Phosphorylation of RUNX1 by Cyclin-Dependent Kinase Reduces Direct Interaction with HDAC1 and HDAC3 and Stimulates Marrow Progenitor Proliferation.

Abstract 2508

Poster Board II-485

RUNX1 helps direct the formation of definitive HSC during embryogenesis and the subsequent maturation of the lymphoid, myeloid, and megakaryocytic lineages in adults. RUNX1 regulates both lineage-specific genes and G1 to S cell cycle progression, the latter in part via induction of cyclin D3 and cdk4 transcription. Recent studies demonstrate that RUNX1 homologs in sea urchin blastocyts or in C. elegans seam cells, a stem cell subset, also stimulate cell proliferation. Of note, RUNX1 activities are commonly perturbed in human AML or ALL cases. RUNX1 residues S48, S303, and S424 fit the consensus, (S/T)PX(R/K), for phosphorylation by cyclin dependent kinases (cdk). Mutation of these residues to the phosphomimetic aspartic acid increases RUNX1 trans-activation, and mutation to alanine reduces trans-activation, without perturbing p300 interaction (Zhang et al 2008). We now demonstrate that cdk phosphorylation of RUNX1 reduces its interaction with HDAC1 and HDAC3. First, co-immunoprecipitation of FLAG-tagged HDAC1 or HDAC3 with myc-tagged RUNX1, RUNX1(tripleA), with S48, S303, and S424 mutated to alanine, or RUNX1(tripleD), with these three residues mutated to aspartic acid, was assessed using extracts from transiently transfected 293T cells. Both HDAC1 and HDAC3 bound RUNX1(tripleD) with greatly reduced affinity compared to RUNX1 or its tripleA variant. Consistent with previous mapping of HDAC1 and HDAC3 interaction to the C-terminal residues 380–432 of RUNX1, when this same assay was repeated using RUNX1(S48D), RUNX1(S303D), or RUNX1(S424D), carrying single residue mutations to aspartic acid, only S424D reduced HDAC1 binding, and S424D had a greater effect than S48D or S303D on HDAC3 binding. Second, when GST-RUNX1, GST-RUNX1(424A), or GST-RUNX1(424D) fusion proteins isolated from bacteria were incubated with in vitro translated, S35-methionine labeled HDAC1 or HDAC3, reduced interaction was again seen to the 424D variant. Finally, incubation of wild-type GST-RUNX1 with cdk1/cyclinB led to reduced interaction with radio-labeled HDAC1 or HDAC3, indicating that cdk phosphorylation and not just change of cdk target residues to aspartic acid reduces HDAC binding to RUNX1. In addition, endogenous HDAC1 in the Ba/F3 hematopoietic cell line co-immunoprecipitated with exogenous RUNX1, and we are attempting to optimize methods to detect interaction between the endogenous proteins. We previously provided data suggesting that RUNX1 cdk phosphorylation stimulates proliferation of the Ba/F3 hematopoietic cell line (Zhang et al 2008). We now find that RUNX1 or RUNX1(tripleD) reproducibly stimulate proliferation of transduced, lineage-negative murine marrow progenitors more potently than RUNX1(tripleA). Together our findings indicate that stimulation of RUNX1 trans-activation activity by cdk-mediated reduction in direct HDAC1 and HDAC3 interaction facilitates hematopoietic progenitor cell proliferation. Future studies will endeavor to determine whether activation of cdk in HSC or myeloid progenitors, for example by Notch or Wnt signaling, stimulates RUNX1 cdk phosphorylation to facilitate induction of cell cycle regulatory genes and cell proliferation. Conversely, reduced RUNX1 cdk phosphorylation may facilitate HSC quiescence.