Isolation of estrogen receptor-binding sites in human genomic DNA.
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 coli 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-CANNNTGACC). 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.
Inoue
S
S
Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
Kondo
S
S
Hashimoto
M
M
Kondo
T
T
Muramatsu
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011960:Receptors, Estrogen / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism
D011960:Receptors, Estrogen; D014157:Transcription Factors; D004247:DNA