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Integrated analysis identifies key determinants of embryonic stem cell identity and homeostasis

ABSTRACT: Despite RNAi-based screens to uncover genes controlling embryonic stem cell (ESC) identity, the pluripotency network remains poorly characterized, as does the precise molecular mechanisms underlying the balance between self-renewal and differentiation. Here we carried out a systematic meta-analysis of published gene expression data to rank-order genes based on their likelihood of regulating ESC identity. Not only did our analysis correctly rank known pluripotency-associated genes atop the list, but it also helped unearth many novel determinants of ESC identity including several components of functionally distinct complexes, as determined using RNAi. We focus on our top-hit Nucleolin, and characterize its mechanistic role in the maintenance of ESC homeostasis by shielding from differentiation-inducing redox imbalance-induced oxidative stress. Notably, we identify a conceptually novel mechanism involving a Nucleolin-dependent bistable switch regulating the homeostatic balance between self-renewal and differentiation in ESCs. Our gene ranks represent a rich and valuable resource for uncovering novel ESC regulators.

ORGANISM(S): Mus musculus

PROVIDER: GSE47872 | GEO | 2014/03/26

SECONDARY ACCESSION(S): PRJNA208208

REPOSITORIES: GEO

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Integrated analysis identifies key determinants of embryonic stem cell identity and homeostasis

Project description:Despite RNAi-based screens to uncover genes controlling embryonic stem cell (ESC) identity, the pluripotency network remains poorly characterized, as does the precise molecular mechanisms underlying the balance between self-renewal and differentiation. Here we carried out a systematic meta-analysis of published gene expression data to rank-order genes based on their likelihood of regulating ESC identity. Not only did our analysis correctly rank known pluripotency-associated genes atop the list, but it also helped unearth many novel determinants of ESC identity including several components of functionally distinct complexes, as determined using RNAi. We focus on our top-hit Nucleolin, and characterize its mechanistic role in the maintenance of ESC homeostasis by shielding from differentiation-inducing redox imbalance-induced oxidative stress. Notably, we identify a conceptually novel mechanism involving a Nucleolin-dependent bistable switch regulating the homeostatic balance between self-renewal and differentiation in ESCs. Our gene ranks represent a rich and valuable resource for uncovering novel ESC regulators. Microarray gene expression profiling in E14Tg2a mESCs after transfection with indicated siRNAs: Ncl siRNA #1 (Invitrogen, 17975-MSS206961) Ncl siRNA #2 (Invitrogen, 17975-MSS275939), and Control siRNA duplex targeting firefly luciferase.

2014-03-26 | E-GEOD-47872 | biostudies-arrayexpress

The Cohesin Complex Cooperates with Pluripotency Transcription Factors in the Maintenance of Embryonic Stem Cell Identity

Project description:Embryonic stem cells (ESCs) cells run a self-renewal gene expression program, requiring the expression of certain transcription factors accompanied by a particular chromosome organization to maintain a balance between pluripotency and the capacity for rapid differentiation. However, how transcriptional regulation is linked to chromosome organization in ESCs remains enigmatic. Here we show that Cohesin exhibits a functional role in maintaining ESC identity through association with the pluripotency transcriptional network. ChIP-seq analyses of the cohesin subunit Rad21 reveal an ESC specific cohesin binding pattern that is characterized by a CTCF independent colocalization of cohesin with pluripotency related transcription factors. Upon ESC differentiation, these binding sites disappear and instead new CTCF independent Rad21 binding sites emerge, which are enriched for binding sites of transcription factors implicated in early differentiation. Furthermore, knock-down of cohesin subunits causes expression changes that are reminiscent of the depletion of key pluripotency transcription factors, demonstrating the functional relevance of the cohesin - pluripotency transcriptional network association. Finally, we show that Nanog physically interacts with the cohesin interacting proteins Stag1 and Wapl, further substantiating this association. Based on these findings we propose that a dynamic placement of cohesin by pluripotency transcription factors contributes to a chromosome organization supporting the ESC expression program. This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2011-05-19 | E-GEOD-24030 | biostudies-arrayexpress

The Cohesin Complex Cooperates with Pluripotency Transcription Factors in the Maintenance of Embryonic Stem Cell Identity

2011-05-19 | GSE24030 | GEO

The THO complex regulates pluripotency gene mRNA export to control embryonic stem cell self-renewal and somatic cell reprogramming (RNA-IP)

Project description:Embryonic stem cell (ESC) self-renewal and differentiation is governed by a comprehensive regulatory network. Although the transcriptional regulation has been extensively investigated, post-transcriptional mechanisms controlling the ESC state are poorly understood. Here we describe a critical role of the THO complex in ESC self-renewal and differentiation. We show that THO preferentially interacts with pluripotency gene transcripts through Thoc5, and is required for self-renewal by regulating their export and expression. During differentiation, THO loses its interaction with those transcripts due to reduced Thoc5 expression, which leads to decreased expression of pluripotency proteins and facilitates differentiation. Finally, THO is also important for the establishment of pluripotency, as its depletion inhibits somatic cell reprogramming and blastocyst development. Together, our data indicates that THO regulates pluripotency gene mRNA export to control ESC self-renewal and differentiation, and uncovers a novel mechanism of post-transcriptional regulation in stem cell fate specification. RNA IP was conducted by use of antibody against Thoc2, the precipitated RNA was used to generate library using illumina Kit, and subsequently sequenced by miSeq

2013-11-30 | E-GEOD-48436 | biostudies-arrayexpress

The THO complex regulates pluripotency gene mRNA export to control embryonic stem cell self-renewal and somatic cell reprogramming (RNA-IP)

2013-11-30 | GSE48436 | GEO

The THO complex regulates pluripotency gene mRNA export to control embryonic stem cell self-renewal and somatic cell reprogramming (microarray)

Project description:Embryonic stem cell (ESC) self-renewal and differentiation are governed by a broad-ranging regulatory network. Although the transcriptional regulatory mechanisms involved have been investigated extensively, post-transcriptional regulation is still poorly understood. Here we describe a critical role of the THO complex in ESC self-renewal and differentiation. We show that THO preferentially interacts with pluripotency gene transcripts through Thoc5, and is required for self-renewal at least in part by regulating their export and expression. During differentiation, THO loses its interaction with those transcripts due to reduced Thoc5 expression, leading to decreased expression of pluripotency proteins that facilitates exit from self-renewal. THO is also important for the establishment of pluripotency, as its depletion inhibits somatic cell reprogramming and blastocyst development. Together, our data indicate that THO regulates pluripotency gene mRNA export to control ESC self-renewal and differentiation, and therefore uncover a role for this aspect of post-transcriptional regulation in stem cell fate specification. mouse J1 cells were transfected with non-targeting (NT), Thoc2, and Thoc5 siRNAs. Total RNA was isolated 96 hours after transfection.

2013-11-30 | E-GEOD-51496 | biostudies-arrayexpress

Transcription factors and Tet1/2 enable Brd4-independent maintenance of stem cell identity

Project description:Histone acetylation and the acetyl-lysine reader Brd4 have recently been implicated in embryonic stem cell (ESC) proliferation and self-renewal. We found that na•ve pluripotent ESCs exhibit increased incorporation of glucose-derived carbons onto acetylated histones and elevations in H3K9ac and Brd4 recruitment at pluripotency gene promoters. Surprisingly, both H3K9 acetyltransferases, GCN5 and PCAF, and Brd4 recruitment were dispensable for proliferation, self-renewal and pluripotency of na•ve ESCs. Na•ve ESCs maintain gene expression by stabilizing Mediator at core pluripotency genes in a Brd4-independent manner. Brd4-independent proliferation could also be achieved in metastable ESCs by overexpression of pluripotency transcription factors. Under all conditions, self-renewal required the DNA methylcytosine oxidases Tet1 and Tet2. These data reveal that there is minimal dependence on Brd4 for self-renewal of na•ve ESCs. Instead, the relative levels of DNA methylation and transcription factor abundance determine the requirement for bromodomain recognition of histone acetylation to the maintenance of stem cell identity.

2018-04-09 | GSE88769 | GEO

DNA-binding pluripotency factors and DNA demethylases can cooperate to maintain pluripotent stem cell identity even in the absence of Brd4 [RNA-seq]

Project description:Histone acetylation and the acetyl-lysine reader Brd4 have recently been implicated in embryonic stem cell (ESC) proliferation and self-renewal. We found that naïve pluripotent ESCs exhibit increased incorporation of glucose-derived carbons onto acetylated histones and elevations in H3K9ac and Brd4 recruitment at pluripotency gene promoters. Surprisingly, both H3K9 acetyltransferases, GCN5 and PCAF, and Brd4 recruitment were dispensable for proliferation, self-renewal and pluripotency of naïve ESCs. Naïve ESCs maintain gene expression by stabilizing Mediator at core pluripotency genes in a Brd4-independent manner. Brd4-independent proliferation could also be achieved in metastable ESCs by overexpression of pluripotency transcription factors. Under all conditions, self-renewal required the DNA methylcytosine oxidases Tet1 and Tet2. These data reveal that there is minimal dependence on Brd4 for self-renewal of naïve ESCs. Instead, the relative levels of DNA methylation and transcription factor abundance determine the requirement for bromodomain recognition of histone acetylation to the maintenance of stem cell identity.

2018-04-10 | GSE88760 | GEO

The THO complex regulates pluripotency gene mRNA export to control embryonic stem cell self-renewal and somatic cell reprogramming (microarray)

2013-11-30 | GSE51496 | GEO

Polycomb-like 2 associates with PRC2 and is required for mouse embryonic stem cell

Project description:Polycomb group (PcG) proteins are highly conserved epigenetic transcriptional repressors important for the control of numerous developmental gene expression programs and have recently been implicated in the modulation of embryonic stem cell (ESC) identity. We identified the PcG protein PCL2 (polycomb-like 2) in a genome-wide screen for novel regulators of self-renewal and pluripotency and predicted that it would play an important role in mouse ESC fate determination. Using multiple biochemical strategies, we provide evidence that PCL2 is a novel Polycomb Repressive Complex 2 (PRC2)-associated protein in mouse ESCs. Knockdown of Pcl2 in ESCs resulted in heightened self-renewal characteristics, defects in differentiation and altered patterns of histone methylation. Through integration of global gene expression and promoter occupancy analyses of both PCL2 and PRC2 components EZH2 and SUZ12, we have predicted PCL2 target genes and formulated regulatory networks describing the role of PCL2 both in modulating transcription of ESC self-renewal genes in undifferentiated ESCs as well as developmental regulators during early commitment and differentiation. Cells were stably expressing Pcl2 shRNA or shRNA mismatch control sequences. Hybridizations of three biological replicates for both the control and Pcl2 shRNA clone were performed.

2010-02-22 | E-GEOD-16364 | biostudies-arrayexpress

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