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HNF4A regulation of transcription profiles in the mouse cecal epithelial cells

ABSTRACT: Purpose: HNF4A is a highly conserved nuclear receptor that has been associated with inflammatory bowel diseases. The goal of this study is to determine transcriptional regulation by HNF4A in the cecal IECs.. Methods: Intestinal epithelial cells (IECs) were sorted from the ceca of young adult WT and HNF4A IEC-specific KO mice and sequenced for transcriptome. Differentially expressed genes comparing the WT and HNF4A IEC-KO were determined. Results: Epithelial HNF4A regulates expression of hundreds of genes. HNF4A is particularly required for the expression of a set of immune signaling molecules that are critical for the IEC-intrapithelial lymphocyte crosstalk. Those molecules include members of the butyrophilin-like molecules (Btnl1 and Btnl6) and MHC-I like molecule H2-T3 etc. Expression of the immune signaling molecules was validated by qPCR. Further comparison with HNF4A ChIP-seq datasets confirmed that those genes are also direct HNF4A targets.

ORGANISM(S): Mus musculus

PROVIDER: GSE199415 | GEO | 2022/06/30

REPOSITORIES: GEO

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Project description:Age-related decline in brain endothelial cell (BEC) function critically contributes to cerebrovascular and neurodegenerative disease. Comprehensive atlases of the BEC transcriptome have become available but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting (MACS)-based mouse BEC enrichment protocol compatible with high-resolution mass-spectrometry and analysed the profiles of protein abundance changes across multiple time points between 3 and 18 months of age and identified Arf6 as one of the most prominently downregulated vesicle-mediated transport protein during BEC aging. To understand the role of ARF6 in human ECs, first we compared GFP-AAV treated human iECs differentiated from ARF6-KO vs WT iPSCs, next we compared ARF6-GFP-AAV vs GFP-AAV treated iECs differentiated from WT iPSCs. During the ARF6-KO vs WT iEC comparison we found 983 vs 741 proteins significantly down- and upregulated, respectively. Enrichment analyses of significantly downregulated proteins revealed mRNA processing among the most significantly affected biological processes. During the ARF6-GFP-AAV vs GFP-AAV treated WT iEC comparison we found 1106 vs 1218 proteins significantly down- and upregulated, respectively. Enrichment analyses of significantly upregulated proteins revealed endocytic recycling, retrograde transport (endosome to Golgi), and ER-Golgi vesicle-mediated transport among the most significantly affected biological processes. Specifically, ARF6 and its binding-protein GGA2, DNM1L and several subunits of the Conserved oligomeric Golgi complex (COG) were upregulated. Our approach uncovered changes not picked up by transcriptomic studies such as accumulation of vesicle cargo and receptor ligands including Apoe, a major regulator of brain lipid metabolism. Proteomic analysis of BECs from Apoe deficient mice revealed a signature of accelerated aging. To explore the role of APOE in human endothelial cells, in this experiment we compared human iECs differentiated from APOE-KO and WT iPSCs and found 326 significantly altered proteins. Enrichment analyses of significantly downregulated proteins revealed vesicle-mediated transport and vesicle fusion to be among the most significantly affected biological processes. Specifically, among the 34 significantly altered vesicle-mediated transport proteins 26 were downregulated. Accordingly, we found reduced levels of endocytosis of FM1-43FX in newly formed vesicles in APOE-KO iECs.

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HNF4A regulation of transcription profiles in the mouse cecal epithelial cells

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