An investigation of regulation of MKRN3 monoallelic expression
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Age at pubertal initiation is associated with the risk of many severe health outcomes later in life. “Reactivation” of the hypothalamic-pituitary-gonadal axis, signified via an increase in the amplitude and frequency of gonadotropin-releasing hormone, (GnRH) marks the commencement of puberty. For decades, physicians and scientists have attempted to unravel the mysterious mechanisms underlying the onset of this transitional period of reproductive maturation. In 2013, loss-of-function mutations in maternally imprinted Makorin RING Finger 3 (MKRN3) were determined to be the one of the causes of central precocious puberty (CPP), establishing MKRN3 as the first inhibitory gene with mutations in humans indicated in the upstream regulation of GnRH. MKRN3 accomplishes this suppression through its function as an E3 ligase, marking components involved in the transcription of kisspeptin and neurokinin B, two stimulatory regulators of GnRH, for degradation by the proteasome. MKRN3 remains the most common genetic cause of CPP to date. As a maternally imprinted gene, loss-of-function mutations in MKRN3 have only been shown to produce a phenotype in patients when inherited paternally. However, the mechanisms conferring monoallelic expression of MKRN3 are not well understood – nor are the downstream implications of MKRN3 imprinting abnormalities on the development, or even simply, association, with pubertal disorders. It has been determined that differential methylation – a common epigenetic modification involved in the demarcation of imprinted genes – plays a role in the imprinting mechanism of MKRN3. The purpose of this study is to characterize the differentially methylated region (DMR) and to expand understanding of how this region may serve to mark the maternal allele for repression. This will set the foundation for subsequent studies into the potential contribution of methylation defects in the MKRN3 DMR toward the onset of pubertal disorders. Two potential regulatory sites decorated with CpG islands were identified: the proximal-promoter and a potential enhancer. In classical imprinting studies, differential methylation of the proximal-promoter has been shown to mark alleles on a parent-specific basis for transcriptional inhibition – subsequently silencing expression dependent on parental origin. Additionally, many studies describe a similar process with an upstream or downstream enhancer region being the site influencing transcriptional activity of the alleles on a parent-of-origin basis; preliminary studies indicate a region 3.7 kb upstream of the MKRN3 transcription start site which may act as an enhancer for MKRN3. In this study, DNA was extracted from whole, peripheral blood of individuals with normal pubertal development, followed by bisulfite conversion and PCR amplification, with clean-up and submission for next generation sequencing. Sequencing analysis allowed for the quantification of the methylation profile of each sample at its respective regulatory site. The proximal-promoter was found to be almost entirely methylated while the enhancer exhibited differential methylation, but with notable variability between samples. We identified this enhancer region as the presumptive DMR of MKRN3. Additional research is required to confirm the status of the proposed enhancer as the DMR regulating the paternal-specific expression of MKRN3.