The HNF1 C-terminal domain contributes to transcriptional activity and modulates nuclear localisation.
HNF1 is a homeoprotein that regulates the expression of a large number of liver specific genes. By performing transient expression assays with a series of C-terminal deletion mutants and with a LexA-HNF1 fusion protein, we show that the C-terminal half of HNF1 is necessary and sufficient for in vivo transcriptional activity, and we map the residues essential for this activity. However, since our data for some mutants showed discrepancies with previous in vitro studies, we undertook a more careful analysis of the mutant proteins using gel retardation assays and immunoblots made with nuclear extracts from transfected cells. We show that progressive C-terminal deletions drastically increase the amount of protein that accumulates in transfected cells. Immunofluorescence microscopy reveals that mutants containing between 348 and 416 residues accumulate outside the nuclear membrane, while longer mutants are nuclear like the 628 amino acid long wild type HNF1. A mutant with 289 residues is predominantly nuclear. Since the only obvious candidate for a nuclear localisation signal is located within this last mutant, we suggest that certain C-terminal deletions expose a sequence that blocks nuclear transport.
Sourdive
D J
DJ
CNRS UA 1644, Département des Biotechnologies, Institut Pasteur, Paris, France.
Chouard
T
T
Yaniv
M
M
D015153:Blotting, Western; D002467:Cell Nucleus / Q000737:chemistry; D004268:DNA-Binding Proteins; D017353:Gene Deletion; D015870:Gene Expression; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D008099:Liver / Q000648:ultrastructure; D009687:Nuclear Proteins; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D009687:Nuclear Proteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta