Transcriptional regulation of the human gonadotropin-releasing hormone (GnRH) II and GnRH receptor genes
TRANSCRIPTIONAL REGULATION OF THE HUMAN GONADOTROPIN-RELEASING HORMONE (GnRH) II AND GnRH RECEPTOR GENES
Ruby L. C. Hoo
for the degree of Doctor of Philosophy at The University of Hong Kong
in December 2003
The human gonadotropin-releasing hormone receptor (hGnRHR) gene is regulated by multiple promoters and several cell-specific cis-elements. In this study, a proximal promoter responsible for a cluster of 3' CAP sites from -635 to -538 was identified by 5' and 3' progressive deletion ofa 2.3 kb 5' flanking region of the hGnRHR gene followed by transient luciferase reporter gene assays. This proximal promoter, located at -677/-558 (relative to ATG), was able to drive a 13.1?.6 fold increase in reporter gene activity in an orientation-dependent manner. Site-directed mutation analysis
revealed that the sequences residing -607/-568 were crucial for the basal promoter activity as any mutations within this region would completely abrogate promoter function. Within the core promoter element, two pyrimidine-rich initiator elements (Inr) that interact with the same protein factors with different affinities were evident by gel mobility shift assays. Southwestern blot analysis showed that multiple nuclear factors of sizes ranging from 36-150 kDa were able to interact specifically with the core proximal promoter element and so as a previously identified distal promoter of the hGnRHR gene. Taken together, our results demonstrated that these two promoters share common protein factors to regulate transcription initiations at two different regions.
Recent studies of the hGnRH II gene demonstrated that its expression is regulated by cAMP, gonadotropins and estrogen, and a minimal promoter was identified at -1124 to -750 (relative to ATG). In this report, an intronic silencer element (-620/-650) was identified as deletion of this 30 bp led to drastic increases in the promoter activities. This intronic silencer was found to be position-dependent and orientation-independent. By site-directed mutagenesis, a NF-kB motif and a hGII-SNOG motif, were found to be crucial for the repressor's function. However, by mobility shift and supershift assays, instead of NF -kB subunits, Sp 1 and Sp3 were found to interact with the NF -kB site. Based on this observation, we hypothesized that the in vivo competition of Sp lISp3 proteins with the NF-kB subunits may play an important role in inducing gene repression. Interestingly, different protein factors from TE671 and JEG-3 cells were found to interact with the Ml-SNOG motif. It is possible that these cells use different mechanisms to mediate the functions of the hGnRH II silencer element.
In summary, we have identified two Inr elements within the proximal promoter of the hGnRHR gene that are responsible for the transcription initiation of a cluster of 3' CAP sites residing -635 to -538. In addition, NF-kB and hGII-SNOG motifs were identified to be crucial for the repressor function of the hGnRH II gene.
School:The University of Hong Kong
School Location:China - Hong Kong SAR
Source Type:Master's Thesis
Keywords:luteinizing hormone releasing receptors genetic regulation
Date of Publication:01/01/2004