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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE201907 | GEO | 2023/01/25

REPOSITORIES: GEO

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

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2022-05-01 | E-MTAB-8329 | biostudies-arrayexpress

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

2015-08-02 | GSE64946 | GEO

Dynamic and flexible bridging of H3K9me3-nucleosomes via HP1β-dimerization establishes a plastic state of condensed chromatin

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2019-12-15 | GSE136659 | GEO

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Neural Stem Cells Direct Axon Guidance via their Radial Fiber Scaffold

Project description:To determine the genes regulated by Fezf2 during neocortical development, we performed RNA-seq on Fezf2 +/+; Emx1-cre (Fezf2 wildtype) and Fezf2 fl/+; Emx1-Cre (Fezf2 Knockout) neocortices isolated from the P0 mice. After RNA isolation from the dissected tissue the samples were processed for library preperation using Illumina Poly-A RNA kit. Fezf2 can reprogram the upper layer neocortical neurons to form corticospinal tract. To understand the key transcriptomic changes driven by Fezf2 that drive this reprogramming, we performed in RNA-seq on the cells over-expressing Fezf2 and Gfp or Gfp. At E15.5, in utero electroporations targeting cortical plate, were performed in pregnant mice with Fezf2 and Gfp plasmids or with Gfp plasmid alone, which was used as control. At P3, the Gfp positive cells were FAC sorted from Fezf2 and Gfp, or Gfp cortices and processed for library preparations using NUGEN Ovation® V2 system. Libraries were sequenced using Illumina 2000 platform to generate 75bp single reads. At least 10 million uniquely mapped reads were obtained for each sample. To determine the genes directly regulated by FEZF2, we performed the ChIP-Seq on N2A cells that were transfected to over express FEZF2 using pCAG- Fezf2-V5 and pCAG-V5. After 48 hr, of transfection, cells were fixed and processed for ChIP seq. Libraries were prepared using Illumina TruSeq ChIP Libary Preparation Kit.

2020-12-05 | GSE152611 | GEO

Comparison of the DNA methylation of in vitro cultured bovine SAM-treated blastocysts and non-treated blastocysts

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2016-02-29 | E-GEOD-75430 | biostudies-arrayexpress

Heterochromatin diversity modulates genome compartmentalization and loop extrusion barriers [Repli-seq]

Project description:Two dominant processes organizing chromosomes are loop extrusion and the compartmental segregation of active and inactive chromatin. The molecular players involved in loop extrusion during interphase, cohesin and CTCF, have been extensively studied and experimentally validated. However, neither the molecular determinants nor the functional roles of compartmentalization are well understood. Here, we distinguish three inactive chromatin states using contact frequency profiling, comprising two types of heterochromatin and a previously uncharacterized inactive state exhibiting a neutral interaction preference. We find that heterochromatin marked by long continuous stretches of H3K9me3, HP1α and HP1β correlates with a conserved signature of strong compartmentalization and is abundant in HCT116 colon cancer cells. We demonstrate that disruption of DNA methyltransferase activity dramatically remodels genome compartmentalization as a consequence of the loss of H3K9me3 and HP1 binding. Interestingly, H3K9me3-HP1α/β is replaced by the neutral inactive state and retains late replication timing. Furthermore, we show that H3K9me3-HP1α/β heterochromatin is permissive to loop extrusion by cohesin but refractory to CTCF, explaining a paucity of visible loop extrusion-associated patterns in Hi-C. Accordingly, CTCF loop extrusion barriers are reactivated upon loss of H3K9me3-HP1α/β, not as a result of canonical demethylation of the CTCF binding motif but due to an intrinsic resistance of H3K9me3-HP1α/β heterochromatin to CTCF binding. Together, our work reveals a dynamic structural and organizational diversity of the inactive portion of the genome and establishes new connections between the regulation of chromatin state and chromosome organization, including an interplay between DNA methylation, compartmentalization and loop extrusion.

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