Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification

AbstractN6-methyladenosine (m6A) on chromosome-associated regulatory RNAs (carRNAs), including repeat RNAs, plays important roles in tuning the chromatin state and transcription, but the intrinsic mechanism remains unclear. Here, we report that YTHDC1 plays indispensable roles in the self-renewal and differentiation potency of mouse embryonic stem cells (ESCs), which highly depends on the m6A-binding ability. Ythdc1 is required for sufficient rRNA synthesis and repression of the 2-cell (2C) transcriptional program in ESCs, which recapitulates the transcriptome regulation by the LINE1 scaffold. Detailed analyses revealed that YTHDC1 recognizes m6A on LINE1 RNAs in the nucleus and regulates the formation of the LINE1-NCL partnership and the chromatin recruitment of KAP1. Moreover, the establishment of H3K9me3 on 2C-related retrotransposons is interrupted in Ythdc1-depleted ESCs and inner cell mass (ICM) cells, which consequently increases the transcriptional activities. Our study reveals a role of m6A in regulating the RNA scaffold, providing a new model for the RNA-chromatin cross-talk.

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Base editing in bovine embryos reveals a species-specific role of SOX2 in regulation of pluripotency

10.1101/2021.11.10.468023 ◽

2021 ◽

Author(s):

Lei Luo ◽

Yan Shi ◽

Huanan Wang ◽

Zizengchen Wang ◽

Yanna Dang ◽

...

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Rna Seq ◽

Bovine Embryos ◽

Base Editing ◽

Sox2 Expression ◽

Nanog Expression ◽

Species Specific ◽

Lineage Specific Genes

The emergence of the first three lineages during development are orchestrated by a network of transcription factors, which are best characterized in mice. However, the role and regulation of these factors are not completely conserved in other mammals, including human and cattle. Here, we establish a gene inactivation system by introducing premature codon with cytosine base editor in bovine embryos with a robust efficiency. Of interest, SOX2 is universally localized in early blastocysts but gradually restricted into the inner cell mass in cattle. SOX2 knockout results in a failure of the establishment of pluripotency. Indeed, OCT4 level is significantly reduced and NANOG was barely detectable. Furthermore, the formation of primitive endoderm is compromised with few SOX17 positive cells. Single embryo RNA-seq reveals a dysregulation of 2074 genes, among which 90% are up-regulated in SOX2-null blastocysts. Intriguingly, more than a dozen lineage-specific genes, including OCT4 and NANOG, are down-regulated. Moreover, SOX2 expression is sustained in the trophectoderm in absence of CDX2 in bovine late blastocysts. Overall, we propose that SOX2 is dispensable for OCT4 and NANOG expression and disappearance of SOX2 in the trophectoderm depends on CDX2 in cattle, which are all in sharp contrast with results in mice.

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287 ACTIVE MITOCHONDRIA ARE PRESENT IN MOUSE AND MONKEY EMBRYONIC STEM CELL LINES

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab287 ◽

2008 ◽

Vol 20 (1) ◽

pp. 223 ◽

Cited By ~ 1

Author(s):

T. Lonergan ◽

A. Harvey ◽

J. Zhao ◽

B. Bavister ◽

C. Brenner

Keyword(s):

Cell Lines ◽

Complex I ◽

Inner Cell Mass ◽

Es Cells ◽

Cell Mass ◽

Embryonic Stem ◽

Mouse Fibroblast ◽

Es Cell ◽

Cell Content ◽

Stem Cell Lines

The inner cell mass (ICM) of the blastocyst develops into the fetus after uterine implantation. Prior to implantation, ICM cells synthesize ATP by glycolytic reactions. We now report that cells of the ICM in 3.5-day-old mouse embryos have too few mitochondria to be visualized with either Mitotracker red (active mitochondria) or an antibody against complex I of OXPHOS. By comparison, all of the surrounding trophectoderm cells reveal numerous mitochondria throughout their cytoplasm. It has largely been assumed that embryonic stem (ES) stem cells derived from the ICM also have few mitochondria, and that replication of mitochondria in the ES cells does not begin until they commence differentiation. We further report that mouse E14 ES cells and monkey ORMES 7 ES cells have considerable numbers of active mitochondria when cultured under standard conditions, i.e., 5% CO2 in air. Both the mouse E14 and monkey ES cell lines expressed two markers of undifferentiated cells, Oct-4 and SSEA-4, and monkey ES cells expressed the undifferentiated cell marker Nanog; however, Oct-4 is nonspecific in monkey ES cells because trophectoderm also expresses this marker, unlike in mice. Ninety-nine percent of the E14 cells examined, and 100% of the ORMES 7 cells, have a visible mitochondrial mass when stained with either Mitoracker red or with an antibody against OXPHOS complex I. The ATP content in the mouse E14 cells (4.13 pmoles ATP/cell) is not significantly different (P = 0.76) from that in a mouse fibroblast control (3.75 pmoles ATP/cell). Cells of the monkey ORMES 7 cell line had 61% of the ATP/cell content (7.55 pmoles ATP/cell) compared to the monkey fibroblast control (12.38 pmoles ATP/cell). Both cell lines expressed two proteins believed to indicate competence of mitochondria to replicate: PolG, the polymerase used to replicate the mitochondrial genome, and TFAM, a nuclear-encoded transcription factor reported to regulate several aspects of mitochondrial function. Both proteins were found to co-localize in the mitochondria. We conclude that when the ICMs are isolated from blastocysts and used to establish these two ES cell lines in cell culture, mitochondrial biosynthesis is activated.

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104. PROLIFERATION ABILITY, TELOMERASE ACTIVITY AND MOLECULAR CHARACTERIZATION OF PLURIPOTENT CELL LINES FROM IVF AND PARTHENOGENTIC PIG EMBRYOS

Reproduction Fertility and Development ◽

10.1071/srb09abs104 ◽

2009 ◽

Vol 21 (9) ◽

pp. 23

Author(s):

T. A. L. Brevini ◽

G. Pennarossa ◽

L. Attanasio ◽

B. Gasparrini ◽

F. Gandolfi

Keyword(s):

Cell Lines ◽

Doubling Time ◽

Inner Cell Mass ◽

Es Cells ◽

Cell Mass ◽

Telomerase Activity ◽

Self Renewal ◽

Pluripotency Markers ◽

Proliferation Ability ◽

Receptor Beta

Porcine pluripotent ES cell lines are a promising tool for biotechnology, biomedical and developmental biology studies. However, no conclusive results have been obtained to derive genuine ES cells in the pig. Here we compare derivation efficiency of putative ES cells from IVF versus parthenogenetic pig embryos. We describe proliferation ability and doubling time, we study pluripotency markers and telomerase activity (TA) of the cell lines obtained. Pig oocytes were either fertilized in vitro or parthenogenetically activated. Blastocysts were subjected to immuno-surgery. Inner cell mass were plated and outgrowth expansion was monitored daily. Self renewal molecules were studied by RT-PCR and/or immunocytochemistry for up to 42 passages. TA was measured every five passages. The results obtained indicate that stable cell lines can be generated from IVF and parthenogenetic embryos. The latter appeared less resilient to immuno-surgery but demonstrated a higher ability to produce outgrowths. 77% of the parthenogenetic lines vs only 33% of the IVF ones expressed pluripotency markers and displayed high TA. Regardless to their origin, colonies showed a latency growth period in the 48 hours after plating, they grew exponentially between day 3 and 6 and then, proliferation rate was greatly reduced. Doubling time was estimated to be 31.5 hours. In both IVF and parthenogenetic cell lines, positivity for Oct-4, Nanog, Sox-2, Rex-1, SSEA-4, Alkaline phosphatase, TRA-1-81 and STAT3 was detected; no signal for LIF-Receptor beta and gp130 was shown. These results indicate that the main pluripotency network related molecules are expressed in the porcine species, while a classical LIF-Receptor beta- gp130-STAT3 activation pathway does not appear to be involved in the maintenance of self renewal. Finally, every cell lines expressed high TA, which was turned down once cells were induced to differentiate, indicating a physiologically normal control of TA in these cells.

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Repression of Nanog Gene Transcription by Tcf3 Limits Embryonic Stem Cell Self-Renewal

Molecular and Cellular Biology ◽

10.1128/mcb.00368-06 ◽

2006 ◽

Vol 26 (20) ◽

pp. 7479-7491 ◽

Cited By ~ 203

Author(s):

Laura Pereira ◽

Fei Yi ◽

Bradley J. Merrill

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Molecular Mechanisms ◽

Inner Cell Mass ◽

Cell Mass ◽

Regulatory Region ◽

Embryonic Stem ◽

Dual Function ◽

Self Renewal ◽

Nanog Gene

ABSTRACT The dual function of stem cells requires them not only to form new stem cells through self-renewal but also to form lineage-committed cells through differentiation. Embryonic stem cells (ESC), which are derived from the blastocyst inner cell mass, retain properties of self-renewal and the potential for lineage commitment. To balance self-renewal and differentiation, ESC must carefully control the levels of several transcription factors, including Nanog, Sox2, and Oct4. While molecular mechanisms promoting transcription of these genes have been described, mechanisms preventing excessive levels in self-renewing ESC remain unknown. By examining the function of the TCF family of transcription factors in ESC, we have found that Tcf3 is necessary to limit the steady-state levels of Nanog mRNA, protein, and promoter activity in self-renewing ESC. Chromatin immunoprecipitation and promoter reporter assays showed that Tcf3 bound to a promoter regulatory region of the Nanog gene and repressed its transcriptional activity in ESC through a Groucho interaction domain-dependent process. The absence of Tcf3 caused delayed differentiation of ESC in vitro as elevated Nanog levels persisted through 5 days of embryoid body formation. These new data support a model wherein Tcf3-mediated control of Nanog levels allows stem cells to balance the creation of lineage-committed and undifferentiated cells.

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Hypoblast Formation in Bovine Embryos Does Not Depend on NANOG

Cells ◽

10.3390/cells10092232 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2232 ◽

Cited By ~ 1

Author(s):

Claudia Springer ◽

Valeri Zakhartchenko ◽

Eckhard Wolf ◽

Kilian Simmet

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Model Organism ◽

Cell Number ◽

Blastocyst Stage ◽

Bovine Embryo ◽

Lineage Differentiation ◽

Species Specific ◽

And Control

The role of the pluripotency factor NANOG during the second embryonic lineage differentiation has been studied extensively in mouse, although species-specific differences exist. To elucidate the role of NANOG in an alternative model organism, we knocked out NANOG in fibroblast cells and produced bovine NANOG-knockout (KO) embryos via somatic cell nuclear transfer (SCNT). At day 8, NANOG-KO blastocysts showed a decreased total cell number when compared to controls from SCNT (NT Ctrl). The pluripotency factors OCT4 and SOX2 as well as the hypoblast (HB) marker GATA6 were co-expressed in all cells of the inner cell mass (ICM) and, in contrast to mouse Nanog-KO, expression of the late HB marker SOX17 was still present. We blocked the MEK-pathway with a MEK 1/2 inhibitor, and control embryos showed an increase in NANOG positive cells, but SOX17 expressing HB precursor cells were still present. NANOG-KO together with MEK-inhibition was lethal before blastocyst stage, similarly to findings in mouse. Supplementation of exogenous FGF4 to NANOG-KO embryos did not change SOX17 expression in the ICM, unlike mouse Nanog-KO embryos, where missing SOX17 expression was completely rescued by FGF4. We conclude that NANOG mediated FGF/MEK signaling is not required for HB formation in the bovine embryo and that another—so far unknown—pathway regulates HB differentiation.

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