state, Smad4 undergoes continuous shuttling between the cytoplasm and the nucleus due to the combined activities of an N-terminal NLS (nuclear localization signal) and an NES (nuclear export signal) located in its linker region. Cell culture experiments suggest that Smad4 nucleocytoplasmic shuttling plays an important role in TGF-β signalling. In the present study we have investigated the role of Smad4 shuttling in vivo using gene targeting to engineer two independent mutations designed to eliminate Smad4 nuclear export. As predicted this results in increased levels of Smad4 in the nucleus of homozygous ES cells (embryonic stem cells) and primary keratinocytes, in the presence or absence of ligand. Neither mutation affects Smad4 expression levels nor its ability to mediate transcriptional activation in homozygous cell lines. Remarkably mouse mutants lacking the Smad4 NES develop normally. Smad4 NES mutants carrying one copy of a Smad4 null allele also fail to display developmental defects. The present study clearly demonstrates that Smad4 nucleocytoplasmic shuttling is not required for embryonic development or tissue homoeostasis in normal, healthy adult mice.

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MOZ and BMI1 play opposing roles during Hox gene activation in ES cells and in body segment identity specification in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1422872112 ◽

2015 ◽

Vol 112 (17) ◽

pp. 5437-5442 ◽

Cited By ~ 14

Author(s):

Bilal N. Sheikh ◽

Natalie L. Downer ◽

Belinda Phipson ◽

Hannah K. Vanyai ◽

Andrew J. Kueh ◽

...

Keyword(s):

Gene Expression ◽

Es Cells ◽

Gene Activation ◽

Embryonic Stem ◽

Mrna Levels ◽

Body Segment ◽

Initial Activation ◽

Activation Phase

Hox genes underlie the specification of body segment identity in the anterior–posterior axis. They are activated during gastrulation and undergo a dynamic shift from a transcriptionally repressed to an active chromatin state in a sequence that reflects their chromosomal location. Nevertheless, the precise role of chromatin modifying complexes during the initial activation phase remains unclear. In the current study, we examined the role of chromatin regulators during Hox gene activation. Using embryonic stem cell lines lacking the transcriptional activator MOZ and the polycomb-family repressor BMI1, we showed that MOZ and BMI1, respectively, promoted and repressed Hox genes during the shift from the transcriptionally repressed to the active state. Strikingly however, MOZ but not BMI1 was required to regulate Hox mRNA levels after the initial activation phase. To determine the interaction of MOZ and BMI1 in vivo, we interrogated their role in regulating Hox genes and body segment identity using Moz;Bmi1 double deficient mice. We found that the homeotic transformations and shifts in Hox gene expression boundaries observed in single Moz and Bmi1 mutant mice were rescued to a wild type identity in Moz;Bmi1 double knockout animals. Together, our findings establish that MOZ and BMI1 play opposing roles during the onset of Hox gene expression in the ES cell model and during body segment identity specification in vivo. We propose that chromatin-modifying complexes have a previously unappreciated role during the initiation phase of Hox gene expression, which is critical for the correct specification of body segment identity.

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Abstract 578: Hex is Essential for Cardiac Myogenesis in Differentiating Embryonic Stem Cells

Circulation ◽

10.1161/circ.116.suppl_16.ii_104-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Ruri Kaneda ◽

Yu Liu ◽

Robert J Schwartz ◽

Michael D Schneider

Keyword(s):

Transcription Factors ◽

Es Cells ◽

Protein A ◽

Embryonic Stem ◽

Chimeric Protein ◽

List Type ◽

Promoter Regions ◽

Chip Technology ◽

Cardiac Myogenesis ◽

Direct Target

Background: We previously demonstrated that Sox17, an Sry-box-containing transcription factor that interacts with the Wnt/ β-catenin pathway, is essential for cardiac myogenesis in differentiating embryonic stem (ES) cells. Sox17 shRNA blocks cardiac myogenesis and selectively impairs the induction of Hex, a member of the homeobox family of transcription factors, many of which are involved in developmental processes. However, whether Hex is a direct target of Sox17 or not and the function of Hex in mouse ES cell differentiation to cardiomyocytes have not been defined. Hypothesis: Hex is a direct target of Sox17, and Hex is essential for cardiac myogenesis in ES cells. Methods: Cells were subjected to lentiviral transduction for a chimeric protein, contains Sox17 fused to protein A, using a protocol that reconstructs the native Sox17 expression pattern. Protein A-TEV-tagged Chromatin Immuno-precipitation (PAT-ChIP) technology was used to purify the DNA fragments bound to Sox17. Chromatin-immunoprecipitated DNA was subjected to PCR for promoter regions of Hex that contain putative Sox-binding motifs. Lentiviral vectors encoding shRNAs that knock down Hex were transduced into AB2.2 cells. Transduced cells, distinguished by expression of EGFP, were flow-sorted and subjected to embryonic body (EB) culture. Total RNA was extracted from EBs at 10 time points. Real-time QRT-PCR was carried out for representative genes related to mesoderm formation, mesoderm patterning, and cardiac myogenesis. Spontaneously beating EBs were scored. Results: Several Hex promoter regions containing predicted Sox17 binding sites were confirmed as potential direct targets of Sox17. The prevalence of beating EBs and the expression of cardiogenic transcription factors (Nkx2.5, Tbx5, Mef2c, Gata4 and myocardin) and cardiac structural genes (Ryr2 and α-MHC) both were suppressed by Hex shRNA. Hex shRNA did not impair the progressive down-regulation of Sox2 and Oct4 (master regulators of pluripotency) or the induction of Brachyury/T and Mesp1/2 (markers of primitive and precardiac mesoderm, respectively). Conclusion: Hex is potentially a direct target of Sox17, and is essential for cardiac myogenesis in differentiating ES cells at the stage of cardiac specification.

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Ten-Eleven Translocation 1 (Tet1) Is Regulated by O-Linked N-Acetylglucosamine Transferase (Ogt) for Target Gene Repression in Mouse Embryonic Stem Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m113.460386 ◽

2013 ◽

Vol 288 (29) ◽

pp. 20776-20784 ◽

Cited By ~ 87

Author(s):

Feng-Tao Shi ◽

Hyeung Kim ◽

Weisi Lu ◽

Quanyuan He ◽

Dan Liu ◽

...

Keyword(s):

Gene Expression ◽

Large Scale ◽

Target Genes ◽

Es Cells ◽

Ectopic Expression ◽

Embryonic Stem ◽

Dna Demethylation ◽

Mouse Es Cells ◽

Chromatin Regulators ◽

Glcnac Transferase

As a member of the Tet (Ten-eleven translocation) family proteins that can convert 5-methylcytosine (5mC) to 5-hydroxylmethylcytosine (5hmC), Tet1 has been implicated in regulating global DNA demethylation and gene expression. Tet1 is highly expressed in embryonic stem (ES) cells and appears primarily to repress developmental genes for maintaining pluripotency. To understand how Tet1 may regulate gene expression, we conducted large scale immunoprecipitation followed by mass spectrometry of endogenous Tet1 in mouse ES cells. We found that Tet1 could interact with multiple chromatin regulators, including Sin3A and NuRD complexes. In addition, we showed that Tet1 could also interact with the O-GlcNAc transferase (Ogt) and be O-GlcNAcylated. Depletion of Ogt led to reduced Tet1 and 5hmC levels on Tet1-target genes, whereas ectopic expression of wild-type but not enzymatically inactive Ogt increased Tet1 levels. Mutation of the putative O-GlcNAcylation site on Tet1 led to decreased O-GlcNAcylation and level of the Tet1 protein. Our results suggest that O-GlcNAcylation can positively regulate Tet1 protein concentration and indicate that Tet1-mediated 5hmC modification and target repression is controlled by Ogt.

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Non-essential function of KRAB zinc finger gene clusters in retrotransposon suppression

10.1101/2020.01.17.910679 ◽

2020 ◽

Cited By ~ 2

Author(s):

Gernot Wolf ◽

Alberto de Iaco ◽

Ming-An Sun ◽

Melania Bruno ◽

Matthew Tinkham ◽

...

Keyword(s):

Zinc Finger ◽

Zinc Finger Protein ◽

Germ Line ◽

Es Cells ◽

Gene Activation ◽

Embryonic Stem ◽

Gene Clusters ◽

Endogenous Retrovirus ◽

Segmental Duplications ◽

Transposon Silencing

AbstractThe Krüppel-associated box zinc finger protein (KRAB-ZFP) family amplified and diversified in mammals by segmental duplications, but the function of the majority of this gene family remains largely unexplored due to the inaccessibility of the gene clusters to conventional gene targeting. We determined the genomic binding sites of 61 murine KRAB-ZFPs and genetically deleted in mouse embryonic stem (ES) cells five large KRAB-ZFP gene clusters encoding nearly one tenth of the more than 700 mouse KRAB-ZFPs. We demonstrate that clustered KRAB-ZFPs directly bind and silence retrotransposons and block retrotransposon-borne enhancers from gene activation in ES cells. Homozygous knockout mice generated from ES cells deleted in one of two KRAB-ZFP clusters were born at sub-mendelian frequencies in some matings, but heterozygous intercrosses could also yield knockout progeny with no overt phenotype. We further developed a retrotransposon capture-sequencing approach to assess mobility of the MMETn family of endogenous retrovirus like elements, which are transcriptionally activated in KRAB-ZFP cluster KOs, in a pedigree of KRAB-ZFP cluster KO and WT mice. We identified numerous somatic and several germ-line MMETn insertions, and found a modest increase in activity in mutant animals, but these events were detected in both wild-type and KO mice in stochastic and highly variable patterns. Our data suggests that the majority of young KRAB-ZFPs play a non-essential role in transposon silencing, likely due to the large redundancy with other KRAB-ZFPs and other transposon restriction pathways in mice.One Sentence SummaryMegabase-scale deletions of KRAB-ZFP gene clusters in mice leads to retrotransposon activation.

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