Defining a minimal estrogen receptor DNA binding domain.
The estrogen receptor (ER) is a transcriptional regulator which binds to cognate palindromic DNA sequences known as estrogen response elements (EREs). A 66 amino acid core region which contains two zinc fingers and is highly conserved among the nuclear receptors is essential for site specific DNA recognition. However, it remains unclear how many flanking amino acids in addition to the zinc finger core are required for DNA binding. Here, we have characterized the minimal DNA binding region of the human ER by analysing the DNA binding properties of a series of deletion mutants expressed in bacteria. We find that the 66 amino acid zinc finger core of the DBD fails to bind DNA, and that the C-terminal end of the minimal ER DBD required for binding to perfectly palindromic EREs corresponds to the limit of 100% amino acid homology between the chicken and human receptors, which represents the boundary between regions C and D in the ER. Moreover, amino acids of region D up to 30 residues C-terminal to the zinc fingers greatly stabilize DNA binding by the DBD to perfectly palindromic EREs and are absolutely required for formation of gel retardation complexes by the DBD on certain physiological imperfectly palindromic EREs. These results indicate that in addition to the zinc finger core, amino acids C-terminal to the core in regions C and D play a key role in DNA binding by the ER, particularly to imperfectly palindromic response elements. The ER DBD expressed in E. coli binds as a dimer to ERE palindromes in a highly cooperative manner and forms only low levels of monomeric protein-DNA complexes on either palindromic or half-palindromic response elements. Conversion of ER amino acids 222 to 226, which lie within region C, to the corresponding residues of the human RAR alpha abolishes formation of dimeric protein-DNA complexes. Conversely, replacement of the same region of RAR alpha with ER residues 222 to 226 creates a derivative that, unlike the RAR alpha DBD, binds cooperatively to an ERE, indicating that this region is important for dimerization in the ER.
Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité de Génie Génétique et de Biologie Moléculaire de l'INSERM, Faculté de Médecine, Strasbourg, France.
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Mader
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Chambon
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White
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism*; D018168:Receptors, Retinoic Acid; D012091:Repetitive Sequences, Nucleic Acid; D014212:Tretinoin
D002352:Carrier Proteins; D010446:Peptide Fragments; D011960:Receptors, Estrogen; D018168:Receptors, Retinoic Acid; D014212:Tretinoin; D004247:DNA