HNF-1α and endodermal transcription factors cooperatively activateFabpl: MODY3 mutations abrogate cooperativity

Abstract

Hepatocyte nuclear factor (HNF)-1alpha plays a central role in intestinal and hepatic gene regulation and is required for hepatic expression of the liver fatty acid binding protein gene (Fabpl). An Fabpl transgene was directly activated through cognate sites by HNF-1alpha and HNF-1beta, as well as five other endodermal factors: CDX-1, C/EBPbeta, GATA-4, FoxA2, and HNF-4alpha. HNF-1alpha activated the Fabpl transgene by as much as 60-fold greater in the presence of the other five endodermal factors than in their absence, accounting for up to one-half the total transgene activation by the group of six factors. This degree of synergistic interaction suggests that multifactor cooperativity is a critical determinant of endodermal gene activation by HNF-1alpha. Mutations in HNF-1alpha that result in maturity onset diabetes of the young (MODY3) provide evidence for the in vivo significance of these synergistic interactions. An R131Q HNF-1alpha MODY3 mutant exhibits complete loss of synergistic activation in concert with the other endodermal transcription factors despite wild-type transactivation ability in their absence. Furthermore, whereas wild-type HNF-1alpha exhibited pairwise cooperative synergy with each of the other five factors, the R131Q mutant could synergize only with GATA-4 and C/EBPbeta. Selective loss of synergy with other endodermal transcription factors accompanied by retention of native transactivation ability in an HNF-1alpha MODY mutant suggests in vivo significance for cooperative synergy.

Cooperative interactions among intestinal GATA factors in activating the rat liver fatty acid binding protein gene

“…However, a lactase transgene was more sensitive to GATA activation than the sucrase transgene, leading to the proposal that GATA factors play a unique role in intestinal gene expression between birth and weaning (30). Fabp1 is expressed at a high and constant level from birth to weaning, with expression rising somewhat in the adult tissue (22), perhaps due to the large number of transactivating factors that bind to the proximal promoter (11), and reflecting the requirement for lipid metabolism in enterocytes in both suckling and adult animals.…”

“…The construction of the Fabp1-transgene and GATAexpression plasmids has been described (11), as has construction of the plasmid containing a GATA site upstream of a minimal promoter (47). Expression plasmids for GATA:Renilla luciferase (luc) fusion proteins were constructed as follows.…”

“…Transient transfections were performed by the calcium phosphate precipitation method essentially as described in Ref. 11. Briefly, the transfection mixtures contained an Fabp1 reporter plasmid, GATA factor expression plasmid(s), and pGL3 control plasmid (Promega) that was included to normalize for differences in transfection efficiency.…”

“…Enterocytic Fabp1 expression remains high throughout the preweaning period and adulthood and is primarily regulated at the transcriptional level (2,22). We have utilized a transgene constructed from rat Fabp1 nucleotides Ϫ596 to ϩ21 to study enterocytic gene regulation (11). A transgene constructed of Fabp1 nucleotides Ϫ596 to ϩ21 relative to the start site of transcription is active in all small intestinal epithelial cells, including enterocytes (41).…”

“…A transgene constructed of Fabp1 nucleotides Ϫ596 to ϩ21 relative to the start site of transcription is active in all small intestinal epithelial cells, including enterocytes (41). There are three consensus GATA sites in the proximal Fabp1 promoter, and GATA factors activate the Fabp1 transgene in cultured cells through interaction with these sites (11). GATA-4 and GATA-5 activate the Fabp1 transgene in cultured cells through preferential interaction of the most proximal of the three GATA binding sites present in the promoter (12).…”