SVEP1 promoter regulation by methylation of CpG sites

SVEP1 gene encodes a cell adhesion molecule (CAM) that was previously shown to be expressed by cells related to skeletal tissues. Here we focus on SVEP1 expression regulation in pre-osteoblastic MBA-15 and mammary adenocarcinoma DA3 cells. We show that SVEP1 message and protein are highly expressed by MBA-15 when compared with DA3 cells. DNA methylation of CpGs sites is an epigenetic mechanism associated with gene silencing. Therefore, we analyzed the methylation status of a region potentially harbors SVEP1 promoter and further activity alterations induced by estrogen (17βE2) and TNFα. We also mapped in silico the transcription binding sites namely TFIIB, NF-κB, ERE, AP1 and Sp1 at the putative promoter. Treatments with demethylation reagents, 5′-aza-deoxy-Cytidine (5′-aza-dC), or histone deacetylase inhibitor, Trichostatin A (TSA) resulted with an elevation of SVEP1 mRNA expression in both cell types. Methylation levels of specific CpGs sites located at transcription binding sites were assessed using sodium bisulfite genomic DNA sequencing, methylated DNA immunoprecipitation (meDIP) and Methylation-Specific PCR (MSP). Our results show that the putative promoter of SVEP1 is hypermethylated in DA3— compared with MBA-15 cells, thus regulating SVEP1 expression levels. In addition, by affecting SVEP1 promoter methylation status, 17βE2 and TNFα regulate ectopic SVEP1 promoter and mRNA expression. Our data sheds light on understanding the cell-type specific promoter status for regulation of the SVEP1. Since SVEP1 protein mediates cellular adhesion, this data might be beneficial for the future characterization of SVEP1 expression in the interactions existing in bone.

► SVEP1 is a cell adhesion molecule, mediates cellular adhesion.
► We studied the gene regulation in pre-osteoblastic and mammary adenocarcinoma cells.
► The results show that demethylation reagents or histone deacetylase inhibitor caused mRNA expression elevation.
► Methylation levels of the promoter were assessed by three different methods.
► Two regulators 17βE2 and TNFα were studied for their effect on methylation status of the promoter.