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Identification of HNF-4α as a key transcription factor to promote ChREBP expression in response to glucose.

ABSTRACT: Transcription factor carbohydrate responsive element binding protein (ChREBP) promotes glycolysis and lipogenesis in metabolic tissues and cancer cells. ChREBP-α and ChREBP-β, two isoforms of ChREBP transcribed from different promoters, are both transcriptionally induced by glucose. However, the mechanism by which glucose increases ChREBP mRNA levels remains unclear. Here we report that hepatocyte nuclear factor 4 alpha (HNF-4α) is a key transcription factor for glucose-induced ChREBP-α and ChREBP-β expression. Ectopic HNF-4α expression increased ChREBP transcription while knockdown of HNF-4α greatly reduced ChREBP mRNA levels in liver cancer cells and mouse primary hepatocytes. HNF-4α not only directly bound to an E-box-containing region in intron 12 of the ChREBP gene, but also promoted ChREBP-β transcription by directly binding to two DR1 sites and one E-box-containing site of the ChREBP-β promoter. Moreover, HNF-4α interacted with ChREBP-α and synergistically promoted ChREBP-β transcription. Functionally, HNF-4α suppression reduced glucose-dependent ChREBP induction. Increased nuclear abundance of HNF-4α and its binding to cis-elements of ChREBP gene in response to glucose contributed to glucose-responsive ChREBP transcription. Taken together, our results not only revealed the novel mechanism by which HNF-4α promoted ChREBP transcription in response to glucose, but also demonstrated that ChREBP-α and HNF-4α synergistically increased ChREBP-β transcription.

SUBMITTER: Meng J

PROVIDER: S-EPMC4814918 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The hepatocyte nuclear factor 4α (HNF-4α; also known as NR2A1) is a member of the nuclear receptor (NR) family of transcription factors, which have conserved DNA-binding domains and ligand-binding domains. HNF-4α is the most abundant DNA-binding protein in the liver, where some 40% of the actively transcribed genes have a HNF-4α response element. These regulated genes are largely involved in the hepatic gluconeogenic program and lipid metabolism. In the pancreas HNF-4α is also a master regulator, controlling an estimated 11% of islet genes. HNF-4α protein mutations are linked to maturity-onset diabetes of the young, type 1 (MODY1) and hyperinsulinaemic hypoglycaemia. Previous structural analyses of NRs, although productive in elucidating the structure of individual domains, have lagged behind in revealing the connectivity patterns of NR domains. Here we describe the 2.9 Å crystal structure of the multidomain human HNF-4α homodimer bound to its DNA response element and coactivator-derived peptides. A convergence zone connects multiple receptor domains in an asymmetric fashion, joining distinct elements from each monomer. An arginine target of PRMT1 methylation protrudes directly into this convergence zone and sustains its integrity. A serine target of protein kinase C is also responsible for maintaining domain-domain interactions. These post-translational modifications lead to changes in DNA binding by communicating through the tightly connected surfaces of the quaternary fold. We find that some MODY1 mutations, positioned on the ligand-binding domain and hinge regions of the receptor, compromise DNA binding at a distance by communicating through the interjunctional surfaces of the complex. The overall domain representation of the HNF-4α homodimer is different from that of the PPAR-γ-RXR-α heterodimer, even when both NR complexes are assembled on the same DNA element. Our findings suggest that unique quaternary folds and interdomain connections in NRs could be exploited by small-molecule allosteric modulators that affect distal functions in these polypeptides.

Project description:AimThe study aimed to develop a scalable dual-fluorescence assay in cells to enable the functional interpretation of HNF-4α missense variants identified in exome sequencing, which can be used to guide clinical diagnosis.MethodsUsing mOrange2 and GFP fluorescence proteins to track the expression of HNF-4α (HNF-4α-mOrange2) and reporter activity under the control of the HNF-1α promoter (pHNF1A-GFP), respectively, we designed a dual-fluorescence assay to evaluate the expression level, cellular localization, and transcriptional function of HNF-4α simultaneously in live cells. To assess the scalable characteristic of the assay, a small library containing five previously reported mutations and wild-type HNF-4α was constructed. Cells infected with this library were sorted into different populations through fluorescence-activated cell sorting (FACS) according to the transcription activity and expression abundance. Cloning and Sanger sequencing were used to detect the mutations of the different groups. High content screening (HCS) assay was used for the validation of individual mutants in the function and expression point of view.ResultsHNF-4α-mOrange2 exhibited nuclear localization and transactivation capability on the HNF-1α promoter as physical HNF-4α does. The expression of HNF-4α-mOrange2 shows a 6-fold induction of GFP expression compared to the control without HNF-4α-mOrange2, which was significantly abolished by the known loss-of-function mutant M373R. The different performances of wild-type and mutant M373R made them distinguishable in the FACS system, empowering the scalable capability of this assay for classifying large numbers of variants combining functional stratification and sequencing. Further application of the assay in the small library showed that three cell populations were seen grouped as Normal (same transactivation as wild type), Reducedexp_nor (reduced transactivation with normal or higher expression), and Reducedexp_low (reduced transactivation with lower expression). Subsequently, Sanger sequencing showed that wild-type HNF-4α was in the Normal group, two mutations (M373R and G79C) were enriched in the Reducedexp_nor group, and three mutations (C115S, L272P, and F83C) belonged to the Reducedexp_low group. These results were validated by further imaging data using HCS assay for individual mutation.ConclusionsOur study proposes a scalable and informative approach for the characterization of the variants in HNF-4α genes in a quantitative and high-throughput manner.

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Identification of HNF-4α as a key transcription factor to promote ChREBP expression in response to glucose.

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