Iron is an essential element in biological systems, but excess iron promotes the formation of reactive oxygen species, resulting in cellular toxicity. Several iron-related genes are highly expressed in the liver, a tissue in which hepatocyte nuclear factor 4α (HNF4α) plays a critical role in controlling gene expression. Therefore, the role of hepatic HNF4α in iron homeostasis was examined using liver-specific HNF4α-null mice (Hnf4a(ΔH) mice). Hnf4a(ΔH) mice exhibit hypoferremia and a significant change in hepatic gene expression. Notably, the expression of transferrin receptor 2 (Tfr2) mRNA was markedly decreased in Hnf4a(ΔH) mice. Promoter analysis of the Tfr2 gene showed that the basal promoter was located at a GC-rich region upstream of the transcription start site, a region that can be transactivated in an HNF4α-independent manner. HNF4α-dependent expression of Tfr2 was mediated by a proximal promoter containing two HNF4α-binding sites located between the transcription start site and the translation start site. Both the GC-rich region of the basal promoter and the HNF4α-binding sites were required for maximal transactivation. Moreover, siRNA knockdown of HNF4α suppressed TFR2 expression in human HCC cells. These results suggest that Tfr2 is a novel target gene for HNF4α, and hepatic HNF4α plays a critical role in iron homeostasis.

Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) and its partners hypoxia-inducible factors (HIF)-1α and HIF-2α are candidate factors for the well-known link between the liver, metabolic dysfunction and elevation in circulating lipids and glucose. Methods: Hepatocyte-specific ARNT-null (LARNT), HIF-1α-null (LHIF1α) and HIF-2α-null (LHIF2α) mice were created.

The glucocorticoid receptor (GR) is a nuclear receptor critical to the regulation of energy metabolism and inflammation. The actions of GR are dependent on cell type and context. Here, we demonstrate the role of liver lineage-determining factor hepatocyte nuclear factor 4A (HNF4A) in defining liver specificity of GR action. In mouse liver, the HNF4A motif lies adjacent to the glucocorticoid response element (GRE) at GR binding sites within regions of open chromatin. In the absence of HNF4A, the liver GR cistrome is remodeled, with loss and gain of GR recruitment evident. Loss of chromatin accessibility at HNF4A-marked sites associates with loss of GR binding at weak GRE motifs. GR binding and chromatin accessibility are gained at sites characterized by strong GRE motifs, which show GR recruitment in non-liver tissues. The functional importance of these HNF4A-regulated GR sites is indicated by an altered transcriptional response to glucocorticoid treatment in the Hnf4a-null liver.

Hepatocyte nuclear factor 4A (HNF4A/NR2a1), a transcriptional regulator of hepatocyte identity, controls genes that are crucial for liver functions, primarily through binding to enhancers. In mammalian cells, active and primed enhancers are marked by monomethylation of histone 3 (H3) at lysine 4 (K4) (H3K4me1) in a cell type-specific manner. How this modification is established and maintained at enhancers in connection with transcription factors (TFs) remains unknown. Using analysis of genome-wide histone modifications, TF binding, chromatin accessibility and gene expression, we show that HNF4A is essential for an active chromatin state. Using HNF4A loss and gain of function experiments in vivo and in cell lines in vitro, we show that HNF4A affects H3K4me1, H3K27ac and chromatin accessibility, highlighting its contribution to the establishment and maintenance of a transcriptionally permissive epigenetic state. Mechanistically, HNF4A interacts with the mixed-lineage leukaemia 4 (MLL4) complex facilitating recruitment to HNF4A-bound regions. Our findings indicate that HNF4A enriches H3K4me1, H3K27ac and establishes chromatin opening at transcriptional regulatory regions.

The CIDE (cell death-inducing DFF45-like effector) family composed of CIDEA, CIDEB, CIDEC/FSP27 (fat-specific protein 27), has a critical role in growth of lipid droplets. Of these, CIDEB and CIDEC2/FSP27B are abundant in the liver, and the steatotic livers, respectively. Hepatocyte nuclear factor 4α (HNF4α) has an important role in lipid homeostasis because liver-specific HNF4α-null mice (Hnf4aΔHep mice) exhibit hepatosteatosis. We investigated whether HNF4α directly regulates expression of CIDE family genes. Expression of Cideb and Fsp27b was largely decreased in Hnf4aΔHep mice, while expression of Cidea was increased. Similar results were observed only in CIDEC2, the human orthologue of the Fsp27b, in human hepatoma cell lines in which HNF4α expression was knocked down. Conversely, overexpression of HNF4α strongly induced CIDEC2 expression in hepatoma cell lines. Furthermore, HNF4α transactivated Fsp27b by direct binding to an HNF4α response element in the Fsp27b promoter. In addition, Fsp27b is known to be transactivated by CREBH that is regulated by HNF4α, and expression of CREBH was induced by HNF4α in human hepatoma cells. Co-transfection of HNF4α and CREBH resulted in synergistic transactivation and induction of Fsp27b compared to that of HNF4α or CREBH alone. These results suggest that HNF4α, in conjunction with CREBH, plays an important role in regulation of Fsp27b expression.

Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor required for normal insulin secretion and maintenance of β-cell number in the pancreas. HNF1α is also expressed in pancreatic α-cells, but its role in these cells is unknown. The aim of this study was to clarify the role of HNF1α in α-cells. Male Hnf1a+/- mice with a mixed background were backcrossed to outbred ICR mice. Glucose tolerance, glucagon and insulin secretion, islet histology, and gene expression were investigated in ICR Hnf1a-/- and Hnf1a+/+ mice. Regulation of Slc5a1 (encoding sodium glucose cotransporter 1 [SGLT1]) expression by HNF1α and the effect of SGLT1 inhibition on glucagon secretion were also explored. ICR Hnf1a-/- mice were glucose intolerant and exhibited impaired glucose-stimulated insulin secretion. The β-cell area of ICR mice was decreased in Hnf1a-/- mice, but the α-cell area in the pancreas was similar between Hnf1a-/- and Hnf1a+/+ mice. Hnf1a-/- mice showed higher fasting glucagon levels and exhibited inadequate suppression of glucagon after glucose load. In addition, glucagon release in response to hypoglycemia was impaired in Hnf1a-/- mice, and glucagon secretion after 1.1 mM glucose administration, was also decreased in Hnf1a-/- islets. Slc5a1 expression was decreased in Hnf1a-/- islets, while HNF1α activated the Slc5a1 promoter in αTC1-6 cells. Inhibition of SGLT1 suppressed 1.1 mM glucose-stimulated glucagon secretion in islets and αTC1-6 cells, but SGLT1 inhibition had no additional inhibitory effect in HNF1α-deficient cells. Our findings indicate that HNF1α modulates glucagon secretion in α-cells through the regulation of Slc5a1.