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

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.

scholarly journals Acceleration of mesoderm development and expansion of hematopoietic progenitors in differentiating ES cells by the mouse Mix-like homeodomain transcription factor

Blood ◽  
2006 ◽  
Vol 107 (8) ◽  
pp. 3122-3130 ◽  
Author(s):  
Stephen Willey ◽  
Angel Ayuso-Sacido ◽  
Hailan Zhang ◽  
Stuart T. Fraser ◽  
Kenneth E. Sahr ◽  
...  
Keyword(s):  
Es Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitors ◽  
Adult Type ◽  
Mesoderm Development ◽  
Molecular Events ◽  
Organ Systems

Abstract The cellular and molecular events underlying the formation and differentiation of mesoderm to derivatives such as blood are critical to our understanding of the development and function of many tissues and organ systems. How different mesodermal populations are set aside to form specific lineages is not well understood. Although previous genetic studies in the mouse embryo have pointed to a critical role for the homeobox gene Mix-like (mMix) in gastrulation, its function in mesoderm development remains unclear. Hematopoietic defects have been identified in differentiating embryonic stem cells in which mMix was genetically inactivated. Here we show that conditional induction of mMix in embryonic stem cell–derived embryoid bodies results in the early activation of mesodermal markers prior to expression of Brachyury/T and acceleration of the mesodermal developmental program. Strikingly, increased numbers of mesodermal, hemangioblastic, and hematopoietic progenitors form in response to premature activation of mMix. Differentiation to primitive (embryonic) and definitive (adult type) blood cells proceeds normally and without an apparent bias in the representation of different hematopoietic cell fates. Therefore, the mouse Mix gene functions early in the recruitment and/or expansion of mesodermal progenitors to the hemangioblastic and hematopoietic lineages.

scholarly journals The Grb2/Mek Pathway Represses Nanog in Murine Embryonic Stem Cells

2006 ◽  
Vol 26 (20) ◽  
pp. 7539-7549 ◽  
Author(s):  
Takashi Hamazaki ◽  
Sarah M. Kehoe ◽  
Toru Nakano ◽  
Naohiro Terada
Keyword(s):  
Es Cells ◽  
Tyrosine Phosphatase ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Cell Aggregation ◽  
Cell Aggregates ◽  
Es Cell ◽  
Sodium Vanadate ◽  
Primitive Endoderm ◽  
Endoderm Differentiation

ABSTRACT The homeobox gene Nanog is a key intrinsic determinant of self renewal in embryonic stem (ES) cells, and its repression leads ES cells to selectively differentiate into primitive endoderm. Although Nanog repression occurs at the outermost layer of ES cell aggregates independent of the leukemia inhibitory factor (LIF)/STAT3 pathway, it is largely undetermined what external cues and intracellular signals cause the event. Of interest, addition of the tyrosine phosphatase inhibitor, sodium vanadate, selectively repressed Nanog transcription without any detectable changes in upstream transcriptional regulators Oct3/4 and Sox2. Furthermore, sodium vanadate induced primitive endoderm differentiation, even in the inner cells of ES cell aggregates. Expression of Gata6 and Zfp42, two putative downstream Nanog effectors, was also increased and decreased by the addition of sodium vanadate, respectively, but these changes were eliminated by exogenous Nanog expression. The effects of sodium vanadate were abrogated by Grb2 deficiency or by the addition of the Mek inhibitor, PD98059. Indeed, PD98059 prevented Nanog repression induced by ES cell aggregation as well. Furthermore, transfection of a constitutive active Mek mutant into ES cells induced Nanog repression and primitive endoderm differentiation. These data indicate that the Grb2/Mek pathway primarily mediates Nanog gene repression upon ES cell differentiation into primitive endoderm.

scholarly journals Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression

2019 ◽  
Author(s):  
Michael J Reimer ◽  
Kirthi Pulakanti ◽  
Linzheng Shi ◽  
Alex Abel ◽  
Mingyu Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Critical Role ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Intermediate Phenotype ◽  
Gene Promoters ◽  
Dna Hypermethylation ◽  
Tet Proteins ◽  
Tet Protein

Abstract Background: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency¬. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments. Results: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 ¬alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs. Conclusions: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.

The putative nuclear receptor mediator TIF1alpha is tightly associated with euchromatin

1999 ◽  
Vol 112 (11) ◽  
pp. 1671-1683 ◽  
Author(s):  
E. Remboutsika ◽  
Y. Lutz ◽  
A. Gansmuller ◽  
J.L. Vonesch ◽  
R. Losson ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Receptors ◽  
Target Genes ◽  
Es Cells ◽  
Embryonic Stem ◽  
Chromosomal Protein ◽  
Metaphase Chromosomes ◽  
Interphase Nuclei ◽  
Ec Cells ◽  
Early Mouse

Ligand-dependent transcriptional regulation by nuclear receptors is believed to be mediated by intermediary factors (TIFs) acting on remodelling of the chromatin structure and/or the activity of the transcriptional machinery. The putative transcriptional mediator TIF1alpha is a nuclear protein kinase that has been identified via its interaction with liganded nuclear receptors, including retinoic acid (RAR), retinoid X (RXR) and estrogen (ER) receptors. Here, we demonstrate that TIF1alpha is a non-histone chromosomal protein tightly associated with highly accessible euchromatic regions of the genome. Immunofluorescence confocal microscopy reveals that TIF1alpha exhibits a finely granular distribution in euchromatin of interphase nuclei, while it is mostly excluded from condensed chromatin and metaphase chromosomes. Immunoelectron microscopy shows that, in contrast to the heterochromatin protein HP1alpha, most of TIF1alpha is associated with euchromatin, where it is preferentially localised on regions known to be sites for RNA polymerase II (perichromatin fibrils and borders between euchromatin and heterochromatin). Early mouse embryos as well as embryonal carcinoma (EC) and embryonic stem (ES) cells express high levels of TIF1alpha. These levels dramatically decrease during organogenesis and upon differentiation of P19 EC cells, indicating that TIF1alpha is preferentially expressed in undifferentiated pluripotent cells in the course of development. Therefore, TIF1alpha could belong to a novel class of chromatin-associated TIFs that facilitate the access of transregulators (e.g. liganded nuclear receptors) to their cognate sites in target genes, thereby participitating in the epigenetic control of transcription during embryonic development and cell differentiation.

scholarly journals Histone Code Modifications on Pluripotential Nuclei of Reprogrammed Somatic Cells

2004 ◽  
Vol 24 (13) ◽  
pp. 5710-5720 ◽  
Author(s):  
Hironobu Kimura ◽  
Masako Tada ◽  
Norio Nakatsuji ◽  
Takashi Tada
Keyword(s):  
Transcriptional Activation ◽  
Es Cells ◽  
Gene Activation ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Gene Activity ◽  
Current Evidence ◽  
Hybrid Cells ◽  
Promoter Regions ◽  
Somatic Genome

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.

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

2013 ◽  
Vol 288 (29) ◽  
pp. 20776-20784 ◽  
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.

scholarly journals Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to differences in gene expression

2019 ◽  
Author(s):  
Michael J Reimer ◽  
Kirthi Pulakanti ◽  
Linzheng Shi ◽  
Alex Abel ◽  
Mingyu Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Critical Role ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Intermediate Phenotype ◽  
Gene Promoters ◽  
Dna Hypermethylation ◽  
Tet Proteins ◽  
Tet Protein

Abstract Background: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency¬. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments. Results: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 ¬alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs. Conclusions: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.

scholarly journals Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression

2019 ◽  
Author(s):  
Michael J Reimer ◽  
Kirthi Pulakanti ◽  
Linzheng Shi ◽  
Alex Abel ◽  
Mingyu Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Critical Role ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Intermediate Phenotype ◽  
Gene Promoters ◽  
Dna Hypermethylation ◽  
Tet Proteins ◽  
Tet Protein

Abstract Background: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency¬. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments. Results: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 ¬alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs. Conclusions: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.

Wnt/(beta)-catenin signaling regulates the expression of the homeobox gene Cdx1 in embryonic intestine

Development ◽  
2000 ◽  
Vol 127 (17) ◽  
pp. 3805-3813 ◽  
Author(s):  
H. Lickert ◽  
C. Domon ◽  
G. Huls ◽  
C. Wehrle ◽  
I. Duluc ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Intestinal Epithelium ◽  
Es Cells ◽  
Embryonic Stem ◽  
Early Period ◽  
Homeobox Gene ◽  
Beta Catenin ◽  
Mammalian Development

During mammalian development, the Cdx1 homeobox gene exhibits an early period of expression when the embryonic body axis is established, and a later period where expression is restricted to the embryonic intestinal endoderm. Cdx1 expression is maintained throughout adulthood in the proliferative cell compartment of the continuously renewed intestinal epithelium, the crypts. In this study, we provide evidence in vitro and in vivo that Cdx1 is a direct transcriptional target of the Wnt/(beta)-catenin signaling pathway. Upon Wnt stimulation, expression of Cdx1 can be induced in mouse embryonic stem (ES) cells as well as in undifferentiated rat embryonic endoderm. Tcf4-deficient mouse embryos show abrogation of Cdx1 protein in the small intestinal epithelium, making Tcf4 the likely candidate to transduce Wnt signal in this part of gut. The promoter region of the Cdx1 gene contains several Tcf-binding motifs, and these bind Tcf/Lef1/(beta)-catenin complexes and mediate (beta)-catenin-dependent transactivation. The transcriptional regulation of the homeobox gene Cdx1 in the intestinal epithelium by Wnt/(beta)-catenin signaling underlines the importance of this signaling pathway in mammalian endoderm development.

scholarly journals Tet Proteins Can Convert 5-Methylcytosine to 5-Formylcytosine and 5-Carboxylcytosine

Science ◽  
2011 ◽  
Vol 333 (6047) ◽  
pp. 1300-1303 ◽  
Author(s):  
Shinsuke Ito ◽  
Li Shen ◽  
Qing Dai ◽  
Susan C. Wu ◽  
Leonard B. Collins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Enzymatic Activity ◽  
Genomic Dna ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Dependent Manner ◽  
Tet Proteins ◽  
Activity Dependent ◽  
Catalyzed Oxidation

5-methylcytosine (5mC) in DNA plays an important role in gene expression, genomic imprinting, and suppression of transposable elements. 5mC can be converted to 5-hydroxymethylcytosine (5hmC) by the Tet (ten eleven translocation) proteins. Here, we show that, in addition to 5hmC, the Tet proteins can generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) from 5mC in an enzymatic activity–dependent manner. Furthermore, we reveal the presence of 5fC and 5caC in genomic DNA of mouse embryonic stem cells and mouse organs. The genomic content of 5hmC, 5fC, and 5caC can be increased or reduced through overexpression or depletion of Tet proteins. Thus, we identify two previously unknown cytosine derivatives in genomic DNA as the products of Tet proteins. Our study raises the possibility that DNA demethylation may occur through Tet-catalyzed oxidation followed by decarboxylation.

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