Date of Award

Winter 2000

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Committee Director

Ke-Wen Dong

Committee Member

Frank Castora

Committee Member

Xi Jiang

Committee Member

Sergio Oehninger

Abstract

Using the human placental choriocarcinoma JEG-3 cell line as an in vitro human placental model, I studied the mechanisms of the tissue-specific expression and steroid hormone regulation of the hGnRH gene in the human placenta. The results showed that all of the previously identified four elements are required for the full activity of the hGnRH upstream promoter in JEG-3 cells, while the element 4 (FP4, −987/−968) is the most important. Studies performed with 5′ end deletion of this region confirmed these observations. Further, supershift assay using Oct-1 antibody demonstrated the involvement of Oct-1 in the FP4 DNA-protein interaction in JEG-3 cells.

Transient transfection studies showed that hERα and hERβ mediate inhibitory effects of estradiol on the hGnRH upstream promoter in JEG-3 cells in a receptor-mediated and dose-dependent manner, while hERβ acts to a lesser extent than hERα. Also, hERα and hERβ exhibit distinctive actions in directing the effects of estrone, estradiol, and estriol on the upstream promoter.

Furthermore, mutagenesis studies confirmed the negative (−991 to −935) and positive (−827 to −730) estrogen responsive elements in the hGnRH upstream promoter. However, gel shift assay using hERα protein did not cause any shifting, suggesting that ER may not be directly involved in the DNA-protein binding.

In addition, progesterone exhibited a stimulatory effect on the hGnRH upstream promoter activity in JEG-3 in a receptor-mediated and dose-dependent manner. PR-B mediated a more potent stimulatory effect than PR-A. Moreover, exogenous expressions of coactivators SRC-1 and CBP, independently and synergistically, upregulated the PR-A and PR-B mediated progesterone effects on the hGnRH upstream promoter in JEG-3 cells.

Taken together, multiple cis-regulatory elements and trans-acting factors involved in the regulation of the hGnRH upstream promoter activity in JEG-3 cells. Different steroid hormone receptor isoforms mediate distinctive effects on the hGnRH upstream promoter activities. Although several hormone responsive elements have been confirmed, there is no evidence for direct binding of hormone receptors with the upstream promoter. Further studies are needed to identify the trans-regulatory proteins important for the expression and regulation of the hGnRH gene in the placental cells.

Comments

A Dissertation Submitted to the Faculty of Old Dominion University and Eastern Virginia Medical School in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Science.

DOI

10.25777/4z9a-cc55

ISBN

9780493178394

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