Abstract 1017: Post-translational Modification Of GATA-4 Involved In The Differentiation Of Monkey ES Cell Into Cardiac Myocytes

Transplantation of embryonic stem (ES) cells into infracted myocardium has been shown to preserve left ventricular function in rodents. Before application of ES cell therapy in humans, however, it is critical to perform pre-clinical studies in large animals such as primates. Characteristics of cynomolgus monkey ES cells are similar with those of human ES cells, but quite different from those of mouse ES cells. Differentiation of Embryonic stem (ES) cells into cardiac myocytes requires activation of a cardiac-specific gene program. Histone acetytrans-ferases (HATs) and Histone deactylases (HDACs) govern gene expression patterns by being recruited to the target genes through association with specific transcription factors. One of the HATs, p300, serves as a coactivator of cardiac-specific transcription factors such as GATA-4. The HAT activity of p300 is required for actylation and DNA binding of GATA-4 and its full transcription activity as well as for promotion of a transcriptionally active chromatin configuration. The role of HATs and HDACs in post-translational modification of GATA-4 during the differentiation of monkey ES cells into cardiac myocytes remained unknown. In an ES cell model of developing embryonic bodies, an acetylated form of GATA-4 and its DNA binding increased concomitantly with the expression of p300 during the differentiation of ES cells into cardiac myocytes. Treatment of ES cells with trichostatin A (TSA), a specific HDAC inhibitor, induced acetylation of histone-3/4 near GATA sites within the atrial natriuretic factor promoter. In addition, TSA augmented the increase in an acetylated form of GATA-4 and its DNA binding during the ES cell differentiation. TSA facilitate the expression of endogenous cardiac β-myosing heavy chain during the differentiation. These findings demonstrate that acetylation of GATA-4 as well as of histone are involved in the differentiation of monkey ES cells into cardiac myocytes.

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The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media

Development ◽

10.1242/dev.129.2.539 ◽

2002 ◽

Vol 129 (2) ◽

pp. 539-549 ◽

Cited By ~ 5

Author(s):

Carrie A. Adelman ◽

Subrata Chattopadhyay ◽

James J. Bieker

Keyword(s):

Embryoid Body ◽

Es Cells ◽

Expression Patterns ◽

Embryonic Stem ◽

Dominant Negative ◽

Erythroid Cell ◽

Specific Gene ◽

Smad Pathway ◽

Cellular Expression ◽

Novel Approach

Erythroid cell-specific gene regulation during terminal differentiation is controlled by transcriptional regulators, such as EKLF and GATA1, that themselves exhibit tissue-restricted expression patterns. Their early expression, already in evidence within multipotential hematopoietic cell lines, has made it difficult to determine what extracellular effectors and transduction mechanisms might be directing the onset of their own transcription during embryogenesis. To circumvent this problem, we have taken the novel approach of investigating whether the ability of embryonic stem (ES) cells to mimic early developmental patterns of cellular expression during embryoid body (EB) differentiation can address this issue. We first established conditions whereby EBs could form efficiently in the absence of serum. Surprisingly, in addition to mesoderm, these cells expressed hemangioblast and hematopoietic markers. However, they did not express the committed erythroid markers EKLF and GATA1, nor the terminally differentiated β-like globin markers. Using this system, we determined that EB differentiation in BMP4 was necessary and sufficient to recover EKLF and GATA1 expression and could be further stimulated by the inclusion of VEGF, SCF, erythropoietin and thyroid hormone. EBs were competent to respond to BMP4 only until day 4 of differentiation, which coincides with the normal onset of EKLF expression. The direct involvement of the BMP/Smad pathway in this induction process was further verified by showing that erythroid expression of a dominant negative BMP1B receptor or of the inhibitory Smad6 protein prevented induction of EKLF or GATA1 even in the presence of serum. Although Smad1, Smad5 and Smad8 are all expressed in the EBs, BMP4 induction of EKLF and GATA1 transcription is not immediate. These data implicate the BMP/Smad induction system as being a crucial pathway to direct the onset of EKLF and GATA1 expression during hematopoietic differentiation and demonstrate that EB differentiation can be manipulated to study induction of specific genes that are expressed early within a lineage.

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258. Differential expression of H2A and H3 variant histones in mouse preimplantation embryos and R1 ES cells

Reproduction Fertility and Development ◽

10.1071/srb08abs258 ◽

2008 ◽

Vol 20 (9) ◽

pp. 58

Author(s):

G. R. Kafer ◽

SA Lehnert ◽

P. L. Kaye ◽

R. J. Moser

Keyword(s):

Messenger Rna ◽

Expression Profiles ◽

Es Cells ◽

Expression Patterns ◽

Embryonic Stem ◽

Histone Variants ◽

Histone Variant ◽

Preimplantation Embryos ◽

Es Cell

Histone variants replace canonical histones in nucleosomes to serve numerous biological processes. This integration alters DNA properties to ultimately regulate gene expression. Previous mouse studies have indicated that some variants (H2AZ and H3.3) are essential for survival, but here we document and correlate histone expression patterns with key developmental events. Using quantitative reverse-transcribed PCR (qRT–PCR) we investigated the expression of 7 genes coding for H2A variants and 4 genes coding for H3 variants in mouse preimplantation embryos and in pluripotent R1 ES cells. Messenger RNA was extracted from pools of 3 embryos flushed from superovulated mice. Embryos were collected at five stages, zygotes, 2-cell embryos, morulae, blastocysts and hatching blastocysts (20 h, 44 h, 68 h, 92 h and 116 h post hCG respectively). The expression of H2A variant genes typically peaked within blastocysts. H2AZ and H2AX expression was 80 – 95% higher in blastocysts than other stages. Conversely, genes coding for H3 variants showed elevated expression in zygotes, where H3.3 expression was 85 – 95% higher and CENPA was ~75% higher than in later preimplantation stages. The expression profiles of histone remodellers SWI/SNF and CAF-1 correlated with the variants they are known to remodel (H2A and H3 variants respectively), suggesting an increased integration of those variants into nucleosomes. We also compared blastocyst and embryonic stem cell (ES cell) expression patterns. R1 ES cells express all histone variants, including H2A.Bbd, H3.1 and H3.2 which were not expressed in preimplantation embryos. Further, expression levels of every histone variant investigated differed significantly between R1 ES cells and hatching blastocysts (ANOVA, P < 0.05, n = 3 experiments). We conclude that histone variant expression reflects preimplantation developmental demands. Further, histone code expression profiles show significant change upon extended cell culture and maintenance of pluripotency as indicated by comparing in vivo hatching blastocysts to the R1 ES cell line.

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Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions

10.1101/299073 ◽

2018 ◽

Cited By ~ 4

Author(s):

Daniel Strebinger ◽

Cédric Deluz ◽

Elias T. Friman ◽

Subashika Govindan ◽

Andrea B. Alber ◽

...

Keyword(s):

Transcription Factors ◽

Cell Fate ◽

Es Cells ◽

Embryonic Stem ◽

Chromatin Accessibility ◽

Directed Differentiation ◽

Es Cell ◽

Endogenous Fluctuations ◽

Cell Fate Decisions ◽

Oct4 Protein

AbstractSOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. However, how temporal fluctuations in their expression levels bias lineage commitment is unknown. Here we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cell towards both neuroectodermal and mesendodermal fates. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.

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A Rapid and Highly Efficient Genotyping Pipeline for Screening Gene Targeted Mouse Embryonic Stem Cells

10.21203/rs.3.rs-748320/v1 ◽

2021 ◽

Author(s):

Roger Caothien ◽

Charles Yu ◽

Lucinda Tam ◽

Robert Newman ◽

Brian Nakao ◽

...

Keyword(s):

Animal Models ◽

Gene Targeting ◽

Fluorescent Protein ◽

Es Cells ◽

Expression Patterns ◽

Embryonic Stem ◽

Rapid Screening ◽

Es Cell ◽

Screening Process ◽

Targeting Vector

Abstract Gene targeting in mouse ES cells replaces or modifies genes of interest; conditional alleles, reporter knock-ins, and amino acid changes are common examples of how gene targeting is used. For example, enhanced green fluorescent protein or Cre recombinase is placed under the control of endogenous genes to define promoter expression patterns. The most important step in the process is to demonstrate that a gene targeting vector is correctly integrated in the genome at the desired chromosomal location. The rapid identification of correctly targeted ES cell clones is facilitated by proper targeting vector construction, rapid screening procedures, and advances in cell culture. The addition of magnetic activated cell sorting (MACS) technology and multiplex droplet digital PCR (ddPCR) to the ES cell screening process can achieve a greater than 60% assurance that ES clones are correctly targeted. In a further refinement of the process, drug selection cassettes are removed from ES cells with adenovirus technology. This improved workflow reduces the time needed to generate preclinical animal models. Faster access to animal models for therapeutic target identification and experimental validation can accelerate the development of therapies for human disease.

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XY female mice resulting from a heritable mutation in the primary testis-determining gene, Tdy

Development ◽

10.1242/dev.109.3.635 ◽

1990 ◽

Vol 109 (3) ◽

pp. 635-646 ◽

Cited By ~ 13

Author(s):

R. Lovell-Badge ◽

E. Robertson

Keyword(s):

Y Chromosome ◽

Southern Blotting ◽

Es Cells ◽

Embryonic Stem ◽

Chromosome Analysis ◽

Specific Gene ◽

Es Cell ◽

Karyotypic Analysis ◽

Testis Determination ◽

Xy Female

Chimeric mice constructed with XY embryonic stem (ES) cells that had been multiply infected with a retroviral vector were used in a genetic screen to look for mutations affecting the sex determination pathway in mice. From a small number of chimeras screened one was identified that gave rise to a low proportion of XY females amongst his offspring. Analysis of the segregating patterns of retroviral insertions demonstrated that the mutation was found in a subset of the offspring derived from one originally infected ES cell. However, the mutation appeared to have occurred subsequent to the infection. Some of the XY females proved to be fertile, and the mutant phenotype was found to segregate exclusively with the Y chromosome. Analysis of the offspring also confirmed the absence of any retroviral insertion that could be correlated with the mutation. Further characterisation of the Y chromosome carrying the mutation by karyotypic analysis, and by Southern blotting with a range of Y-specific DNA probes suggested that there has been no gross deletion or rearrangement of the Y carrying the mutation. There also appeared to be no loss of Y-specific gene functions apart from that of testis determination. Moreover, the mutation is complemented by Sxr', the minimum portion of the mouse Y known to carry Tdy. From the phenotype and deduced location of the mutation, we conclude that it is within the Tdy locus. This is the first such mutation to be described in mice.

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Increased Apoptosis and Skewed Differentiation in Mouse Embryonic Stem Cells Lacking the Histone Methyltransferase Mll2

Molecular Biology of the Cell ◽

10.1091/mbc.e06-11-1060 ◽

2007 ◽

Vol 18 (6) ◽

pp. 2356-2366 ◽

Cited By ~ 66

Author(s):

Sandra Lubitz ◽

Stefan Glaser ◽

Julia Schaft ◽

A. Francis Stewart ◽

Konstantinos Anastassiadis

Keyword(s):

Gene Expression ◽

Es Cells ◽

Expression Patterns ◽

Embryonic Stem ◽

Lineage Commitment ◽

Es Cell ◽

Histone 3 ◽

Caspase Inhibition ◽

Transcriptional Memory

Epigenetic regulation by histone methyltransferases provides transcriptional memory and inheritable propagation of gene expression patterns. Potentially, the transition from a pluripotent state to lineage commitment also includes epigenetic instructions. The histone 3 lysine 4 methyltransferase Mll2/Wbp7 is essential for embryonic development. Here, we used embryonic stem (ES) cell lines deficient for Mll2 to examine its function more accurately. Mll2−/− ES cells are viable and retain pluripotency, but they display cell proliferation defects due to an enhanced rate of apoptosis. Apoptosis was not relieved by caspase inhibition and correlated with decreased Bcl2 expression. Concordantly, Mll2 binds to the Bcl2 gene and H3K4me3levels are reduced at the binding site when Mll2 is absent. In vitro differentiation showed delays along representative pathways for all three germ layers. Although ectodermal delays were severe and mesodermal delays persisted at about three days, endodermal differentiation seemed to recover and overshoot, concomitant with prolonged Oct4 gene expression. Hence, Mll2 is not required for ES cell self-renewal or the complex changes in gene expression involved in lineage commitment, but it contributes to the coordination and timing of early differentiation decisions.

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Zic3 Is Required for Maintenance of Pluripotency in Embryonic Stem Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e06-07-0624 ◽

2007 ◽

Vol 18 (4) ◽

pp. 1348-1358 ◽

Cited By ~ 85

Author(s):

Linda Shushan Lim ◽

Yuin-Han Loh ◽

Weiwei Zhang ◽

Yixun Li ◽

Xi Chen ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Embryonic Stem Cells ◽

Regulatory Networks ◽

Molecular Mechanisms ◽

Es Cells ◽

Embryonic Stem ◽

Es Cell ◽

Lineage Specification ◽

Mouse Es Cells

Embryonic stem (ES) cell pluripotency is dependent upon sustained expression of the key transcriptional regulators Oct4, Nanog, and Sox2. Dissection of the regulatory networks downstream of these transcription factors has provided critical insight into the molecular mechanisms that regulate ES cell pluripotency and early differentiation. Here we describe a role for Zic3, a member of the Gli family of zinc finger transcription factors, in the maintenance of pluripotency in ES cells. We show that Zic3 is expressed in ES cells and that this expression is repressed upon differentiation. The expression of Zic3 in pluripotent ES cells is also directly regulated by Oct4, Sox2, and Nanog. Targeted repression of Zic3 in human and mouse ES cells by RNA interference–induced expression of several markers of the endodermal lineage. Notably, the expression of Nanog, a key pluripotency regulator and repressor of extraembryonic endoderm specification in ES cells, was significantly reduced in Zic3 knockdown cells. This suggests that Zic3 may prevent endodermal marker expression through Nanog-regulated pathways. Thus our results extend the ES cell transcriptional network beyond Oct4, Nanog, and Sox2, and further establish that Zic3 plays an important role in the maintenance of pluripotency by preventing endodermal lineage specification in embryonic stem cells.

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Biological Analysis of AML1/RUNX1 Arginine-Mutants by Means of Hematopoietic Rescue Experiments of Runx1-deficient Mouse Embryonic Stem Cells,

Blood ◽

10.1182/blood.v118.21.3382.3382 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3382-3382

Author(s):

Shinsuke Mizutani ◽

Masafumi Taniwaki ◽

Tsukasa Okuda

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Biological Significance ◽

Deficient Mouse ◽

Es Cell ◽

Wild Type ◽

Post Translational Modification ◽

Cell Clones ◽

Arginine Residues

Abstract Abstract 3382 Runt-Related Transcription Factor 1 (RUNX1; also called as Acute Myeloid Leukemia 1: AML1) is one of the most frequently mutated genes associated with human acute leukemia, and encodes DNA binding subunit of the Core-Binding Factor (CBF) transcription complex whose activity is essential for the development of definitive hematopoiesis. RUNX1 serves as a transcriptional activator as well as a repressor to its target genes, depending on the cellular context, mediated through its interaction with co-factors. Increasing evidence obtained these days suggests that post-translational modification of RUNX1, including phosphorylation, methylation, or acetylation on its target amino acid residues, is important for proper and fine tuning of this RUNX1-function, likely by altering its association with functional cofactors. However, biological significance of these modifications has not yet been examined in detail. As an initial effort towards systematic comprehension how these modifications influence RUNX1 function, we tried to evaluate RUNX1 methylation in vitro in this study. Arginine residues just douwnstream to the Runt-domain of RUNX1 were recently reported to be methylated to inhibit corepressor-binding thus enhances its trans-activating activity. In order to elucidate the biological effects of this post-translational modification, we manufactured arginine-to-lysine substitutions at the sites within the mouse cDNA. When these arginine-mutants were exogenously expressed in mammalian cell lines, they showed reduced trans-activating activity detected by a dual-luciferase assay on known reporter constructs in comparison to the wild-type Runx1, confirming previous reports. We then introduced the mutant cDNA into Runx1-deficient mouse embryonic stem (ES) cells by means of a knock-in strategy at the disrupted Runx1 gene locus. These ES cell clones were subjected to the in vitro differentiation to hematopoietic lineages. Wild-type ES cells are known to differentiate into hematopoietic cell lineages via embryoid body formation in a semi-solid culture system, whereas ES cells of Runx1-deficient genotype lose the ability to undergo hematopoietic differentiation. This phenomenon is recognized to be an in vitro phenocopy of the Runx1-deficient mice that suffer from embryonic death due to complete block of fetal liver hematopoiesis. Initial study so far showed that the Runx1-deficient ES cell clones restored the ability to develop hematopoietic cells including macrophages in culture when the arginine-mutant cDNA was re-expressed from the knock-in allele, as is the case for the control Runx1-deficient ES cells with the knocked-in wild-type Runx1. These results suggest that this arginine-to-lysine mutation is dispensable, at least, for the in vitro hematopoietic function of wild-type Runx1 although its trans-activating activity is somewhat impaired. We are currently focusing on introducing this mutation into mouse germ line, and the resultant genome-modified mice should show us the biological significance of the methylation-modification to this important molecule in the context of an entire animal. Disclosures: No relevant conflicts of interest to declare.

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Orphan Nuclear Receptor GCNF Is Required for the Repression of Pluripotency Genes during Retinoic Acid-Induced Embryonic Stem Cell Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.25.19.8507-8519.2005 ◽

2005 ◽

Vol 25 (19) ◽

pp. 8507-8519 ◽

Cited By ~ 123

Author(s):

Peili Gu ◽

Damien LeMenuet ◽

Arthur C.-K. Chung ◽

Michael Mancini ◽

David A. Wheeler ◽

...

Keyword(s):

Transcription Factors ◽

Retinoic Acid ◽

Cell Differentiation ◽

Embryonic Development ◽

Nuclear Receptor ◽

Es Cells ◽

Embryonic Stem ◽

Orphan Nuclear Receptor ◽

Es Cell ◽

Pluripotency Genes

ABSTRACT Embryonic stem (ES) cell pluripotency and differentiation are controlled by a network of transcription factors and signaling molecules. Transcription factors such as Oct4 and Nanog are required for self-renewal and maintain the undifferentiated state of ES cells. Decreases in the expression of these factors indicate the initiation of differentiation of ES cells. Inactivation of the gene encoding the orphan nuclear receptor GCNF showed that it plays an important role in the repression of Oct4 expression in somatic cells during early embryonic development. GCNF −/− ES cells were isolated to study the function of GCNF in the down-regulation of pluripotency genes during differentiation. Loss of repression of ES cell marker genes Oct4, Nanog, Sox2, FGF4, and Stella was observed upon treatment of GCNF −/− ES cells with retinoic acid. The loss of repression of pluripotency genes is either a direct or indirect consequence of loss of GCNF. Both the Oct4 and Nanog genes are direct targets of GCNF repression during ES cell differentiation and early mouse embryonic development. In contrast Sox2 and FGF4 are indirectly regulated by GCNF through Oct4. These findings establish a central role for GCNF in the repression of pluripotency gene expression during retinoic acid-induced ES cell differentiation.

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Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells.

The Journal of Cell Biology ◽

10.1083/jcb.126.3.701 ◽

1994 ◽

Vol 126 (3) ◽

pp. 701-711 ◽

Cited By ~ 59

Author(s):

J M Metzger ◽

W I Lin ◽

L C Samuelson

Keyword(s):

Cardiac Myocytes ◽

Cardiac Myocyte ◽

Contractile Activity ◽

Es Cells ◽

Embryonic Stem ◽

Functional Assay ◽

Contractile Apparatus ◽

Es Cell ◽

Cardiac Contractile

Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types including spontaneously contracting cardiac myocytes. We have utilized the ES cell differentiation culture system to study the development of the cardiac contractile apparatus in vitro. Difficulties associated with the cellular and developmental heterogeneity of this system have been overcome by establishing attached cultures of differentiating ES cells, and by the micro-dissection of the contracting cardiac myocytes from culture. The time of onset and duration of continuous contractile activity of the individual contracting myocytes was determined by daily visual inspection of the cultures. A functional assay was used to directly measure force production in ES cell-derived cardiac myocyte preparations. The forces produced during spontaneous contractions in the membrane intact preparation, and during activation by Ca2+ subsequent to chemical permeabilization of the surface membranes were determined in the same preparation. Results showed a transition in contractile sensitivity to Ca2+ in ES cell-derived cardiac myocytes during development in vitro. Cardiac preparations isolated from culture following the initiation of spontaneous contractile activity showed marked sensitivity of the contractile apparatus to activation by Ca2+. However, the Ca2+ sensitivity of tension development was significantly decreased in preparations isolated from culture following prolonged continuous contractile activity in vitro. The alteration in Ca2+ sensitivity obtained in vitro paralleled that observed during murine cardiac myocyte development in vivo. This provides functional evidence that ES cell-derived cardiac myocytes recapitulate cardiogenesis in vitro. Alterations in Ca2+ sensitivity could be important in optimizing the cardiac contractile response to variations in the myoplasmic Ca2+ transient during embryogenesis. The potential to stably transfect ES cells with cardiac regulatory genes, together with the availability of a functional assay using control and genetically modified ES cell-derived cardiac myocytes, will permit determination of the functional significance of altered cardiac gene expression during cardiogenesis in vitro.

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