Identification of Two Regulatory Elements within the Promoter Region of the Mouse Connexin 43 Gene

doi:10.1074/jbc.270.8.3863

Identification of Two Regulatory Elements within the Promoter Region of the Mouse Connexin 43 Gene (*)

From the (1)Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, and the Departments of
(2)Obstetrics and Gynaecology and
(3)Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

↵^¶ Current address: Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia V5Z 1A1, Canada.
↵** Current address: Cephalon Inc., West Chester, PA 19380-4245.

Abstract

To define the minimal sequences required for expression of the connexin 43 gene (cx43) in myometrial cells, we generated 5′ deletion constructs of a fragment extending 1686 base pairs upstream and 162 base pairs downstream of the transcription start site and determined their ability to drive expression of the chloramphenicol acetyltransferase reporter gene in transfected myometrial cell lines. Our investigation revealed two cis-acting regulatory elements within this fragment. Deletion of a region extending from −102 to −92 led to an increase of the promoter activity by greater than 10-fold, indicating a presence of a repressor element. Deletion of a region extending from −72 to −62 caused a decrease of the promoter activity of a similar extent, implying the existence of a positive element. Electrophoretic mobility shift assays demonstrated that synthetic oligonucleotides derived from these two small regions can each bind with a nuclear protein(s) prepared from myometrial cells, and an introduction of three and two base substitutions into each of these oligomers was sufficient to abolish their protein binding capability. These same mutations, when incorporated in the chloramphenicol acetyltransferase constructs, diminished regulatory functions of the negative and positive elements, and the protein(s) that bind to these functional elements was found in several tissues known to express cx43 gene.

Footnotes

↵^§ These authors contributed equally to this work.
↵^§§ Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada and a Howard Hughes International Scholar.
↵^¶¶ Career Scientist of the Ontario Ministry of Health.
↵* This work was supported by grants from the Medical Research Council of Canada (to S. J. L. and to J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17892[GenBank].
↵¹ The abbreviations used are:

bp

base pair(s)

EMSA

electrophoretic mobility shift assay

kb

kilobase pair(s)

CAT

chloramphenicol acetyltransferase.
- Received November 9, 1994.