Structural basis of natural promoter recognition by a unique nuclear receptor, HNF4alpha. Diabetes gene product

HNF4alpha (hepatocyte nuclear factor 4alpha) plays an essential role in the development and function of vertebrate organs, including hepatocytes and pancreatic beta-cells by regulating expression of multiple genes involved in organ development, nutrient transport, and diverse metabolic pathways. As such, HNF4alpha is a culprit gene product for a monogenic and dominantly inherited form of diabetes, known as maturity onset diabetes of the young (MODY). As a unique member of the nuclear receptor superfamily, HNF4alpha recognizes target genes containing two hexanucleotide direct repeat DNA-response elements separated by one base pair (DR1) by exclusively forming a cooperative homodimer. We describe here the 2.0 angstroms crystal structure of human HNF4alpha DNA binding domain in complex with a high affinity promoter element of another MODY gene, HNF1alpha, which reveals the molecular basis of unique target gene selection/recognition, DNA binding cooperativity, and dysfunction caused by diabetes-causing mutations. The predicted effects of MODY mutations have been tested by a set of biochemical and functional studies, which show that, in contrast to other MODY gene products, the subtle disruption of HNF4alpha molecular function can cause significant effects in afflicted MODY patients.     

Naturally occurring mutations in the human HNF4alpha gene impair the function of the transcription factor to a varying degree

The hepatocyte nuclear factor (HNF)4alpha, a member of the nuclear receptor superfamily, regulates genes that play a critical role in embryogenesis and metabolism. Recent studies have shown that mutations in the human HNF4alpha gene cause a rare form of type 2 diabetes, maturity onset diabetes of the young (MODY1). To investigate the properties of these naturally occurring HNF4alpha mutations we analysed five MODY1 mutations (R154X, R127W, V255M, Q268X and E276Q) and one other mutation (D69A), which we found in HepG2 hepatoma cells. Activation of reporter genes in transfection assays and DNA binding studies showed that the MODY1-associated mutations result in a variable reduction in function, whereas the D69A mutation showed an increased activity on some promoters. None of the MODY mutants acted in a dominant negative manner, thus excluding inactivation of the wild-type factor as a critical event in MODY development. A MODY3-associated mutation in the HNF1alpha gene, a well-known target gene of HNF4alpha, results in a dramatic loss of the HNF4 binding site in the promoter, indicating that mutations in the HNF4alpha gene might cause MODY through impaired HNF1alpha gene function. Based on these data we propose a two-hit model for MODY development.     

Anatomy of a homeoprotein revealed by the analysis of human MODY3 mutations

Hepatocyte nuclear factor 1alpha (HNF1alpha) is an atypical dimeric homeodomain-containing protein that is expressed in liver, intestine, stomach, kidney, and pancreas. Mutations in the HNF1alpha gene are associated with an autosomal dominant form of non-insulin-dependent diabetes mellitus called maturity-onset diabetes of the young (MODY3). More than 80 different mutations have been identified so far, many of which involve highly conserved amino acid residues among vertebrate HNF1alpha. In the present work, we investigated the molecular mechanisms by which MODY3 mutations could affect HNF1alpha function. For this purpose, we analyzed the properties of 10 mutants resulting in amino acid substitutions or protein truncation. Some mutants have a reduced protein stability, whereas others are either defective in the DNA binding or impaired in their intrinsic trans-activation potential. Three mutants, characterized by a complete loss of trans-activation, behave as dominant negatives when transfected with the wild-type protein. These data define a clear causative relationship between MODY3 mutations and functional defects in HNF1alpha trans-activation. In addition, our analysis sheds new light on the structure of a homeoprotein playing a key role in pancreatic beta cell function.