Regulation of platelet myosin light chain (MYL9) by RUNX1: implications for thrombocytopenia and platelet dysfunction in RUNX1 haplodeficiency.
Mutations in transcription factor RUNX1 are associated with familial platelet disorder, thrombocytopenia, and predisposition to leukemia. We have described a patient with thrombocytopenia and impaired agonist-induced platelet aggregation, secretion, and glycoprotein (GP) IIb-IIIa activation, associated with a RUNX1 mutation. Platelet myosin light chain (MLC) phosphorylation and transcript levels of its gene MYL9 were decreased. Myosin IIA and MLC phosphorylation are important in platelet responses to activation and regulate thrombopoiesis by a negative regulatory effect on premature proplatelet formation. We addressed the hypothesis that MYL9 is a transcriptional target of RUNX1. Chromatin immunoprecipitation (ChIP) using megakaryocytic cells revealed RUNX1 binding to MYL9 promoter region -729/-542 basepairs (bp), which contains 4 RUNX1 sites. Electrophoretic mobility shift assay showed RUNX1 binding to each site. In transient ChIP assay, mutation of these sites abolished binding of RUNX1 to MYL9 promoter construct. In reporter gene assays, deletion of each RUNX1 site reduced activity. MYL9 expression was inhibited by RUNX1 short interfering RNA (siRNA) and enhanced by RUNX1 overexpression. RUNX1 siRNA decreased cell spreading on collagen and fibrinogen. Our results constitute the first evidence that the MYL9 gene is a direct target of RUNX1 and provide a mechanism for decreased platelet MYL9 expression, MLC phosphorylation, thrombocytopenia, and platelet dysfunction associated with RUNX1 mutations.
Jalagadugula
Gauthami
G
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Mao
Guangfen
G
Kaur
Gurpreet
G
Goldfinger
Lawrence E
LE
Dhanasekaran
Danny N
DN
Rao
A Koneti
AK
D001483:Base Sequence; D001791:Blood Platelet Disorders / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D001792:Blood Platelets / Q000378:metabolism*; D002478:Cells, Cultured; D050676:Core Binding Factor Alpha 2 Subunit / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000235:genetics* / Q000378:metabolism; D010974:Platelet Aggregation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D013921:Thrombocytopenia / Q000235:genetics* / Q000378:metabolism / Q000473:pathology
D050676:Core Binding Factor Alpha 2 Subunit; D018994:Myosin Light Chains; D034741:RNA, Small Interfering; C493728:RUNX1 protein, human