Title

The connexin43 promoter: Characterization of cis and trans-acting elements

Date of Award

1999

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Rudolf Werner, Committee Chair

Abstract

Connexin43 is the major gap junction protein in myocardium and myometrium. In the heart, connexin43 is constitutively expressed whereas in the uterus its 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, different regions of the rat connexin43 promoter were analyzed by in vivo DNA footprinting. The results showed that a small stretch of the proximal promoter, corresponding to the sequence between nucleotides -34 and -71 with respect to the transcription start site, was involved in both the tissue-specific regulation and the estrogen-mediated upregulation of the gene. A uterine-specific DNA-protein complex was detected by Electromobility Shift Assay (EMSA) with a probe containing this sequence of the cx43 promoter (nucleotides between -34 and -71). This complex seems to involve the binding of a 35 kDa protein (Southwestern blot). In addition, a 90 kDa estrogen-inducible uterine protein was also shown to bind to this probe by Southwestern blot, even though no estrogen-specific complex could be observed by EMSA.Screening of an estrogen-induced myometrial expression library with this same probe did not yield a clone corresponding to either of these proteins, but led to the discovery of a novel transcription factor, or co-factor, that I named ini.Ini is a ubiquitously expressed, estrogen-inducible, 110-amino acid (12.4 kDa) protein containing a RING-finger-like motif (amino acids 4 to 49), and a 4-cysteine zinc finger motif (amino acids 57 to 74), which is capable of directly binding to a region of the cx43 promoter, located between nucleotides -34 and -71, in vitro, as shown by EMSA and Southwestern blot analysis. Ini also contains a potential nuclear localization signal (amino acids 102 to 110), which may mediate its translocation to the nucleus. Ini has been shown to be localized in the nucleus of RL clone9 cells.Ini's sequence is one of the most highly conserved sequences known, being identical in all mammals analyzed (mouse, rat and human) and highly conserved in plants and even in yeast. This suggests that the ini protein serves an important cellular function. This was confirmed by the finding that knockout of the ini gene in Schizosaccharomyces pombe results in a lethal phenotype.Ini seems to up-regulate transcription by a mechanism that does not require an AP-1 site, since a three tandem copy of its binding site, lacking the nucleotides -34 to -48 (AP-1 site), was able to up-regulate the SV40 minimal promoter activity by 6 to 7-fold when ini was overexpressed in transiently transfected HeLa cells.I propose a model for ini-mediated activation of transcription. In this model ini's most conserved domain (the amino-terminal RING-finger like domain) is involved in mediating protein-protein interactions with common trans-activators of the eukaryotic transcription machinery. I believe it is this protein-protein interaction which unmasks ini's DNA binding motif (amino acids 57 to 110) thereby allowing it to interact with the DNA sequences on the promoter of the genes that ini regulates. This interaction allows ini to recruit other activators to the transcription start site. More work is required to test this hypothesis.

Keywords

Biology, Molecular; Biology, Genetics

Link to Full Text

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