Anatomy of a Homeoprotein Revealed by the Analysis of Human MODY3 Mutations

Martine Vaxillaire,Amar Abderrahmani,Philippe Boutin,Bernard Bailleul,Philippe Froguel,Moshe Yaniv,Marco Pontoglio

doi:10.1074/jbc.274.50.35639

Abstract

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.

Highlights

Hepatocyte nuclear factor 1␣ (HNF1␣ or LF-B1)1 was initially characterized as a transcriptional regulator of a large set of hepatic genes, including albumin, ␤ fibrinogen, and ␣1-antitrypsin [1,2,3]
Mutations in the HNF1␣ gene are associated with an autosomal dominant form of non-insulin-dependent diabetes mellitus called maturity-onset diabetes of the young (MODY3)
We investigated the molecular mechanisms by which MODY3 mutations could affect HNF1␣ function

Summary

The abbreviations used are

HNF1␣, hepatocyte nuclear factor 1␣; MODY, maturity-onset diabetes of the young; RSV, Rous sarcoma virus; CAT, chloramphenicol acetyltransferase; PBS, phosphate-buffered saline. Ͼ80 different mutations (missense, nonsense, or frameshift) have been identified in the HNF1␣ sequence in association with the MODY3 phenotype (28 –33) These mutations are localized along the different functional domains of the protein and involve highly conserved amino acid residues, suggesting that MODY3 is accounted for by impaired HNF1␣ function. The exact nature of the molecular defects and the mechanisms that explain the dominant transmission of the mutations are unknown These mutations could generate proteins that might interfere with the function of the protein encoded by the wild-type allele from the other homologous chromosome. To investigate this issue, we analyzed the transcriptional activity of a number of representative MODY3 mutations. Several but not all of the mutants behaved as dominant negative effectors on the wild-type protein

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION