MODY1 mutation Q268X in hepatocyte nuclear factor 4alpha allows for dimerization in solution but causes abnormal subcellular localization.

Abstract

Recent studies have shown that mutations in the hepatocyte nuclear factor (HNF)-4alpha gene give rise to maturity-onset diabetes of the young, type 1 (MODY1). HNF-4, an orphan member of the nuclear receptor superfamily, contains a DNA-binding domain (DBD) and a putative ligand-binding domain (LBD) that can act independently of each other. The first MODY1 mutation identified creates a stop codon at amino acid 268 in the LBD of HNF-4 (Q268X) that leaves the DBD intact, suggesting that the mutant protein may retain some of the properties of the wild-type protein. To determine the functional properties of this mutant, we constructed HNF4.Q268X and tested it in vitro and in vivo for DNA binding, protein dimerization, and transactivation activity. Results of an electrophoretic mobility shift assay showed that HNF4.Q268X neither binds DNA alone nor binds it as a dimer with wild-type HNF-4 (HNF4.wt). In contrast, a co-immunoprecipitation assay showed that HNF4.Q268X is capable of dimerizing in solution with HNF4.wt. Transient transfection assays, however, indicated that HNF4.Q268X does not affect transactivation by HNF4.wt in vivo, supporting the argument against a dominant negative effect. Additional results suggest that the lack of a dominant negative effect could be due to a striking differential subcellular localization of the HNF4.Q268X protein: HNF4.Q268X could be extracted from transfected cells only when treated with SDS. Taken together, our results suggest that the MODY1 phenotype is due to a loss of functional HNF-4 protein that is aggravated in tissues that express relatively low amounts of HNF-4, such as pancreas.

Analysis of the HNF4α gene in Caucasian Type II diabetic nephropathic patients

“…The loss of functional HNF4a protein in the termination mutations is thought to be exacerbated in tissues where there is low HNF4a expression (e. g. pancreas). Recent characterisation of the E276Q mutation suggests that haploinsufficiency as well as a weak dominant negative effect contribute to the Type II diabetic phenotype [46]. Our finding of a 50 % reduction in activation and binding in the D7 promoter mutation is consistent with this hypothesis.…”

“…Functional characterisation of the two previously identified premature termination mutations (Q268X, R154X) in MODY1 families, indicate that the diabetic phenotype is due to the loss of functional HNF4a protein, as opposed to a gain of function from the mutant protein [6,46]. The loss of functional HNF4a protein in the termination mutations is thought to be exacerbated in tissues where there is low HNF4a expression (e. g. pancreas).…”

Mutations in the human genes encoding the transcription factors of the hepatocyte nuclear factor (HNF)1 and HNF4 families: functional and pathological consequences

“…Most similar data were obtained for the nonsense mutant, Q268X, by showing that this mutant lacks DNA binding, has some altered localization in the cell compartment and has lost its transactivation potential. More importantly, a dominant negative action on the wild-type factor has been excluded by experiments in several laboratories (Stoffel & Duncan 1997, Sladek et al 1998, Lausen et al 2000. Thus, the MODY mutants R154X and Q268X encode transcription factors whose activities are more or less destroyed by the mutation and it is most likely that the frameshift mutants F75fsdelT and K99fsdelAA that have not yet been functionally characterized are similar loss-of-function mutations.…”

Disease allele-dependent small-molecule sensitivities in blood cells from monogenic diabetes

“…2) in which affected members possess a Q268X nonsense mutation (16). This mutation truncates the ligand-binding domain and leads to impaired DNA binding, abnormal cellular trafficking, and loss of transcriptional activity (8,17). Sequencing and RT-PCR confirmed the presence of a heterozygous C-to-T substitution in codon 268 exclusively in the diabetic subjects, and low levels of HNF4α expression.…”