Cloning, novel promoter sequence, and estrogen regulation of a rat oxytocin receptor gene

Tracy L. Bale, Daniel Dorsa

Research output: Contribution to journalArticle

96 Citations (Scopus)

Abstract

Expression of the oxytocin receptor (OR) gene in vivo is known to be regulated by estradiol (E2). We have cloned and sequenced 4 kilobases (kb) of 5'-flanking DNA of the rat OR gene and identified an internal segment of 1260 nucleotides that was absent in an initial publication of this promoter and an additional 2 kb of upstream sequence. This novel internal region is located between two large tg nucleotide repeats. PCR amplification using genomic DNA verified that this sequence is present in the rat genome. To explain transcriptional effects of E2, a palindromic estrogen response element (ERE) that is active in estrogen receptor binding was identified within this new sequence, approximately 4 kb 5' of the translational start site. The ability of E2 to enhance transcription of this promoter was tested in transfection experiments in MCF7 cells. E2 only weakly induced transcription of a truncated construct. Mutational analysis of the ERE in the context of a basal promoter indicated that it functions as an enhancer, and that mutation of two bases eliminates this activity. Further support of the efficacy of this response was shown in mobility gel shift assays in which the OR ERE bound estrogen receptor present in uterine extracts. Receptor binding studies using 125I-ornithine vasotocin in MCF7 cells revealed that E2 dramatically up-regulated endogenous ORs. Western blot analysis confirmed this increase in OR protein with E2 treatment of MCF7 cells. These studies have identified a novel region of the rat OR promoter containing an upstream palindromic ERE that imparts E2 inducibility of OR gene transcription.

Original languageEnglish (US)
Pages (from-to)1151-1158
Number of pages8
JournalEndocrinology
Volume138
Issue number3
DOIs
StatePublished - 1997
Externally publishedYes

Fingerprint

Oxytocin Receptors
Response Elements
Organism Cloning
MCF-7 Cells
Estrogens
Estrogen Receptors
Genes
Nucleotides
Vasotocin
Ornithine
Electrophoretic Mobility Shift Assay
Transfection
Publications
Estradiol
Western Blotting
Gels
Genome
Polymerase Chain Reaction
Mutation
rat oxytocin receptors

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Cloning, novel promoter sequence, and estrogen regulation of a rat oxytocin receptor gene. / Bale, Tracy L.; Dorsa, Daniel.

In: Endocrinology, Vol. 138, No. 3, 1997, p. 1151-1158.

Research output: Contribution to journalArticle

@article{b052429462fe4faf85b4e5deb9de9ab2,
title = "Cloning, novel promoter sequence, and estrogen regulation of a rat oxytocin receptor gene",
abstract = "Expression of the oxytocin receptor (OR) gene in vivo is known to be regulated by estradiol (E2). We have cloned and sequenced 4 kilobases (kb) of 5'-flanking DNA of the rat OR gene and identified an internal segment of 1260 nucleotides that was absent in an initial publication of this promoter and an additional 2 kb of upstream sequence. This novel internal region is located between two large tg nucleotide repeats. PCR amplification using genomic DNA verified that this sequence is present in the rat genome. To explain transcriptional effects of E2, a palindromic estrogen response element (ERE) that is active in estrogen receptor binding was identified within this new sequence, approximately 4 kb 5' of the translational start site. The ability of E2 to enhance transcription of this promoter was tested in transfection experiments in MCF7 cells. E2 only weakly induced transcription of a truncated construct. Mutational analysis of the ERE in the context of a basal promoter indicated that it functions as an enhancer, and that mutation of two bases eliminates this activity. Further support of the efficacy of this response was shown in mobility gel shift assays in which the OR ERE bound estrogen receptor present in uterine extracts. Receptor binding studies using 125I-ornithine vasotocin in MCF7 cells revealed that E2 dramatically up-regulated endogenous ORs. Western blot analysis confirmed this increase in OR protein with E2 treatment of MCF7 cells. These studies have identified a novel region of the rat OR promoter containing an upstream palindromic ERE that imparts E2 inducibility of OR gene transcription.",
author = "Bale, {Tracy L.} and Daniel Dorsa",
year = "1997",
doi = "10.1210/en.138.3.1151",
language = "English (US)",
volume = "138",
pages = "1151--1158",
journal = "Endocrinology",
issn = "0013-7227",
publisher = "The Endocrine Society",
number = "3",

}

TY - JOUR

T1 - Cloning, novel promoter sequence, and estrogen regulation of a rat oxytocin receptor gene

AU - Bale, Tracy L.

AU - Dorsa, Daniel

PY - 1997

Y1 - 1997

N2 - Expression of the oxytocin receptor (OR) gene in vivo is known to be regulated by estradiol (E2). We have cloned and sequenced 4 kilobases (kb) of 5'-flanking DNA of the rat OR gene and identified an internal segment of 1260 nucleotides that was absent in an initial publication of this promoter and an additional 2 kb of upstream sequence. This novel internal region is located between two large tg nucleotide repeats. PCR amplification using genomic DNA verified that this sequence is present in the rat genome. To explain transcriptional effects of E2, a palindromic estrogen response element (ERE) that is active in estrogen receptor binding was identified within this new sequence, approximately 4 kb 5' of the translational start site. The ability of E2 to enhance transcription of this promoter was tested in transfection experiments in MCF7 cells. E2 only weakly induced transcription of a truncated construct. Mutational analysis of the ERE in the context of a basal promoter indicated that it functions as an enhancer, and that mutation of two bases eliminates this activity. Further support of the efficacy of this response was shown in mobility gel shift assays in which the OR ERE bound estrogen receptor present in uterine extracts. Receptor binding studies using 125I-ornithine vasotocin in MCF7 cells revealed that E2 dramatically up-regulated endogenous ORs. Western blot analysis confirmed this increase in OR protein with E2 treatment of MCF7 cells. These studies have identified a novel region of the rat OR promoter containing an upstream palindromic ERE that imparts E2 inducibility of OR gene transcription.

AB - Expression of the oxytocin receptor (OR) gene in vivo is known to be regulated by estradiol (E2). We have cloned and sequenced 4 kilobases (kb) of 5'-flanking DNA of the rat OR gene and identified an internal segment of 1260 nucleotides that was absent in an initial publication of this promoter and an additional 2 kb of upstream sequence. This novel internal region is located between two large tg nucleotide repeats. PCR amplification using genomic DNA verified that this sequence is present in the rat genome. To explain transcriptional effects of E2, a palindromic estrogen response element (ERE) that is active in estrogen receptor binding was identified within this new sequence, approximately 4 kb 5' of the translational start site. The ability of E2 to enhance transcription of this promoter was tested in transfection experiments in MCF7 cells. E2 only weakly induced transcription of a truncated construct. Mutational analysis of the ERE in the context of a basal promoter indicated that it functions as an enhancer, and that mutation of two bases eliminates this activity. Further support of the efficacy of this response was shown in mobility gel shift assays in which the OR ERE bound estrogen receptor present in uterine extracts. Receptor binding studies using 125I-ornithine vasotocin in MCF7 cells revealed that E2 dramatically up-regulated endogenous ORs. Western blot analysis confirmed this increase in OR protein with E2 treatment of MCF7 cells. These studies have identified a novel region of the rat OR promoter containing an upstream palindromic ERE that imparts E2 inducibility of OR gene transcription.

UR - http://www.scopus.com/inward/record.url?scp=0031039952&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031039952&partnerID=8YFLogxK

U2 - 10.1210/en.138.3.1151

DO - 10.1210/en.138.3.1151

M3 - Article

C2 - 9048622

AN - SCOPUS:0031039952

VL - 138

SP - 1151

EP - 1158

JO - Endocrinology

JF - Endocrinology

SN - 0013-7227

IS - 3

ER -