HP1β has distinct subnuclear localizations, chromatin binding features and functions in embryonic stem cells and differentiating cells [ChIP-seq]
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ABSTRACT: In recent years, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, although the role of the major heterochromatin proteins in pluripotency remains largely unknown. Here we identify heterochromatin protein 1-beta (HP1β) to be differentially localized and associated with chromatin in pluripotent and differentiated cells, to be essential for proper ESC differentiation as well as for the maintenance of the pluripotent state. Microscopy analysis supported by biochemical fractionations and chromatin immunoprecipitation (ChIP) experiments demonstrate that unlike its prominent localization in heterochromatic chromocenters in differentiated cells, HP1β is diffuse in the ESC nucleoplasm, poorly bound to chromatin, and highly expressed. The fraction of HP1β that does associate with chromatin in ESCs is highly enriched in genic regions. HP1β-knockout ESCs but not HP1α knockout ESCs exhibit a loss of the morphological and proliferative characteristics of embryonic pluripotent cells. HP1β depletion in ESCs results in reduced expression of pluripotency factors and aberrant differentiation. In contrast, HP1β depletion in differentiated cells perturbs the maintenance of differentiation, and facilitates reprogramming to induced pluripotent stem cells. Our results demonstrate a dual role for HP1β, in ESCs it is essential for pluripotency maintenance, and in differentiated cells for proper differentiation. ChIP-Seq of HP1β in mouse embryonic stem cells.
Project description:Pluripotent Embryonic Stem Cells (ESCs) can be captured in vitro in different states, ranging from unrestricted ‘naïve’ to more developmentally constrained ‘primed’ pluripotency. Complexes involved in epigenetic regulation and key transcription factors have been shown to be involved in specifying these distinct states. In this study, we use proteomic profiling of the chromatin landscape in naive pluripotent ESCs, Epistem cells (EpiSCs) and early differentiated ESCs to survey the chromatin in naïve and primed pluripotency and during differentiation. We provide a comprehensive overview of epigenetic complexes situated on the chromatin and identify proteins associated with the maintenance and loss of pluripotency. The findings presented here indicate major compositional alterations of epigenetic complexes starting from ESC priming onwards. Our results contribute to the understanding of ESC differentiation and provide a framework for future studies of lineage commitment of ESCs.
Project description:In recent years, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, although the role of the major heterochromatin proteins in pluripotency remains largely unknown. Here we identify heterochromatin protein 1-beta (HP1β) to be differentially localized and associated with chromatin in pluripotent and differentiated cells, to be essential for proper ESC differentiation as well as for the maintenance of the pluripotent state. Microscopy analysis supported by biochemical fractionations and chromatin immunoprecipitation (ChIP) experiments demonstrate that unlike its prominent localization in heterochromatic chromocenters in differentiated cells, HP1β is diffuse in the ESC nucleoplasm, poorly bound to chromatin, and highly expressed. The fraction of HP1β that does associate with chromatin in ESCs is highly enriched in genic regions. HP1β-knockout ESCs but not HP1α knockout ESCs exhibit a loss of the morphological and proliferative characteristics of embryonic pluripotent cells. HP1β depletion in ESCs results in reduced expression of pluripotency factors and aberrant differentiation. In contrast, HP1β depletion in differentiated cells perturbs the maintenance of differentiation, and facilitates reprogramming to induced pluripotent stem cells. Our results demonstrate a dual role for HP1β, in ESCs it is essential for pluripotency maintenance, and in differentiated cells for proper differentiation.
Project description:Embryonic stem cell (ESC) fate decisions are regulated by a complex molecular circuitry that requires tight and coordinated gene expression regulations at multiple levels from chromatin organization to mRNA processing. Recently, ribosome biogenesis and translation have emerged as key pathways that efficiently control stem cell homeostasis. However, the molecular mechanisms underlying the regulation of these pathways remain largely unknown to date. Here, we analyzed the expression, in mouse ESCs, of over 300 genes involved in ribosome biogenesis and we identified RSL24D1 as the most differentially expressed between self-renewing and differentiated ESCs. RSL24D1 is highly expressed in multiple mouse pluripotent stem cell models and its expression profile is conserved in human ESCs. RSL24D1 is associated with nuclear pre-ribosomes and is required for the maturation and the synthesis of 60S subunits in mouse ESCs. Interestingly, RSL24D1 depletion significantly impairs global translation, particularly of key pluripotency factors, including POU5F1 and NANOG, as well as components of the polycomb repressive complex 2 (PRC2). Consistently, RSL24D1 is required for mouse ESC self-renewal and proliferation. Taken together, we show that RSL24D1-dependant ribosome biogenesis is required to both sustain the expression of pluripotent transcriptional programs and silence developmental programs, which concertedly dictate ESC homeostasis.
Project description:Dormancy is an essential biological process for the propagation of many life forms through generations and stressful conditions. Early embryos of many mammals are preservable for weeks to months within the uterus under dormancy, which can be induced in vitro through mTOR inhibition. Dormancy features silencing of the genome and abundant heterochromatin formation, which conflicts with the permissive and uncommitted genomic profile of pluripotent cells. Cellular strategies to maintain pluripotency in the fate of this conflict are not known. Here we probed chromatin regulation during embryonic stem cells’ (ESC) entry into dormancy to identify mechanisms that ensure faithful propagation of cellular identity through dormancy. We show a global increase in DNA methylation and loss of chromatin accessibility in dormant ESCs and find that TET DNA demethylases are essential to counteract this trend at key pluripotency regulatory elements, particularly at young LINE1 repeats and active enhancers. Demethylation of these targets by TETs is essential for transcription factor (TF) recruitment and transient chromatin decondensation before hypercompaction. We propose that key regulatory elements are bookmarked coordinately by TETs and TFs in dormancy for maintenance of cellular identity. Perturbation of TET activity compromises embryo survival through dormancy; whereas its enhancement improves survival rates. Our results reveal the first essential chromatin regulator in establishing mammalian embryonic dormancy and pave the way to building its temporal regulatory code in embryonic and adult tissues.
Project description:Chromocenters are established after the 2-cell (2C) stage during mouse embryonic development, but the factors that mediate chromocenter formation remain largely unknown. To identify regulators of 2C heterochromatin establishment, we generated an inducible system to convert embryonic stem cells (ESCs) to 2C-like cells. This conversion is marked by a global reorganization and dispersion of H3K9me3-heterochromatin foci, which are then reversibly formed upon re-entry into pluripotency. Profiling the chromatin-bound proteome (chromatome) by genome capture of ESCs transitioning to 2C-like cells, we uncover chromatin regulators involved in de novo heterochromatin formation. We identified TOPBP1 and investigated its binding partner SMARCAD1. SMARCAD1 and TOPBP1 associate with H3K9me3-heterochromatin in ESCs. Interestingly, the nuclear localization of SMARCAD1 is lost in 2C-like cells. SMARCAD1 or TOPBP1 depletion in mouse embryos lead to developmental arrest, reduction of H3K9me3 and remodeling of heterochromatin foci. Collectively, our findings contribute to comprehending the maintenance of chromocenters during early development.
Project description:The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocyes from ESCs. Here we report that a high density of human embryonic stem cell (ESC)-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells (HLCs) with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells (iPSCs). Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and Activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, as its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model, and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells. Total RNA were isolated from ORMES6 ESC, differentiated cells at IVDS2 and 3, and cells in the central foci (IVDS2-C) and peripheral (IVDS2-P) area of ESC colonies at IVDS2. Each condition was repeated twice and used ORMES6 ESC as control.
Project description:The decline in stem cell function leads to impairments in tissue homeostasis but genetic factors that control differentiation and de-differentiation of stem cells in the context of tissue homeostasis remain to be delineated. Here we show that Tnfaip2 (a target gene of TNFa/NFkB signaling) has an essential role for the differentiation of pluripotent, embryonic stem cells (ESCs). Knockdown of the planarian pseudo-orthologue, Smed-exoc3, impairs pluripotent stem cell differentiation, tissue homeostasis and regeneration in vivo. The study shows that Tnfaip2 deletion impairs changes in lipid metabolism that drive differentiation induction of ESCs. The application of palmitic acid (PA, the most abundant saturated fatty acid in mammalian cells) and palmitoylcarnitine (a mitochondrial carrier of PA) fully restores the differentiation of ESCs as well as the differentiation of pluripotent stem cells and organ maintenance in Smed-exoc3-depleted planarians. Together, these results identify a novel pathway downstream of TNFa/NFkB signaling, which is essential for exit from pluripotency by mediating changes in lipid metabolism.
Project description:Zfp462 is a vertebrate-specific C2H2-type transcription factor that has been found to play a crucial role in chromatin assembly and heterochromatin-mediated transcriptional silencing. However, its exact function in regulating the pluripotency of embryonic stem cells (ESCs) remains poorly understood. In this study, we observe that Zfp462 is highly expressed in undifferentiated ESCs, gradually decreases upon pluripotency exit, but then increases again during lineage differentiation. Knocking out of Zfp462 leads to reduced self-renewal of ESCs, abnormal pluripotency exit, and imbalance of lineage specification/differentiation. Multi-omics integrative analysis suggests that Zfp462 may function as a component of the ESC CORE module and collaborate with Oct4/Sox2/Nanog (OSN) to regulate the expression of pluripotency and developmental genes. Collectively, our research not only reveals the functions of Zfp462 in ESC self-renewal and differentiation but also proposes Zfp462 as a novel player in the pluripotency gene regulatory network (PGRN).
Project description:CDC14 phosphatases are critical components of the cell cycle machinery that drives exit from mitosis in yeast. However, the two mammalian paralogs, CDC14A and CDC14B, are dispensable for cell cycle progression or exit, and their function remains unclear. By generating a double Cdc14a; Cdc14b-null mouse model, we report here that CDC14 phosphatases control cell differentiation in pluripotent cells and their absence results in deficient development of the neural system. Lack of CDC14 impairs neural differentiation from embryonic stem cells (ESCs) accompanied by deficient induction of genes controlled by bivalent promoters. During ESC differentiation, CDC14 directly dephosphorylates and destabilizes Undifferentiated embryonic Transcription Factor 1 (UTF1), a critical regulator of stemness. In the absence of CDC14, increased UTF1 levels prevent the firing of bivalent promoters, resulting in defective induction of the transcriptional programs required for differentiation. These results suggest that mammalian CDC14 phosphatases function during the terminal exit from the cell cycle by modulating the transition from the pluripotent to the differentiated chromatin state, at least partially by controlling chromatin dynamics and transcription in a UTF1-dependent manner.
Project description:Previous investigations of the core gene regulatory circuitry that controls embryonic stem cell (ESC) pluripotency have largely focused on the roles of transcription, chromatin and non- coding RNA regulators. Alternative splicing (AS) represents a widely acting mode of gene regulation, yet its role in the regulation of ESC pluripotency and differentiation is poorly understood. Here, we identify the Muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of AS events that are differentially regulated between ESCs and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ESC-like AS pattern for at least half of these AS events. Among the events is an ESC-specific AS switch in the forkhead family transcription factor FOXP1 that controls pluripotency. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells (iPSCs) during somatic cell reprogramming. mRNA profiles of various embryonic stem cells, tissues and cell lines from human and mouse using high-throughput sequencing data and the role of MBNL proteins in regulation of ESC-differential alternative splicing