Impact of DNA methylation on the regulation of the luteinizing hormone/choriogonadotropin receptor expression

Introduction: The luteinizing hormone/choriogonadotropin receptor (LHCGR) plays a central role in human gonadal maturation and function. However, the regulatory mechanism of the LHCGR gene expression is not fully understood yet. We postulate due to the high frequency of CpG dinucleotides in the core promoter sequence that the transcription of the LHCGR is affected by DNA methylation.

Methods: 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, the obtained plasmids were in vitro methylated 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.

Results: Luciferase reporter assays demonstrated that the LHCGR promoter activity decreases by about 80% through DNA methylation, our data also indicate that the regulation of the LHCGR expression is dependent on specific CpG dinucleotides inside the Sp1 transcription factor binding domains. An in vitro mutagenesis of these CpG sites reduces the influence of promoter methylation to approximately 50%.

Conclusion: he activation of the LHCGR gene is directly impaired by the methylation 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.