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Viral shRNA Knockdown of INS Promotor Activity in EndoC-βH1 Cells

ABSTRACT: Viral shRNA Knockdown of INS Promotor Activity in EndoC-βH1 Cells

PROVIDER: PRJNA445712 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6898972.fastq.gz	Fastqsanger.gz
	SRR6898973.fastq.gz	Fastqsanger.gz
	SRR6898974.fastq.gz	Fastqsanger.gz
	SRR6898975.fastq.gz	Fastqsanger.gz
	SRR6898977.fastq.gz	Fastqsanger.gz

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Viral shRNA Knockdown of INS Promotor Activity in EndoC-βH1 Cells

Project description:To inhibit INS expression, we used shRNA to target the INS promoter. We find that knocking down INS expression with such an shRNA targeting the INS promoter significantly affects expression of 259 genes.

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Genome-wide maps of chromatin state in EndoC-βH1 Cells

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Project description:We have developped a novel human pancreatic beta cell line: EndoC-βH5. EndoC-βH5 cells are ready-to-use and storable cells with physiological insulin secretion. EndoC-βH5 cells were generated by integrative gene transfer of immortalizing transgenes hTERT and SV40 large T along with Herpes Simplex Virus-1 thymidine kinase into human fetal pancreas. Immortalizing transgenes were removed after amplification using CRE activation and remaining non-excized cells eliminated using ganciclovir. Resulting cells were distributed as ready to use EndoC-βH5 cells. We performed comparative transcriptome analysis with EndoC-βH1 cells , extensive functional and immunological assays. RNA-seq confirmed abundant expression of beta cell transcription factors and functional markers, including incretin receptors.

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Project description:EndoC-βH1 is emerging as a critical human β cell model to study the genetic and environmental etiologies of β cell (dys)function and diabetes. Comprehensive knowledge of its molecular landscape is lacking, yet required, for effective use of this model. Here, we report chromosomal (spectral karyotyping), genetic (genotyping), epigenomic (ChIP-seq and ATAC-seq), chromatin interaction (Hi-C and Pol2 ChIA-PET), and transcriptomic (RNA-seq and miRNA-seq) maps of EndoC-βH1. Analyses of these maps define known (e.g., PDX1 and ISL1) and putative (e.g., PCSK1 and mir-375) β cell-specific transcriptional cis-regulatory networks and identify allelic effects on cis-regulatory element use. Importantly, comparison with maps generated in primary human islets and/or β cells indicates preservation of chromatin looping but also highlights chromosomal aberrations and fetal genomic signatures in EndoC-βH1. Together, these maps, and a web application we created for their exploration, provide important tools for the design of experiments to probe and manipulate the genetic programs governing β cell identity and (dys)function in diabetes.

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Dexamethasone effect on gene expression in human pancreatic islets and human pancreatic EndoC-βH1 cells

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RNAsequencing of control and STAT3 knocked down transriptomes of EndoC cells

Project description:Most obese and insulin resistant individuals do not develop diabetes. This is the result of the capacity of β-cells to adapt and produce enough insulin to cover the needs of the organism. The underlying mechanism of β-cell adaptation in obesity, however, remains unclear. Previous studies have suggested a role for STAT3 in mediating β-cell development and human glucose homeostasis, but little is known about its role in β-cells in obesity. We observed enhanced cytoplasmatic expression of STAT3 in severe obese and diabetic subjects. To address the functional role of STAT3 in adult β-cells, we generated mice with tamoxifen-inducible partial or full deletion of STAT3 in β-cells and fed them a high fat diet before analysis. Interestingly, β-cell homozygous and heterozygous STAT3 deficient obese mice showed glucose intolerance when compared to controls. Gene expression analysis by RNA-seq showed reduced expression of mitochondrial genes in STAT3 knocked-down human EndoC-βH1 cells and was confirmed in FACS-purified β-cells from STAT3 deficient mice. Moreover, knockdown of STAT3 impaired mitochondria activity in EndoC-βH1 and human islets, suggesting a mechanism for STAT3-regulated β-cell function. We propose non-canonical STAT3 activity as a marker of β-cell identity, improving glucose induced insulin secretion in obesity.

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