Autoregulation of Xenopus estrogen receptor gene expression is mediated by a novel estrogen response element in the protein coding region
Lee, James H.
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Permalink
https://hdl.handle.net/2142/19105
Description
Title
Autoregulation of Xenopus estrogen receptor gene expression is mediated by a novel estrogen response element in the protein coding region
Author(s)
Lee, James H.
Issue Date
1994
Doctoral Committee Chair(s)
Shapiro, David J.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Chemistry, Biochemistry
Language
eng
Abstract
The induction of the Xenopus estrogen receptor mRNA by its ligand, estradiol-17$\beta$ (E$\sb2$), results in the establishment of a permanent autoregulatory loop. In order to identify the element(s) responsible for the long term regulation of the estrogen receptor gene, we cloned the estrogen receptor promoter and adjacent 5$\sp\prime$-flanking region of the gene. The transcription start was identified by primer extension analysis and confirmed with the polymerase chain reaction. Sequence analysis and transfections of 3 kb of the 5$\sp\prime$ flanking region failed to identify any hormone responsive element(s). A novel estrogen response element (ERE) was identified in the protein coding region of the gene. Located approximately 480 base pairs downstream of the transcription initiation site, this imperfect palindrome (GGTCA GTT TGACG) differs from the consensus ERE (cERE) by a single nucleotide substitution on one arm of the palindrome. Biological function was tested by inserting two copies of the Xenopus estrogen receptor ERE (xERE) into the chloramphenicol acetyltransferase (CAT) reporter plasmid, ATCO, and performing transfection experiments in Xenopus liver cells. After 72 hrs of treatment with 10$\sp{-7}$ M E$\sb2$, CAT expression increased by approximately 4 to 5 fold. In comparison, two copies of the cERE were able to stimulate CAT expression 10 to 11 fold following E$\sb2$ treatment. In order to compare the affinity of the xERE and cERE for the estrogen receptor, competition gel shift assays were performed using labeled cERE and partially purified estrogen receptor. Consistent with the results of the gene transfer experiments, the xERE is approximately 15-fold less effective as a competitor than the cERE. In addition to its unique location and sequence, these experiments suggest that the xERE may play a major role in the establishment and maintenance of the long-term induction of Xenopus estrogen receptor gene expression.
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