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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

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Parallel genome-scale CRISPR-Cas9 screens uncouple human pluripotent stem cell identity versus fitness

Project description:Pluripotent stem cells are defined by their self-renewal capacity, which is the ability of the stem cells to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into any somatic cell lineage. However, understanding the mechanisms that control stem cell fitness versus the pluripotent cell identity is challenging. To investigate the interplay between these two aspects of pluripotency, we performed four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSC self-renewal conditions, and the dissolution of the primed pluripotency identity during early differentiation. Comparative analyses led to the discovery of genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further discovered a core set of factors that control both stem cell fitness and pluripotent identity, including a network of chromatin factors that safeguard pluripotency. Our unbiased and systematic screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide rich datasets for exploring pluripotent cell identity versus cell fitness, and offer a valuable model for categorizing gene function in broad biological contexts.

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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

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

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Project description:Translational control has emerged as a fundamental regulatory layer of proteome complexity that governs cellular identity and functions. As initiation is the rate-limiting step of translation, we carried out an RNAi screen for key translation initiation factors required to maintain embryonic stem cell (ESC) identity. We identified eIF4A2 and defined its mechanistic action through Rps26-independent and -dependent ribosomes in translation initiation activation of mRNAs encoding pluripotency factors and the histone variant H3.3 with demonstrated roles in maintaining stem cell pluripotency. eIF4A2 also mediates translation initiation activation of Ddx6, which acts together with eIF4A2 to restrict the totipotent 2-cell transcription program in ESCs through Zscan4 mRNA degradation and translation repression. Accordingly, knockdown of eIF4A2 disrupts ESC proteome causing the loss of ESC identity. Collectively, we establish a translational paradigm of the protein synthesis of pluripotency transcription factors and epigenetic regulators imposed on their established roles in controlling pluripotency.

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