Hepatocyte nuclear factor 4α (HNF4α) is a master regulator of development and function of digestive tissues. The HNF4A gene uses two separate promoters P1 and P2, with P1 products predominant in adult liver, whereas P2 products prevalent in fetal liver, pancreas, and liver/colon cancer. To date, the mechanisms for the regulation of HNF4A and the dynamic switch of P1-HNF4α and P2-HNF4α during ontogenesis and carcinogenesis are still obscure. Our study validated the previously reported self-stimulation of P1-HNF4α but invalidated the reported synergism between HNF4α and HNF1α. HNF4A-AS1, a long noncoding RNA, is localized between the P2 and P1 promoters of HNF4A. We identified critical roles of P1-HNF4α in regulating the expression of HNF4A-AS1 and its mouse ortholog Hnf4a-os. Paired box 6 (PAX6), a master regulator of pancreas development overexpressed in colon cancer, cooperated with HNF1α to induce P2-HNF4α but antagonized HNF4α in HNF4A-AS1 expression. Thus, PAX6 may be important in determining ontogenic and carcinogenic changes of P2-HNF4α and HNF4A-AS1 in the pancreas and intestine. We also interrogated transactivation activities on multiple gene targets by multiple known and novel HNF4α mutants identified in patients with maturity onset diabetes of the young 1 (MODY1) and liver cancer. Particularly, HNF4α-D78A and HNF4α-G79S, two mutants found in liver cancer with mutations in DNA-binding domain, displayed highly gene-specific transactivation activities. Interestingly, HNF4α-Q277X, a MODY1 truncation mutant, antagonized the transactivation activities of HNF1α and farnesoid X receptor, key regulators of insulin secretion. Taken together, our study provides novel mechanistic insights regarding the transcriptional regulation and transactivation activity of HNF4α in digestive tissues.

Hepatocyte nuclear factor 4-alpha (HNF4α) is a well established master regulator of liver development and function. We identified the in vitro presence of a stable secondary structure, G-quadruplex (G4) in the 5′ UTR of P1-HNF4A, the predominant HNF4α isoform(s) in adult liver. Our data suggest that the cooperation of G4 and the adjacent putative protein-binding sites within the 5′ UTR was necessary and sufficient to mediate a strong translational repression. This was supported by analysis of deleted/mutated 5′UTRs and two native regulatory single-nucleotide polymorphisms in the 5′UTR. Additional results indicated that G4 motifs in the 5′ UTRs of other liver-enriched transcription factors also inhibited protein expression. Moreover, pyridostatin, a G4 ligand, specifically potentiated the translational suppressing effect of P1-HNF4A-5′ UTR. In summary, the present study provides the first evidence of the presence of G4 in human P1-HNF4A-5′ UTR in vitro, and establishes a novel working model of strong inhibition of protein translation via interactions of G4 with potential RNA-binding proteins (RBPs). The protein expression of the tumor suppressor HNF4α may be inhibited by interactions of RBPs with the G4 motif in the 5′ UTR to promote cell proliferation during liver development and carcinogenesis.

Specific basic and aromatic amino acids in the C-terminal tail of Mgm101 play key roles in mediating ssDNA binding, stabilizing the oligomeric rings, and maintaining protein stability in vivo. The C-tail may couple ssDNA binding with ring disassembly, which facilitates the formation of nucleoprotein complexes competent for the repair of mtDNA.

Hepatocyte nuclear factor 4α (HNF4α) and glucocorticoid receptor (GR), master regulators of liver metabolism, are down-regulated in fatty liver diseases. The present study was aimed to elucidate the role of down-regulation of HNF4α and GR in fatty liver and hyperlipidemia. Adult mice with liver-specific heterozygote and knockout (knockout) of HNF4α were fed a low-fat diet (LFD) or a high-fat-high-sugar diet (HFHS) for 15 days. Compared to LFD-fed mice, HFHS-fed wildtype mice had hepatic induction of lipid catabolic genes and down-regulation of lipogenic genes. Compared to HFHS-fed wildtype mice, HNF4α heterozygote mice had down-regulation of lipid catabolic genes, induction of lipogenic genes, and increased hepatic and blood levels of lipids, whereas HNF4α knockout mice had mild hypolipidemia, down-regulation of lipid-efflux genes, but induction of genes for uptake/storage of lipids. Sterol-regulatory-element-binding protein-1c (SREBP-1C), a master lipogenic regulator, was induced in HFHS-fed HNF4α heterozygote mice. In reporter assays, HNF4α potently inhibited the transactivation of mouse and human SREBP-1C promoter by liver X receptor. Surprisingly, nuclear GR proteins were gene-dosage-dependently decreased in HNF4α heterozygote and knockout mice. HFHS-fed mice with liver-specific knockout of GR had increased hepatic lipids and induction of SREBP-1C and PPARγ. In reporter assays, GR and HNF4α synergistically/additively induced lipid catabolic genes. Phosphorylation of AMP-activated protein kinase (AMPK), a key GR modulator, was dramatically decreased in HNF4α knockout mice. Thus, cooperative induction of lipid catabolic genes and suppression of lipogenic genes by HNF4α and GR, modulated by AMPK, may mediate the early resistance to HFHS-induced fatty liver and hyperlipidemia.

HNF4α is a liver‐enriched master regulator down‐regulated in liver cancer and cirrhosis. Restoration of HNF4α expression can treat both liver cancer and liver fibrosis. However, lack of known activating ligand makes HNF4α “undruggable”. The purpose of this study was to elucidate the mechanism of translational regulation of HNF4α1, the adult‐predominant HNF4α isoform, so as to develop a novel approach to restore HNF4α1 expression for cancer therapy. The 5' UTR of both human and mouse HNF4α1 have potential G‐quadruplex (G4), a stable secondary structure known to inhibit transcription and translation. FRET assay of dual‐labeled oligo from human HNF4α1 5' UTR indicated the formation of G4. Luciferase reporter assay showed the wild‐type, but not G4‐mutated, 5' UTR of HNF4α1 strongly inhibited protein translation without affecting mRNA expression, which is further confirmed by Western blot analysis of protein expression of HNF4α1 in the presence/absence of its 5' UTR. In HEK 293 cells, protein expression of mouse Hnf4α1 was completely inhibited by its 5' UTR. Luciferase assays of the deletion/mutation reporter constructs of Hnf4α1 5' UTR suggest G4 motif and its adjacent stem loop may play the key role in suppressing Hnf4α1 protein expression. In summary, the 5' UTR of human and mouse HNF4α1 markedly inhibit HNF4α1 expression at translational levels, most likely due to interaction of G4 motif with stem loop within 5' UTR. Grant Funding Source: NIH Grant CA143656