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RNA-Seq to identify germline stem cell-enriched genes and cystoblast-enriched genes and BMP targeted genes

ABSTRACT: RNA-Seq to identify germline stem cell-enriched genes and cystoblast-enriched genes and BMP targeted genes

PROVIDER: PRJEB28003 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR2719950_1.fastq.gz	Fastqsanger.gz
	ERR2719950_2.fastq.gz	Fastqsanger.gz
	ERR2719951_1.fastq.gz	Fastqsanger.gz
	ERR2719951_2.fastq.gz	Fastqsanger.gz
	ERR2719952_1.fastq.gz	Fastqsanger.gz

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RNA-Seq to identify germline stem cell-enriched genes and cystoblast-enriched genes and BMP targeted genes

Project description:We sequenced the transcriptomes of genetically expanded stem cells that either experience high levels of BMP signalling (expression of constitutively active BMPR, tkvQD.) or low levels (expression of shRNA again the differentiation promoting factor bam). We also sequenced RNA from differentiating daughter cells (cystoblasts) isolated through the expression of bam.GFP. We then performed a differential expression analysis. Our aim was to identify germline stem cell-enriched gene and cystoblast-enriched genes by tkvQD vs bam-GFP. We also wished to identify possible BMP target genes by comparing tkvQD and bam.shRNA. We have subsequently taken a few of the genes identified and validated their roles in germline stem cell self-renewal and differentiation in vivo.

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E2F6 initiates stable epigenetic silencing of germline genes during embryonic development

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Project description:In mammals, many germline genes are repressed by epigenetic mechanisms to prevent their illegitimate expression in embryonic and somatic cells. To advance our understanding of the complete mechanisms restricting the expression of germline genes in mammals, we analyzed the chromatin signature of germline genes and performed a genome-wide CRISPR-Cas9 knock-out screen for genes involved in germline gene repression using a reporter system in which GFP is under the control of the epigenetically repressed Dazl germline promoter in mouse embryonic stem cells (ESCs). We showed that the repression of germline genes mainly depends on the polycomb complex PRC1.6 and DNA methylation, which function additively in mouse ESCs. Furthermore, we identified and validated several novel genes involved in the repression of germline genes, and characterized three of them: Usp7, Shfm1 (also known as Sem1) and Erh. Inactivation of Usp7, Shfm1 or Erh led to the upregulation of germline genes, as well as retrotransposons for Shfm1, in mouse ESCs. Functionally, Usp7 acts at two levels: firstly it associates with PRC1.6 components and represses germline genes independently of DNA methylation, and secondly it facilitates DNA methylation deposition at germline genes for long term repression. In summary, our study provides a global view of the epigenetic mechanisms and novel factors required for silencing germline genes in embryonic cells.

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