Session: MON 515-547-Female Reproductive Endocrinology
Poster Board MON-542
The impact of promoter methylation on the LHCGR expression was analyzed with a cell-culture-based in vitro reporter assay. Therefore, the LHCGR core promoter sequence was cloned into a CpG-free luciferase reporter vector. Afterwards, the plasmids were in vitromethylated and transiently transfected in COS7 cells. The transcription efficiency of the unmethylated and methylated promoter sequence was compared using an enzymatic luciferase-based assay system.
An in vitro mutagenesis of the Sp1 transcription factor binding domains resulted in a significant decrease of luciferase activity to 50 %, demonstrating the importance of this transcription factor for the receptor expression. The luciferase reporter assay demonstrated furthermore that the LHCGR promoter activity decreased by 80 % after complete DNA methylation of the constructs. Preliminary data indicate that the regulation of the LHCGR expression is dependent on specific CpG dinucleotides inside the Sp1 transcription factor binding domains. Replacing these CpG dinucleotides by mutagenesis weakened the effect of methylation on the promoter activity, although due to the methylation of the remaining CpGs still a decrease in activity occurs.
The activation of the LHCGR gene is tightly regulated by the methylation status of its core promoter sequence. We detected an inverse relationship between DNA methylation and the promoter activity of the LHCGR. In future studies, we will complete our in vitro data with in vivo experiments on primary granulosa cells to identify the influence of promoter methylation on the natural expression of the LHCGR gene.
Nothing to Disclose: JG, JS, BT
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