Event Title
Targeted Dinucleotide CpG Methylation in a Uterine Fibroid Cell Line
Description
Uterine fibroids are benign smooth muscle tumors estimated to occur in 25-89% of reproductive age women; 25% of those women suffer from clinically significant symptoms including excessive menstrual bleeding, dysmenorrhea, and infertility. Non-invasive treatment options for fibroids such as hormone regimens are unsuitable for long-term applications, and hysterectomy remains the only permanent treatment for fibroid-associated morbidities. Three molecular subtypes of fibroids account for 80-90% of incidence which are characterized by MED12 mutation (MED12mt), or HMGA1/HMGA2 overexpression (HMGA1hi and HMGA2hi, respectively). Clustering of DNA methylation profiles segregated subtypes from normal tissue and revealed differential hypomethylation in the HMGA2 gene body in HMGA2hi fibroids. HMGA2 overexpression is most commonly reported with translocations at 12q15, but hypomethylation in the HMGA2 gene body is present in HMGA2hi fibroids with no detected translocation suggesting hypomethylation may be an additional mechanism for upregulation of HMGA2. DNA methylation at CpG dinucleotides is a key epigenetic modification with critical roles in gene regulation, genomic integrity, and DNA-protein interactions including CTCF-binding, which we hypothesize to be involved in restructuring the chromatin around fibroid-associated genes. Specific methylation events in uterine fibroid development have not yet been explored. Here, we demonstrate site-specific de novo methylation in the HMGA2 gene body in a uterine fibroid cell line using a fusion protein of nuclease deficient Cas9 (dCas9) and prokaryotic DNA methyltransferase MQ1. Cells were co-transfected with dCas9-MQ and sgRNA transfer plasmids and isolated by FACS for DNA methylation analysis by NGS. This tool can be used to further explore site-specific DNA methylation events.
Targeted Dinucleotide CpG Methylation in a Uterine Fibroid Cell Line
Uterine fibroids are benign smooth muscle tumors estimated to occur in 25-89% of reproductive age women; 25% of those women suffer from clinically significant symptoms including excessive menstrual bleeding, dysmenorrhea, and infertility. Non-invasive treatment options for fibroids such as hormone regimens are unsuitable for long-term applications, and hysterectomy remains the only permanent treatment for fibroid-associated morbidities. Three molecular subtypes of fibroids account for 80-90% of incidence which are characterized by MED12 mutation (MED12mt), or HMGA1/HMGA2 overexpression (HMGA1hi and HMGA2hi, respectively). Clustering of DNA methylation profiles segregated subtypes from normal tissue and revealed differential hypomethylation in the HMGA2 gene body in HMGA2hi fibroids. HMGA2 overexpression is most commonly reported with translocations at 12q15, but hypomethylation in the HMGA2 gene body is present in HMGA2hi fibroids with no detected translocation suggesting hypomethylation may be an additional mechanism for upregulation of HMGA2. DNA methylation at CpG dinucleotides is a key epigenetic modification with critical roles in gene regulation, genomic integrity, and DNA-protein interactions including CTCF-binding, which we hypothesize to be involved in restructuring the chromatin around fibroid-associated genes. Specific methylation events in uterine fibroid development have not yet been explored. Here, we demonstrate site-specific de novo methylation in the HMGA2 gene body in a uterine fibroid cell line using a fusion protein of nuclease deficient Cas9 (dCas9) and prokaryotic DNA methyltransferase MQ1. Cells were co-transfected with dCas9-MQ and sgRNA transfer plasmids and isolated by FACS for DNA methylation analysis by NGS. This tool can be used to further explore site-specific DNA methylation events.