RUNX1/core binding factor A2 regulates platelet 12-lipoxygenase gene (ALOX12): studies in human RUNX1 haplodeficiency.
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 agonist-induced 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.
Kaur
Gurpreet
G
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Jalagadugula
Gauthami
G
Mao
Guangfen
G
Rao
A Koneti
AK
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000096:biosynthesis; D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics* / Q000378:metabolism; D016718:Arachidonic Acid / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D016384:Consensus Sequence; D050676:Core Binding Factor Alpha 2 Subunit / Q000172:deficiency* / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D006238:Haploidy; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D015539:Platelet Activation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D050676:Core Binding Factor Alpha 2 Subunit; D034741:RNA, Small Interfering; D016718:Arachidonic Acid; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001092:Arachidonate 12-Lipoxygenase; D008156:Luciferases; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate