Defining a minimal estrogen receptor DNA binding domain

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Abstract
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 amlno acid zinc finger core of the DBD falls to bind DNA, and that the C-termlnal end of the minimal ER DBD required for binding to perfectly palindromic EREs corresponds to the limit of 100% amlno 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-termlnal 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, amlno acids C-termlnal 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 dlmer to ERE palindromes in a highly cooperative manner and forms only low levels of monomerlc protein - DNA complexes on either palindromic or half-pallndromlc response elements. Conversion of ER amlno acids 222 to 226, which lie within region C, to the corresponding residues of the human RARα abolishes formation of dimeric protein - DNA complexes. Conversely, replacement of the same region of RARα with ER residues 222 to 226