In order to isolate novel estrogen-responsive genes, we utilized a CpG
island library in which the regulatory regions of genes are enriched,
CpG islands were screened for the ability to bind to a recombinant es
trogen receptor protein with a genomic binding site (GBS) cloning meth
od, Six CpG islands were selected, and they contained perfect, imperfe
ct, and/or multiple half-palindromic estrogen-responsive elements (ERE
s). Northern blot analysis of various human cells showed that all thes
e genomic fragments hybridized to specific mRNAs, suggesting that the
genes associated with these EREs might be transcribed in human cells,
Then cDNAs associated with two of them, EB1 and EB9, were isolated fro
m libraries of human placenta and MCF-7 cells derived from a human bre
ast cancer, respectively, Both transcripts were increased by estrogen
in MCF-7 cells, The increase is inhibited by actinomycin D but not by
cycloheximide, indicating that no protein synthesis is required for th
e up-regulation, The cDNA associated with EB1 encodes a 114-amino-acid
protein similar to the cytochrome c oxidase subunit VIIa, named COX7R
P (cytochrome c oxidase subunit VII-related protein), The cDNA associa
ted with EB9 is homologous only to an express sequence tag and was nam
ed EBAG9 (estrogen receptor-binding fragment-associated gene 9). The p
alindromic ERE of EB1 is located in an intron of COX7RP, and that of E
B9 is in the 5' upstream region of the cDNA. Both EREs had significant
estrogen-dependent enhancer activities in a chloramphenicol acetyltra
nsferase assay, when they were inserted into the 5' upstream region of
the chicken beta-globin promoter, We therefore propose that the CPG-G
BS method described here for isolation of the DNA binding site from th
e CpG island library would be useful for identification of novel targe
t genes of certain transcription factors.