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Polycomb Underlies Transcriptional Heterogeneity in Lineage Priming of Embryonic Stem Cells

ABSTRACT: Polycomb Underlies Transcriptional Heterogeneity in Lineage Priming of Embryonic Stem Cells

PROVIDER: PRJNA273633 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR1772450.fastq.gz	Fastqsanger.gz
	SRR1772441.fastq.gz	Fastqsanger.gz
	SRR1772451.fastq.gz	Fastqsanger.gz
	SRR1772442.fastq.gz	Fastqsanger.gz
	SRR1772452.fastq.gz	Fastqsanger.gz

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Polycomb Underlies Transcriptional Heterogeneity in Lineage Priming of Embryonic Stem Cells [ChIP-Seq]

Project description:X-linked Chromatin Immunoprecipitation (ChIP) for the histone modification H3K27me3 was performed on subpopulations of the HexVenus reporter mouse ES cell line (clone HV1.5) grown under self-renewing conditions. Subpopulations were identified and isolated based on the expression of the ES cell surface marker SSEA 1 and the expression of the venus protein. At approximately 70% confluence, cells were trypsinised, resuspended in FACs buffer (10%FCS in PBS) and incubated with a mouse monoclonal antibody to SSEA 1 (MC-480, Developmental Hybridoma Studies Bank, University of Iowa). Cells were then incubated with an Alexa-647 conjugated anti-mouse IgM antibody (Invitrogen) and subpopulations were fractionated by flow cytometry prior to further processing specific to each method. The aim was to determine if subtle changes in priming gene transcription were associated with changes in the magnitude of H3K27me3.

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Polycomb Underlies Transcriptional Heterogeneity in Lineage Priming of Embryonic Stem Cells

Project description:This SuperSeries is composed of the SubSeries listed below.

2016-10-11 | GSE65289 | GEO

Mus musculus

Project description:Polycomb Underlies Transcriptional Heterogeneity in Lineage Priming of Embryonic Stem Cells [gene expression]

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Polycomb Underlies Transcriptional Heterogeneity in Lineage Priming of Embryonic Stem Cells [gene expression]

Project description:Quantitation of polyA mRNA levels in subpopulations of the HexVenus reporter (clone HV5.1) mouse ES cell line growing under self-renewing conditions. Subpopulations were identified and isolated based on the expression of the ES cell surface marker SSEA 1 and the expression of the venus protein. At approximately 70% confluence, cells were trypsinised, resuspended in FACs buffer (10%FCS in PBS) and incubated with a mouse monoclonal antibody to SSEA 1 (MC-480, Developmental Hybridoma Studies Bank, University of Iowa). Cells were then incubated with an Alexa-647 conjugated anti-mouse IgM antibody (Invitrogen) and subpopulations were fractionated by flow cytometry. The aim was to identify genes which are differentially expressed between FACS-sorted HV-S+ and HV+S+ primed mESC populations.

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Mus musculus

Project description:Polycomb Underlies Transcriptional Heterogeneity in Lineage Priming of Embryonic Stem Cells [ChIP-Seq and GRO-Seq]

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Polycomb Underlies Transcriptional Heterogeneity in Lineage Priming of Embryonic Stem Cells [ChIP-Seq and GRO-Seq]

Project description:Chromatin Immunoprecipitation (ChIP) for the histone modification H3K4me3 and H3K27me3 and Genome Wide Run-on seq (GROseq) were performed on subpopulations of the HexVenus reporter mouse ES cell line (clone HV1.5) growing under self-renewing conditions. Subpopulations were identified and isolated based on the expression of the ES cell surface marker SSEA 1 and the expression of the venus protein. At approximately 70% confluence, cells were trypsinised, resuspended in FACs buffer (10%FCS in PBS) and incubated with a mouse monoclonal antibody to SSEA 1 (MC-480, Developmental Hybridoma Studies Bank, University of Iowa). Cells were then incubated with an Alexa-647 conjugated anti-mouse IgM antibody (Invitrogen) and subpopulations were fractionated by flow cytometry prior to further processing specific to each method. The aim was to determine if differential gene expression between these populations is primarily mediated at the level of transcription and if these changes are concomitant with changes in chromatin state.

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Reduced lymphoid lineage priming promotes human hematopoietic stem cell expansion

Project description:Hematopoietic stem cells (HSCs) must balance self-renewal and lineage differentiation to regenerate the hematopoietic system throughout life. HSCs exhibit lineage-associated gene expression that keeps them responsive to demands of mature blood production. However, it is not known whether this process, termed lineage priming, directly influences HSC self-renewal. We investigated the link between stemness and lineage priming by attenuating the early lymphoid transcription factor E47 through ID2 over-expression (OE). Transcriptional profiling of ID2 OE HSCs showed down regulation of B-cell factors including EBF1 and FOXO1 with a concomitant increase in stemness programs and myeloerythroid factors including CEBPA and GATA1. This resulted in myeloid commitment bias from the earliest stages of differentiation. HSC self-renewal was strongly affected by this lineage perturbation resulting in an 11-fold expansion of HSCs. Thus, early lymphoid transcription factors antagonize human HSC self-renewal, providing a direct link between differentiation program priming and the maintenance of stem cell self-renewal.

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Transcriptional heterogeneity and cell cycle regulation as a central determinant of Primitive Endoderm priming

Project description:During embryonic development cells acquire identity at the same time as they are proliferating, implying that an intrinsic facet of cell fate choice requires coupling lineage decisions to rates of cell division. How is the cell cycle regulated to promote or suppress heterogeneity and differentiation? We explore this question combining time lapse imaging with single cell RNAseq in the contexts of self-renewal, priming and differentiation of embryonic stem cells (ESCs) towards the Primitive Endoderm lineage (PrE). Since ESCs are derived from the Inner Cell Mass of the mammalian blastocyst, ESCs in standard culture conditions are transcriptionally heterogeneous containing subfractions that are primed for either of the two ICM lineages, Epiblast and PrE. These subfractions represent dynamic states that can readily interconvert in culture, and the PrE subfraction is functionally primed for endoderm differentiation. Here we find that differential regulation of cell cycle can tip the balance between these primed populations, such that naïve ESC culture conditions promote Epiblast-like expansion and PrE differentiation stimulates the selective proliferation of PrE-primed cells. In endoderm differentiation, we find that this change is accompanied by a counter-intuitive increase in G1 length that also appears replicated in vivo. While FGF/ERK signalling is a known key regulator of ESC and PrE differentiation, we find it is not just responsible for ESC heterogeneity, but also cell cycle synchronisation, required for the inheritance of similar cell cycles between sisters and cousins. Taken together, our results point to a tightly regulated relationship between transcriptional heterogeneity and cell cycle regulation in the context of lineage priming, with primed cell populations providing a pool of flexible cell types that can be expanded in a lineage specific fashion while allowing plasticity during early determination.

2022-08-01 | GSE200534 | GEO

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