Isolation of Estrogen-Responsive Genes with a CpG Island Library

Abstract

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 estrogen receptor protein with a genomic binding site (GBS) cloning method. Six CpG islands were selected, and they contained perfect, imperfect, and/or multiple half-palindromic estrogen-responsive elements (EREs). Northern blot analysis of various human cells showed that all these 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 from libraries of human placenta and MCF-7 cells derived from a human breast 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 the up-regulation. The cDNA associated with EB1 encodes a 114-amino-acid protein similar to the cytochrome c oxidase subunit VIIa, named COX7RP (cytochrome c oxidase subunit VII-related protein). The cDNA associated with EB9 is homologous only to an express sequence tag and was named EBAG9 (estrogen receptor-binding fragment-associated gene 9). The palindromic ERE of EB1 is located in an intron of COX7RP, and that of EB9 is in the 5' upstream region of the cDNA. Both EREs had significant estrogen-dependent enhancer activities in a chloramphenicol acetyltransferase assay, when they were inserted into the 5' upstream region of the chicken beta-globin promoter. We therefore propose that the CpG-GBS method described here for isolation of the DNA binding site from the CpG island library would be useful for identification of novel target genes of certain transcription factors.

Functional Analysis of p53 Binding under Differential Stresses

“…The top 44 remaining loci are presented in Table 1. Genes located near these loci perform a broad range of functions including DNA synthesis and repair (REV3L [48] and PCNA [73]), transcription (XPMC2H [55], HNRPUL1 [45], BANP [67], ATF7 [64], ZNF215 [3], and MYC [21]), mitochondrial function (CA5A [58] and COX7A2L [79]), and mitotic spindle assembly (CDCA8) (16). One target has direct involvement in the secretory functions of the cell (COH1) (41), while another target has a direct link to the unfolded protein response (UPR) (SYVN1) (75).…”

Identification of estrogen-responsive genes based on the DNA binding properties of estrogen receptors using high-throughput sequencing technology

“…In order to isolate estrogen-responsive genes, including EREs in their transcription regulatory regions, we have developed a technique called genomic binding-site cloning [26] . Using this method, several genomic sequences containing EREs were successfully isolated and, subsequently, novel estrogen-responsive genes were identified nearby functional EREs [27,28] . Protein products of these genes include estrogenresponsive finger protein (Efp), cytochrome c oxidase subunit VIIa-related polypeptide (COX7RP), and estrogen receptorbinding fragment-associated antigen 9 (EBAG9).…”

A stabilizing factor for mitochondrial respiratory supercomplex assembly regulates energy metabolism in muscle

“…Here we focus our study on a COX-related molecule, cytochrome c oxidase (COX) subunit 7a-related protein (COX7RP), which was originally identified as an oestrogen-responsive gene in MCF7 cells 22 . COX7RP encodes a 114-amino-acid protein, which is similar to the mitochondrial respiratory enzyme COX subunit 7a (ref.…”