RUNX1/core binding factor A2 regulates platelet 12-lipoxygenase gene (ALOX12): studies in human RUNX1 haplodeficiency

Abstract

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...

Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation and platelet formation and function

“…56,57 ALOX12, a RUNX1 target gene, 58 is severely reduced in dKO megakaryocytes and platelets. ALOX12 and F13A1 are both downregulated in patients bearing CBFA2/RUNX1 mutations and presenting macrothrombocytopenia.…”

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

“…on May 12, 2018. by guest www.bloodjournal.org From is complex, arising from multiple mechanisms mediated by alterations in distinct gene products regulated by RUNX1 in megakaryocytes/ platelets. We have shown that these proteins include 12-lipoxygenase, 45 PKC-, 44 and PF4. 19 Promoters of several genes regulated by RUNX1 have multiple consensus sites for RUNX1 binding, [46][47][48][49] including GPIIb 46 and ALOX-12 45 in megakaryocytic cells.…”

Mechanisms underlying platelet function defect in a pedigree with familial platelet disorder with a predisposition to acute myelogenous leukemia: potential role for candidate RUNX1 targets

“…RUNX1 targets involved in decreased platelet production include the genes for the MPL receptor (MPL) [11], myosin IIA (MYH9), myosin IIB (MYH10), and myosin regulatory light chain (MYL9) [10]. Besides MYL9, other RUNX1 target genes with a role in platelet function have been found, by platelet expression profiling, to be downregulated in one FPD/AML patient; these included the genes for platelet 12-lipoxygenase (ALOX12) and protein kinase C-h (PKCh) [12][13][14][15][16].…”