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Transcriptional Regulation of the Angptl8 Gene by Hepatocyte Nuclear Factor-1 in the Murine Liver.

ABSTRACT: Brief refeeding times (~60?min) enhanced hepatic Angptl8 expression in fasted mice. We cloned the mouse Angptl8 promoter region to characterise this rapid refeeding-induced increase in hepatic Angptl8 expression. Deletion of the -309/-60 promoter region significantly attenuated basal promoter activity in hepatocytes. A computational motif search revealed a potential binding motif for hepatocyte nuclear factor 1?/1? (HNF-1?/?) at -84/-68 bp of the promoter. Mutation of the HNF-1 binding site significantly decreased the promoter activity in hepatocytes, and the promoter carrying the mutated HNF-1 site was not transactivated by co-transfection of HNF-1 in a non-hepatic cell line. Silencing Hnf-1 in hepatoma cells and mouse primary hepatocytes reduced Angptl8 protein levels. Electrophoretic mobility-shift assays confirmed direct binding of Hnf-1 to its Angptl8 promoter binding motif. Hnf-1? expression levels increased after short-term refeeding, paralleling the enhanced in vivo expression of the Angptl8 protein. Chromatin immunoprecipitation (ChIP) confirmed the recruitment of endogenous Hnf-1 to the Angptl8 promoter region. Insulin-treated primary hepatocytes showed increased expression of Angptl8 protein, but knockdown of Hnf-1 completely abolished this enhancement. HNF-1 appears to play essential roles in the rapid refeeding-induced increases in Angptl8 expression. HNF-1? may therefore represent a primary medical target for ANGPTL8-related metabolic abnormalities. The study revealed the transcriptional regulation of the mouse hepatic Angptl8 gene by HNF-1.

SUBMITTER: Watanabe T

PROVIDER: S-EPMC7305314 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Transcriptional Regulation of the Angptl8 Gene by Hepatocyte Nuclear Factor-1 in the Murine Liver.

Watanabe Takuya T Ozawa Atsushi A Masuda Shinnosuke S Yoshino Satoshi S Ishida Emi E Kondo Yuri Y Matsumoto Shunichi S Katano-Toki Akiko A Horiguchi Kazuhiko K Nakajima Yasuyo Y Yamada Eijiro E Tomaru Takuya T Saito Tsugumichi T Ishii Sumiyasu S Shibusawa Nobuyuki N Okada Shuichi S Satoh Tetsurou T Yamada Masanobu M

Scientific reports 20200619 1

Brief refeeding times (~60 min) enhanced hepatic Angptl8 expression in fasted mice. We cloned the mouse Angptl8 promoter region to characterise this rapid refeeding-induced increase in hepatic Angptl8 expression. Deletion of the -309/-60 promoter region significantly attenuated basal promoter activity in hepatocytes. A computational motif search revealed a potential binding motif for hepatocyte nuclear factor 1α/1β (HNF-1α/β) at -84/-68 bp of the promoter. Mutation of the HNF-1 binding site sign ...[more]

PMID: 32561878

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Project description:AIM:To uncover the role of hepatocyte nuclear factor 4 alpha (HNF4?) in regulating hepatic expression of microRNAs. METHODS:Microarray and real-time PCR were used to determine hepatic expression of microRNAs in young-adult mice lacking Hnf4? expression in liver (Hnf4?-LivKO). Integrative genomics viewer software was used to analyze the public chromatin immunoprecipitation-sequencing datasets for DNA-binding of HNF4?, RNA polymerase-II, and histone modifications to loci of microRNAs in mouse liver and human hepatoma cells. Dual-luciferase reporter assay was conducted to determine effects of HNF4? on the promoters of mouse and human microRNAs as well as effects of microRNAs on the untranslated regions (3'UTR) of two genes in human hepatoma cells. RESULTS:Microarray data indicated that most microRNAs remained unaltered by Hnf4? deficiency in Hnf4?-LivKO mice. However, certain liver-predominant microRNAs were down-regulated similarly in young-adult male and female Hnf4?-LivKO mice. The down-regulation of miR-101, miR-192, miR-193a, miR-194, miR-215, miR-802, and miR-122 as well as induction of miR-34 and miR-29 in male Hnf4?-LivKO mice were confirmed by real-time PCR. Analysis of public chromatin immunoprecipitation-sequencing data indicates that HNF4? directly binds to the promoters of miR-101, miR-122, miR-194-2/miR-192 and miR-193, which is associated with histone marks of active transcription. Luciferase reporter assay showed that HNF4? markedly activated the promoters of mouse and human miR-101b/miR-101-2 and the miR-194/miR-192 cluster. Additionally, miR-192 and miR-194 significantly decreased activities of luciferase reporters for the 3'UTR of histone H3F3 and chromodomain helicase DNA binding protein 1 (CHD1), respectively, suggesting that miR-192 and miR-194 might be important in chromosome remodeling through directly targeting H3F3 and CHD1. CONCLUSION:HNF4? is essential for hepatic basal expression of a group of liver-enriched microRNAs, including miR-101, miR-192, miR-193a, miR-194 and miR-802, through which HNF4? may play a major role in the post-transcriptional regulation of gene expression and maintenance of the epigenome in liver.

Project description:HNF-4 (hepatocyte nuclear factor 4) is a key regulator of liver-specific gene expression in mammals. We have shown previously that the activity of the human APOC3 (apolipoprotein C-III) promoter is positively regulated by the anti-inflammatory cytokine TGFbeta (transforming growth factor beta) and its effectors Smad3 (similar to mothers against decapentaplegic 3) and Smad4 proteins via physical and functional interactions between Smads and HNF-4. We now show that the pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) antagonizes TGFbeta for the regulation of APOC3 gene expression in hepatocytes. TNFalpha was a strong inhibitor of the activity of apolipoprotein promoters that harbour HNF-4 binding sites and this inhibition required HNF-4. Using specific inhibitors of TNFalpha-induced signalling pathways, it was shown that inhibition of the APOC3 promoter by TNFalpha involved NF-kappaB (nuclear factor kappaB). Latent membrane protein 1 of the Epstein-Barr virus, which is an established potent activator of NF-kappaB as well as wild-type forms of various NF-kappaB signalling mediators, also inhibited strongly the APOC3 promoter and the transactivation function of HNF-4. TNFalpha had no effect on the stability or the nuclear localization of HNF-4 in HepG2 cells, but inhibited the binding of HNF-4 to the proximal APOC3 HRE (hormone response element). Using the yeast-transactivator-GAL4 system, we showed that both AF-1 and AF-2 (activation functions 1 and 2) of HNF-4 are inhibited by TNFalpha and that this inhibition was abolished by overexpression of different HNF-4 co-activators, including PGC-1 (peroxisome-proliferator-activated-receptor-gamma co-activator 1), CBP [CREB (cAMP-response-element-binding protein) binding protein] and SRC3 (steroid receptor co-activator 3). In summary, our findings indicate that TNFalpha, or other factors that trigger an NF-kappaB response in hepatic cells, inhibit the transcriptional activity of the APOC3 and other HNF-4-dependent promoters and that this inhibition could be accounted for by a decrease in DNA binding and the down-regulation of the transactivation potential of the AF-1 and AF-2 domains of HNF-4.

Dataset Information

Transcriptional Regulation of the Angptl8 Gene by Hepatocyte Nuclear Factor-1 in the Murine Liver.

Publications

Transcriptional Regulation of the Angptl8 Gene by Hepatocyte Nuclear Factor-1 in the Murine Liver.

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