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.
Haploinsufficiency of RUNX1 (also known as CBFA2/AML1) is associated with familial thrombocytopenia, platelet dysfunction, and predisposition to acute leukemia. We have reported on a patient with thrombocytopenia and impaired agonistinduced aggregation, secretion, and protein phosphorylation associated with a RUNX1 mutation. Expression profiling of platelets revealed approximately 5-fold decreased expression of 12-lipoxygenase (12-LO, gene ALOX12), which catalyzes 12-hydroxyeicosatetraenoic acid production from arachidonic acid. We hypothesized that ALOX12 is a direct transcriptional target gene of RUNX1. In present studies, agonist-induced platelet 12-HETE production was decreased in the patient. Four RUNX1 consensus sites were identified in the 2-kb promoter region of ALOX12 (at ؊1498, ؊1491, ؊708, ؊526 from ATG). In luciferase reporter studies in human erythroleukemia cells, mutation of each site decreased activity; overexpression of RUNX1 up-regulated promoter activity, which was abolished by mutation of RUNX1 sites. Gel shift studies, including with recombinant protein, revealed RUNX1 binding to each site. Chromatin immunoprecipitation revealed in vivo RUNX1 binding in the region of interest. siRNA knockdown of RUNX1 decreased RUNX1 and 12-LO proteins. ALOX12 is a direct transcriptional target of RUNX1. Our studies provide further proof of principle that platelet expression profiling can elucidate novel alterations in platelets with inherited dysfunction. (Blood. 2010;115(15):3128-3135) Introduction RUNX1, also known as CBFA2 (core binding factor A2), is a member of a family of transcription factors that regulate the expression of several hematopoietic-specific genes through a highly conserved DNA-binding region called the RUNT homology domain (RHD). 1 The RHD dimerizes with CBF to form a stable complex. The complex acts as an anchor to recruit other cofactors that bind in cis to adjacent sites or interact directly with RUNX1. RUNX1 plays a critical role in normal fetal hematopoiesis. 2,3 Homozygous deletion of RUNX1 results in embryonic lethality related to absence of definitive hematopoiesis. [2][3][4] In humans, haploinsufficiency of RUNX1 is associated with familial thrombocytopenia, platelet dysfunction, and predisposition to acute leukemia. 5 Most of the point mutations identified in RUNX1 occur in the RHD leading to loss of DNA binding. 6,7 We have previously reported 8,9 studies in a patient with mild thrombocytopenia, impaired agonist-induced platelet aggregation, secretion and protein phosphorylation (myosin light chain and pleckstrin), and decreased platelet protein kinase C-(PKC-), associated with a mutation (haplodeficiency) in the conserved region of RUNX1. Expression profiling of patient platelets revealed an approximately 5-fold decreased mRNA expression of platelet-type 12-lipoxygenase (12-LO, gene ALOX12). 10 Lipoxygenases are a family of non-heme iron-containing enzymes that catalyze the incorporation of molecular oxygen into polyunsaturated fatty acids, such as arachidonic acid (AA). Th...
Summary. Background: Platelet factor 4 (PF4) is an abundant protein stored in platelet a-granules. Several patients have been described with platelet PF4 deficiency, including the gray platelet syndrome, characterized by a deficiency of a-granule proteins. Defective granule formation and protein targeting are considered to be the predominant mechanisms. We have reported on a patient with thrombocytopenia and impaired platelet aggregation, secretion, and protein phosphorylation, associated with a mutation in the transcription factor RUNX1. Platelet expression profiling showed decreased transcript expression of PF4 and its non-allelic variant PF4V1. Objectives: To understand the mechanism leading to PF4 deficiency associated with RUNX1 haplodeficiency, we addressed the hypothesis that PF4 is a transcriptional target of RUNX1. Methods/results: Chromatin immunoprecipitation and gel-shift assays with phorbol 12-myristate 13-acetate-treated human erythroleukemia (HEL) cells revealed RUNX1 binding to RUNX1 consensus sites at )1774/)1769 and )157/)152 on the PF4 promoter. In luciferase reporter studies in HEL cells, mutation of each site markedly reduced activity. PF4 promoter activity and PF4 protein level were decreased by small interfering RNA RUNX1 knockdown and increased by RUNX1 overexpression. Conclusions: Our results provide the first evidence that PF4 is regulated by RUNX1 and that impaired transcriptional regulation leads to the PF4 deficiency associated with RUNX1 haplodeficiency. Because our patient had decreased platelet albumin and IgG (not synthesized by megakaryocytes) levels, we postulate additional defects in RUNX1-regulated genes involved in vesicular trafficking. These studies advance our understanding of the mechanisms in a-granule deficiency.
In the majority of patients with an inherited abnormality in platelet function and a bleeding diathesis, the underlying platelet molecular mechanisms are unknown. The usually considered entities, such as thrombasthenia, the Bernard-Soulier syndrome, and storage pool deficiency, occur in a small proportion of patients. A substantial number of patients present with decreased aggregation and secretion of dense granule contents upon activation, and are lumped in the category of primary secretion defects or platelet activation defects. Evidence is now available that defects in platelet signaling mechanisms may be the basis for the platelet dysfunction in some of these patients. This evidence is presented here. If the key components in signal transduction are the surface receptors, the G-proteins, and the effectors, evidence now exists for specific human platelet abnormalities at each of these levels. There is a pressing need for a concerted effort to delineate the molecular mechanisms in the large group of patients with impaired platelet function who represent an untapped reservoir of new information into normal platelet function.
Summary. Background: Gaq (Gene GNAQ) plays a major role in platelet signal transduction but little is known regarding its transcriptional regulation. Objectives: We studied Gaq promoter activity using luciferase reporter gene assays in human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. Methods and results: PMA-treated HEL cells showed enhanced Gaq expression. Reporter (luciferase) gene studies on 5¢ upstream construct (up to )116 bp from ATG) revealed a negative regulatory site at )238/)202 and two positive sites at )203/)138 and )1116/)731. The positive regulatory region )203/)138 contained overlapping Sp1/AP-2/ EGR-1 consensus sites. Gel shift studies on Gaq oligonucleotides 1 ()203/)175) and 2 ()174/)152) using HEL cell extracts demonstrated protein binding that was due to early growth response factor EGR-1 at two sites. Mutations in either EGR-1 site markedly decreased the gene activity, indicating functional relevance. Mutation of consensus E-Box motif ()185/)180) had no effect. Reduction in the expression of endogenous EGR-1 with antisense oligonucleotide to EGR-1 inhibited PMA-induced Gaq transcription. Correspondingly, Egr-1 deficient mouse platelets also showed 50% reduction in the Gaq expression relative to wild-type platelets. Conclusions: These studies suggest that Gaq gene is regulated during PMA-induced megakaryocytic differentiation by EGR-1, an early growth response transcription factor that regulates a wide array of genes and plays a major role in diverse activities, including cell proliferation, differentiation and apoptosis, and in vascular response to injury and atherosclerosis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.