DNA binding and transcription activation specificity of hepatocyte nuclear factor 4.
Hepatocyte nuclear factor 4 (HNF-4) is an orphan intracellular receptor that appears to be a key factor in the regulation of energy metabolism. In order to gain greater understanding of the binding and activation requirements of HNF-4, we performed genetic analysis of the apoCIII promoter, a promoter that has previously been shown to be highly sensitive to HNF-4-induced transcription. We identified two elements within the apoCIII promoter that bind HNF-4, either of which are sufficient to confer promoter induction in response to HNF-4. These two elements are both direct repeat-like in nature, but they differ significantly in motif sequence and the repeats are separated by either 1 or 2 nt. Therefore, to better define the DNA sequence recognition requirements of HNF-4, we utilized PCR-based binding site selection. HNF-4 selected for direct repeat-like elements with either 1 or 2 nt between the repeats. Surprisingly, the strongest selection was for a core motif that included the nucleotides between the repeats. Mutation of the nucleotide between the repeats resulted in a 6-fold reduction in affinity, indicating that the interaction between HNF-4 and the intervening nucleotide(s) is critical for high affinity binding.
Fraser
J D
JD
Ligand Pharmaceuticals Inc., 10255 Science Center Drive, San Diego, CA 92121, USA. jfraser@ligand.com
Martinez
V
V
Straney
R
R
Briggs
M R
MR
D053305:Apolipoprotein C-III; D001056:Apolipoproteins C / Q000235:genetics*; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D053305:Apolipoprotein C-III; D001056:Apolipoproteins C; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D004247:DNA