Intestine-specific ablation of the Hepatocyte Nuclear Factor 4a (Hnf4a) gene in mice has minimal impact on serum lipids and ileum gene expression profile due to upregulation of its paralog Hnf4g

Ablation of the gene encoding the nuclear receptor Hepatocyte Nuclear Factor 4a (Hnf4a) in the liver strongly affects HDL concentration, structure and functionality but the role of this receptor in the intestine, the second organ contributing to serum HDL levels, has been overlooked. In the present study we show that mice with intestine-specific ablation of Hnf4a (H4IntKO) had undetectable levels of ΗΝF4A in ileum, proximal and distal colon but normal expression in liver. H4IntKO mice presented normal serum lipid levels, HDL-C and particle size (α1-α3). The expression of the major HDL biogenesis genes Apoa1, Abca1, Lcat was not affected but there was significant increase in Apoc3 as well as in Hnf4g, a paralog of Hnf4a. RNA-sequencing identified metabolic pathways significantly affected by Hnf4a ablation such as type II diabetes, glycolysis, gluconeogenesis and p53 signaling. Chromatin immunoprecipitation assays showed that HNF4G bound to various apolipoprotein gene promoters in control mice but its binding affinity was reduced in the ileum of H4IntKO mice suggesting a redundancy but also a cooperation between the two factors. In the distal colon of H4IntKO mice, where both HNF4A and HNF4G are absent and in a mouse model of DSS-induced colitis presenting decreased levels of HNF4A, most lipoprotein genes were strongly downregulated. In conclusion, Hnf4a ablation in mice does not significantly affect serum lipid levels or lipoprotein gene expression in ileum possibly due to compensatory effects by its paralog Hnf4g in this tissue.     

In vivo role of the HNF4alpha AF-1 activation domain revealed by exon swapping

The gene encoding the nuclear receptor hepatocyte nuclear factor 4alpha (HNF4alpha) generates isoforms HNF4alpha1 and HNF4alpha7 from usage of alternative promoters. In particular, HNF4alpha7 is expressed in the pancreas whereas HNF4alpha1 is found in liver, and mutations affecting HNF4alpha function cause impaired insulin secretion and/or hepatic defects in humans and in tissue-specific 'knockout' mice. HNF4alpha1 and alpha7 isoforms differ exclusively by amino acids encoded by the first exon which, in HNF4alpha1 but not in HNF4alpha7, includes the activating function (AF)-1 transactivation domain. To investigate the roles of HNF4alpha1 and HNF4alpha7 in vivo, we generated mice expressing only one isoform under control of both promoters, via reciprocal swapping of the isoform-specific first exons. Unlike Hnf4alpha gene disruption which causes embryonic lethality, these 'alpha7-only' and 'alpha1-only' mice are viable, indicating functional redundancy of the isoforms. However, the former show dyslipidemia and preliminary results indicate impaired glucose tolerance for the latter, revealing functional specificities of the isoforms. These 'knock-in' mice provide the first test in vivo of the HNF4alpha AF-1 function and have permitted identification of AF-1-dependent target genes.     

Developmentally regulated N-terminal variants of the nuclear receptor hepatocyte nuclear factor 4alpha mediate multiple interactions through coactivator and corepressor-histone deacetylase complexes

To understand the mechanisms governing the regulation of nuclear receptor (NR) function, we compared the parameters of activation and repression of two isoforms of the orphan receptor hepatocyte nuclear factor (HNF) 4alpha. HNF4alpha7 and HNF4alpha1 differ only in their N-terminal domains, and their expression in the liver is regulated developmentally. We show that the N-terminal activation function (AF)-1 of HNF4alpha1 possesses significant activity that can be enhanced through interaction with glucocorticoid receptor-interacting protein 1 (GRIP-1) and cAMP response element-binding protein-binding protein (CBP). In striking contrast, HNF4alpha7 possesses no measurable AF-1, implying major functional differences between the isoforms. Indeed, although HNF4alpha1 and HNF4alpha7 are able to interact via AF-2 with GRIP-1, p300, and silencing mediator for retinoid and thyroid receptors (SMRT), only HNF4alpha1 interacts in a synergistic fashion with GRIP-1 and p300. Although both isoforms interact physically and functionally with SMRT, the repression of HNF4alpha7 is less robust than that of HNF4alpha1, which may be caused by an increased ability of the latter to recruit histone deacetylase (HDAC) activity to target promoters. Moreover, association of SMRT with HDACs enhanced recruitment of HNF4alpha1 but not of HNF4alpha7. These observations suggest that NR isoform-specific association with SMRT could affect activity of the SMRT complex, implying that selection of HDAC partners is a novel point of regulation for NR activity. Possible physiological consequences of the multiple interactions with these coregulators are discussed.