Regulation of Xenopus laevis estrogen receptor gene expression is mediated by an estrogen response element in the protein coding region

Abstract

To investigate the 17 beta-estradiol induction of the mRNA coding for the Xenopus laevis estrogen receptor (XER), we cloned the promoter and the 5'-flanking region of the ER gene. Transcription initiation sites were identified by primer extension, and confirmed by the polymerase chain reaction. The promoter and 5'-flanking region contain an imperfect TATA box and a potential CAAT box at -51. Sequence analysis and transfections indicated that no functional estrogen response element (ERE) was present in approximately 3 kb of 5'-flanking region. An imperfect ERE, GGTCAGTTTGACG, which differs from the consensus ERE sequence by 1 nucleotide, was detected in the protein coding region of the gene, approximately 480 nucleotides downstream of the transcription initiation site. In transient transfections using a simple promoter containing two copies of this Xenopus estrogen receptor ERE (XERE), we observed an estrogen-dependent increase in CAT activity of four- to five-fold, to a level approximately 20-fold greater than the activity of the control plasmid lacking the XEREs. In competition gel mobility-shift assays, the XERE exhibited a weak, but clearly detectable, ability to compete for binding of human ER to a labeled consensus ERE. Because it exhibits sequence-specific binding to the ER in competition gel mobility-shift assays, and is able to confer estrogen-dependent transcription on a simple synthetic promoter, the novel XERE, located in the protein coding region of the XER gene appears to represent a weak, but functional, ERE.