Title

Isolation and characterization of upstream regulatory elements of the rat connexin43 gene

Date of Award

1994

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Rudolf Werner, Committee Chair

Abstract

Connexin43 is the major gap junction protein in myocardium and myometrium. In the heart, the connexin43 is constitutively expressed whereas in the uterus the expression appears to be induced by estrogen at parturition, when coordinated contraction of the uterine smooth muscle is required for the expulsion of the fetus. To study the basis for this differential regulation of the gene, a genomic clone of the rat connexin43 gene was isolated and characterized. This clone contains the exon encoding the 5$\sp\prime$-untranslated sequence of the connexin43 cDNA, 7kb of upstream sequence, and 8kb of intronic sequence. The coding region is not present in this clone. Linkage between this clone and the coding region was then demonstrated by genomic DNA PCR. The gene structure of the rat connexin43 is proposed to consist of two exons separated by an 8.5kb intron.Sequence analysis of the region upstream from exon I revealed the presence of a TATA box and six half-palindromic estrogen response elements. In addition, there were three half-palindromic progesterone/glucocorticoid response elements and several AP-1 and AP-2 binding sites. The transcription start and acceptor splice sites were mapped with total RNA from rat heart, uterus, ovary and stomach smooth muscle, isolated from both estrogen-injected and control rats. In all cases, the sites were identical suggesting that the differential expression of the connexin43 gene in heart and myometrium is not accomplished by the use of different promoters or by different splicing mechanisms.Functional assays of the promoter/enhancer region of the connexin43 gene were carried out by transient transfection of cell lines with a construct containing 1.3kb upstream region of connexin43 and luciferase as the reporter gene. This construct was found to be expressed both in HeLa cells and the osteosarcoma cell line, ROS 17/2.8. Under estrogen treatment, an additional 10-fold increase in expression was obtained from HeLa cells cotransfected with the construct and estrogen receptor cDNA. Mutations in each of three half-palindromic estrogen response elements caused decreases in estrogen response by 39-56%. In contrast, ROS 17/2.8 cells cotransfected with the same DNAs did not respond to estrogen.Responses of the construct to progesterone, cAMP and TPA were also determined in HeLa cells. Progesterone and TPA stimulated the expression of the construct. Treatment with cAMP had no effect on the luciferase activity. Possible mechanisms for the differential activation of the connexin43 promoter by estrogen in HeLa and ROS 17/2.8 cells, and the progesterone response in HeLa cells, which appears to be contrary to the in vivo situation in myometrium, are discussed.

Keywords

Biology, Molecular; Biology, Cell; Biology, Animal Physiology

Link to Full Text

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