Epigenetic modulation of glycoprotein VI gene expression by DNA methylation

Glycoprotein VI (GPVI) is an important platelet membrane receptor. The expression of GPVI on platelet membranes is increased in patients with coronary heart disease (CHD). DNA methylation is one of the most common post-replication and pre-transcriptional modifications and plays a critical role in the regulation of gene expression. Here, we aimed to reveal how methylation regulates GPVI expression.

Pyrosequencing was used to determine whether the GPVI promoter region in leukocytes from CHD patients is hypomethylated. The expressions of GPVI in CHD patients were detected using qRT-PCR and Western blot. The effect of methylation of the GPVI promoter region on regulating its transcriptional activity was analyzed using in vitro luciferase assay. The expression of P-selectin in platelet-like particles was determined using flow cytometry, and SYK phosphorylation was observed using Western blot.

We found that the GPVI promoter region in leukocytes from CHD patients was hypomethylated and the expression of GPVI at the mRNA and protein level was elevated in CHD patients. We also found that the hypermethylation of GPVI promoter region inhibited the expression of GPVI in the -322 to +75, -539 to +75, and -937 to +75 regions in Dami cells. Moreover, the data showed that the methylation or demethylation regulated the GPVI expression and platelet-like particle activation in Dami cells.

Taken together, these results indicate that DNA methylation regulates GPVI expression and that CpG methylation levels in the promoter region of the GPVI gene may be a biomarker of CHD.