Core binding factor 2 (CBFA2), also known as AML1 and RUNX1, is a transcription factor that regulates the expression of genes involved in hematopoiesis, through highly conserved DNA binding region, called RUNT homology domain (RHD). We have previously reported a patient with a mutation (haplodeficiency) in the conserved region of RUNX1/CBFA2 associated with mild thrombocytopenia and impaired platelet function. Expression profiling of patient platelets revealed ∼5 fold decreased mRNA expression of 12-lipoxygenase (12-LO, gene ALOX12) (
Sun L. et al. J Thromb Haemost, 5:146–154, 2006
). 12-LO catalyzes 12-hydroxyeicosatetraenoic acid (12-HETE) production from arachidonic acid (AA) upon platelet activation. We have performed studies to determine whether ALOX-12 is regulated by CBFA2. We studied 12-HETE production in patient platelets and the regulation of ALOX-12 by CBFA2 in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA) to induce megakaryocytic (MK) transformation. 12-HETE production was decreased in patient platelets in response to 10 U/ml of thrombin (19.5 ng/10 8 platelets, normal subjects: range 29–306, n=9) and 100 μM of arachidonic acid (5.4, normal subjects: 67– 442, n=10). Three CBFA2 consensus binding sites were identified (−1498/−1493, −708/−70, −526/−521 from ATG) by computer analysis within 2 kb of ALOX-12 5′ upstream region. The binding site at −1489 is a 13nt palindromic sequence with two CBFA2 motifs. The other two CBFA2 binding sites at −708 and −526 bp overlap AP2 binding sites. Luciferase reporter studies in HEL cells using a construct carrying ∼ 1600 bp of 5′ upstream region indicate a greater than 10 fold increase in activity in PMA treated HEL cells relative to untreated cells. Truncation at − 873 bp in PMA-treated HEL cells resulted in a ∼10 fold decrease in activity with only minimal decreases with truncations at −705 or −438 to delete the other CBFA2 binding sites. Mutation of each of the putative CBFA2 binding sites individually resulted in 5–10 fold decrease in activity. Gel shift studies using a 30-mer probe (−1507/−1478) and PMA-treated HEL extracts revealed specific protein binding that was eliminated by CBFA2 antibody and by mutating CBFA2 site from TGGGGT to TGCATT. Specific protein binding was observed with probes containing putative CBFA2 sites at −708 and −526, but it was not altered by the antibody. Chromatin immunoprecipitation (ChIP) analysis using HEL cells demonstrated (PCR amplification) in vivo binding of CBFA2 to ALOX-12 promoter in the region −1507/−1478 but not the other two sites.Conclusions: CBFA2 haplodeficiency is associated with decreased platelet 12-HETE production and 12-LO activity. ALOX-12 is regulated by CBFA2 in megakaryocytes/platelets. These findings are important because of the role of 12-lipoxygenase in platelet arachidonate metabolism and function.
