A POU-A related region dictates DNA binding specificity of LFB1/HNF1 by orienting the two XL-homeodomains in the dimer.
LFB1/HNF1 regulates the hepatocyte-specific transcription of several genes, binding as a dimer to cis-acting elements that match the inverted palindrome GTTAATNATTAAC. The DNA binding domain of LFB1/HNF1 is characterized by a unique tripartite structure that includes an unusually long homeodomain (domain C), a region related to the POU-specific A-box (domain B) and a short N-terminal dimerization domain (domain A). We report that a recombinant peptide corresponding to the isolated homeodomain of LFB1/HNF1 binds as a monomer to a half-palindrome binding site, but shows diminished sequence specificity. Domain B, in addition to the homeodomain, is required and sufficient for proper recognition of LFB1/HNF1-responsive sites. A protein consisting of only these latter two domains is a monomer in solution, but forms dimers upon DNA binding. The protein-protein contacts established within the bound dimer restrain the orientation of the two homeodomains with respect to one another, thus contributing in a critical fashion to the recognition of the dyad symmetry-related LFB1/HNF1 sites. The DNA-independent dimerization domain (domain A) is required to increase the affinity of DNA binding, but does not influence the dimer geometry.
Tomei
L
L
Istituto di Ricerche di Biologia Molecolare P. Angeletti, Roma, Italy.
Cortese
R
R
De Francesco
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005801:Genes, Homeobox; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D007700:Kinetics; D008099:Liver / Q000502:physiology; D046911:Macromolecular Substances; D008433:Mathematics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D051538:Hepatocyte Nuclear Factor 1-alpha; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D004247:DNA