Thrombopoietin Initiates Demethylation-Based Transcription of GP6 during Megakaryocyte Differentiation.

Glycoprotein VI (GPVI) is an essential platelet receptor for collagens that is exclusively expressed in the megakaryocytic lineage. Transcription of the human gene GP6 is driven largely by GATA-1, Sp1 and Fli-1. However, the mere presence of these is not sufficient to initiate transcription during megakaryocyte differentiation, and other mechanisms are suggested to be involved in the regulation of megakaryocyte-specific expression of GPVI. In this study, we show that GPVI expression during megakaryocytic differentiation is dependent on CpG demethylation that can be initiated by thrombopoietin (TPO). Sodium bisulfite genomic sequencing established that a CpG-rich island within the GP6 promoter region is fully methylated at 10 CpG sites in GPVI non-expressive cell lines, such as UT-7/EPO and C8161, but completely unmethylated in GPVI expressive cell lines, including UT-7/TPO and CHRF288-11. To further confirm the relationship between CpG demethylation and expression of GPVI in primary cells, we treated human cord blood cells with TPO. The GP6 promoter is highly methylated in cord blood mononuclear cells (progenitors) but not in CD41+enriched cells obtained after TPO differentiation. Furthermore, when UT-7/EPO-Mpl cells, which stably express human c-mpl, were treated with TPO, demethylation of the GP6 promoter was induced. In every case, demethylation of the GP6 promoter correlated with an increase in mRNA level. Thus, megakaryocyte-specific expression of the GP6 gene is regulated, in part, by CpG demethylation which can be directly initiated by TPO. Our results establish, for the first time, a role for TPO in dynamic changes in CpG methylation status that are involved in the epigenetic regulation of megakaryocyte-specific gene expression.