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Nucleic Acids Research, 1991, Vol. 19, No. 15 4091-4096
© 1991

MOLECULAR BIOLOGY

Isolation of estrogen receptor-binding sites in human genomic DNA

Satoshi Inoue, Shigeru Kondo, Makoto Hashimoto, Takashi Kondo and Masami Muramatsu*

Department of Biochemistry, Faculty of Medicine, The University of Tokyo Hongo, Bunkyo-ku, Tokyo 113, Japan

* To whom correspondence should be addressed

Received June 3, 1991. Revised July 11, 1991. Accepted July 11, 1991.

Total genomic DNA digested by restriction enzymes was mixed with the DNA-binding domain of the estrogen receptor (ER-DBD) that was expressed in Escherichia coil and the fragments that bound to it were selected by nitrocellulose filter. These fragments were cloned into a plasmid vector and amplified. This selection process was repeated six times and five fragments ranging from 0.2 to 2 kb were isolated. Interestingly, each of these fragments had a perfect palindromic estrogen responsive element (ERE) (GGT CANNNTGAC) More surprisingly, one of the fragments was found to be derived from the same locus as a fragment obtained by another similar but independent experiment. The results indicate that the ER-DBD region can bind by itself specifically to the perfect palindromic ERE with a 3 base pair spacing but it does not bind strongly enough to the half palindromic EREs or to the imperfect palindromic EREs. Chloramphenicol acetyltransferase assay has shown that some of these fragments have estrogen-dependent enhancer activity, suggesting the existence of a target gene near these fragments. The method described here may be generally applicable for screening and isolation of other transcription factor-binding sites in genomic DNA.


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