Regulation of platelet myosin light chain (MYL9) by RUNX1: implications for thrombocytopenia and platelet dysfunction in RUNX1 haplodeficiency

Abstract

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.

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

“…The arrest of endomitosis is linked to MK maturation and RUNX1 has a role in this process by regulating genes involved in platelet functions, including myosin light chain 9 (MYL9/MLC2) 22 24,25 suggesting that RUNX1 could also regulate the switch from mitosis to endomitosis by targeting MYH10. Using an in silico approach with the ChipMapper online software, we identified a putative conserved RUNX1 transcription factor-binding site in the MYH10 promoter region (Fig.…”

Myeloid neoplasms with germ line RUNX1 mutation

“…RUNX1 also directly regulates the expression of myosin light chain 9 (MYL9) in megakaryocytes. Thus, downregulation of MYL9 due to RUNX1 haploinsufficiency could affect abnormal platelet production and function [31]. Platelet storage pool deficiency and low levels of MPL receptor expression have also been reported [25,32].…”

Surface CD3 expression proceeds through both myosin regulatory light chain 9 (MYL9)-dependent and MYL9-independent pathways in Jurkat cells

“…It was recently suggested that the MYL9 gene is a direct target of RUNX1 and provides a mechanism for decreased platelet MYL9 expression, myosin light chain (MLC) phosphorylation, thrombocytopenia, and platelet dysfunction associated with RUNX1 mutations (Jalagadugula et al, 2010). However, the functions of MYL9 in blood cells have not yet been clarified.…”