title: Thrombopoietin initiates demethylation-based transcription of GP6 during megakaryocyte differentiation.
abstract: 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-binding protein 1 (GATA-1), specificity protein 1 (Sp1), and Friend leukemia integration 1 (Fli-1). In this report, we show that GPVI expression during megakaryocytic differentiation is dependent on cytosine-phosphate-guanosine (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-nonexpressive 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-myeloproliferative leukemia virus ligand (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.
authors.affiliation: Roon Research Center for Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis and Thrombosis, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Kanaji
authors.forename: Taisuke
authors.initials: T
authors.lastname: Kanaji
authors.forename: Beatrice
authors.initials: B
authors.lastname: Jacquelin
authors.forename: Mei
authors.initials: M
authors.lastname: Chang
authors.forename: Diane J
authors.initials: DJ
authors.lastname: Nugent
authors.forename: Norio
authors.initials: N
authors.lastname: Komatsu
authors.forename: Masaaki
authors.initials: M
authors.lastname: Moroi
authors.forename: Kenji
authors.initials: K
authors.lastname: Izuhara
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Kunicki
mesh_terms: D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D019175:DNA Methylation / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000235:genetics*; D013926:Thrombopoietin / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; D013926:Thrombopoietin