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Effect of depletion of Hp1α (Cbx5) and Hp1β (Cbx1) on gene expression of mouse embryonic stem cells (mESCs)

ABSTRACT: Effect of depletion of Hp1α (Cbx5) and Hp1β (Cbx1) on gene expression of mouse embryonic stem cells (mESCs)

PROVIDER: PRJNA833445 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18976342_1.fastq.gz	Fastqsanger.gz
	SRR18976342_2.fastq.gz	Fastqsanger.gz
	SRR18976343_1.fastq.gz	Fastqsanger.gz
	SRR18976343_2.fastq.gz	Fastqsanger.gz
	SRR18976344_1.fastq.gz	Fastqsanger.gz

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Effect of depletion of Hp1α (Cbx5) and Hp1β (Cbx1) on gene expression of mouse embryonic stem cells (mESCs)

Project description:To investigate the function of HP1α and HP1β proteins in the regulation of heterochromatin in mESCs, we isolated blastocysts from matings of mice homozygous for Hp1βF/F; Hp1αF/F in a single well of a 96-well plate in ESC medium. After 2 days, when blastocysts hatched, they were trypsinized and cells were seeded in the same well for other 2 days. Expanded mESC clones were further tested for their genotype by PCR. A single clone of Hp1βF/F; Hp1αF/F was transfected with either pCAG-Cre-GFP or pCAG-GFP as a control. GFP positive cells were sorted by flow cytometry and seeded into 10 cm plates. Individual clones were isolated and expanded. Deletion of Hp1β or Hp1α floxed alleles in pCAG-Cre-GFP transfected clones was tested by Western Blotting with appropriate antibodies.

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HP1β has distinct subnuclear localizations, chromatin binding features and functions in embryonic stem cells and differentiating cells [ChIP-seq]

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. ChIP-Seq of HP1β in mouse embryonic stem cells.

2015-08-02 | E-GEOD-64946 | biostudies-arrayexpress

HP1β dependent phase separation regulates embryonic stem cells exit from naïve pluripotent state

Project description:Liquid-liquid phase separation has drawn great attention as a physical process that mediates the formation of membraneless organelles in cells to coordinate biological reactions in space and time. Previously, it was proposed that the formation of phase-separated droplets is a unique property of HP1α. Here, we show that HP1β, but not HP1α and HP1ɤ, forms nucleosome dependent phase separated droplets. We demonstrate that H3K9me3 is required for HP1β dependent phase separation. Importantly, HP1β dependent phase separation regulates heterochromatin formation, thus controlling embryonic stem cells differentiation. In response to pluripotency exit, HP1β is phosphorylated at serine 89 recruiting KAP1, a pluripotency regulator, and sequestering it in the heterochromatin compartment. This induces changes in the level of pluripotency factors and triggers pluripotency exit. We propose that such a trapping mechanism creates a fast and dynamic mode of remote control of gene expression to regulate cell fate decisions solely based on membraneless organelles.

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HP1β has distinct subnuclear localizations, chromatin binding features and functions in embryonic stem cells and differentiating cells [ChIP-seq]

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BRD4 represses CBX5

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HP1β has distinct subnuclear localizations, chromatin binding features and functions in embryonic stem cells and differentiating cells [expression]

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Maternal RNF114 plays critical roles in activation of the early zygotic genome through promoting ubiquitin-mediated degradation of Chromobox protein CBX5

Project description:Zygotic genomic activation (ZGA) is a landmark event in the maternal-to-zygotic transition (MZT), and the regulation of ZGA by maternal factors remains to be elucidated. In this study, the depletion of maternal RNF114 led to 2-cell embryos developmental arrest in mice. RNF114 was proven to play an important role in major ZGA using ethynyl uridine (EU) incorporation and transcriptome analysis. To study the underlying mechanism, we performed protein profiling in mature oocytes and found a potential substrate for RNF114, Chromobox protein CBX5, whose ubiquitination and degradation was regulated by RNF114. Furthermore, the overexpression of CBX5 prevented embryonic development and impeded major ZGA. In summary, our study reveals that maternal RNF114, as a ubiquitin E3 ligase, plays a precise role in mediating the degradation of repressive protein CBX5 during MZT, the misregulation of which may impede the appropriate activation of major ZGA in mouse embryos.

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HP1? has distinct subnuclear localizations, chromatin binding features and functions in embryonic stem cells and differentiating cells [expression]

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