VPB0365 - Differential Autoregulation of RUNX1 by Isoforms RUNX1 B and RUNX1 C in Megakaryocytic Cells

Background: Haplodeficiency of RUNX1, a key hematopoietic transcription factor, causes impaired megakaryocyte (MK) differentiation, thrombocytopenia, platelet dysfunction, and predisposition to AML. Two major RUNX1 isoforms RUNX1C and RUNX1B differ by 14 AA and are expressed from distinct promoters, P1 and P2, respectively. Little is known about the differential effects of RUNX1 isoforms.

Aims: We studied the effects of RUNX1B and RUNX1C on RUNX1 autoregulation.

Methods: Studies were performed on RUNX1 regulation in PMA-treated megakaryocytic HEL cells and in HeLa cells, which express negligible RUNX1.

Results: ChIP studies using HEL cells and PCR amplification showed RUNX1 binds to 5 consensus sites in P1 and one site in P2 promoters (1-1.5 Kb). In luciferase reporter promoter studies, mutating RUNX1 binding sites 1 and 2 in P1 promoter reduced activity; mutating sites 3 and 4 increased activity, indicating their functional effects. Mutating binding site in P2 promoter increased activity. In co-transfection studies in HeLa cells (with negligible RUNX1), increasing RUNX1C concentration increased P1 promoter activity and RUNX1B reduced promoter activity. With constant expression of RUNX1C, increasing RUNX1B reduced P1 promoter activity. Moreover, RUNX1B expression reduced P2 promoter activity, while RUNX1C increased this activity. Thus, RUNX1B and RUNX1C have differential autoregulatory effects on RUNX1 promoters. We then examined RUNX1 protein levels in HEL cells by immunoblotting. RUNX1B overexpression decreased (~50%) RUNX1C; RUNX1C overexpression increased RUNX1B (2-fold). In studies in HEL cells using RUNX1C-specific antibody, and expression plasmids for hemagglutinin-tagged-RUNX1B and c-Myc-tagged RUNX1C, RUNX1B overexpression decreased RUNX1C; RUNX1C overexpression increased RUNX1B. Preliminary studies suggest that the isoforms regulate MYL9 (RUNX1 target) expression differentially.

Conclusion(s): RUNX1B and RUNX1C autoregulate RUNX1 expression differentially in an isoform-specific manner in megakaryocytic cells. This may be important in understanding altered downstream gene expression and in modulating RUNX1 for therapeutic purposes in inherited RUNX1 haplodeficiency states. (Supported by NIH grants R01HL109568 and R01137376)