Calpain Regulation of Megakaryopoiesis through a Positive Regulatory Loop Involving P-TEFb, GATA-1, and RUNX1.

Abstract 566

GATA-1 and RUNX1 cooperate in programming megakaryocytic development through the critical intermediation of the active P-TEFb kinase complex (Cdk9/cyclin T1). RUNX1 on its own traps P-TEFb in inactive chromatin loops and causes RNA pol II (RNAP II) stalling. GATA-1 by contrast remodels chromatin loops and promotes RNAP II elongation. Thus, P-TEFb most likely integrates and resolves conflicting signals from RUNX1 and GATA-1 to coordinate orderly activation of megakaryocytic target genes during development. P-TEFb activity is tightly regulated by a large network of interacting factors including Cdk9, cyclin T1, HEXIM1 and 7SK snRNA, RNA processing factors, and transcriptional regulators. During megakaryocytic differentiation, global activation of P-TEFb involves dissociation of HEXIM1 and recruitment of GATA-1, in a manner dependent on Cdk9 activity. The current studies address factors that regulate this dramatic reconfiguration of the P-TEFb complex during initiation of megakaryocytic differentiation. Candidate factors were identified based on two criteria: participation in the P-TEFb complex and specific upregulation in megakaryocytic differentiation. Notably, analysis of a P-TEFb interactome database identified the protease calpain 2 and its cofactor calpain S1 as participants in this complex (Jeronimo CD. Et. al. Mol Cell 2007). We confirmed a physical interaction by coimmunoprecipitation of endogenous calpain 2 and cyclin T1. Analysis of gene expression databases revealed three striking features of calpain 2: 1) strong upregulation early in megakaryocytic differentiation, 2) defective upregulation in GATA-1-deficient megakaryocytes, and 3) defective upregulation in megakaryocytes expressing GATA-1s, a mutant form associated with Down syndrome-associated megakaryocytic disorders (DS-TMD and DS-AMKL). The role of calpain in megakaryocytic differentiation of primary human CD34+ progenitors was assessed by shRNA knockdown (kd) of calpain S1, a required cofactor for calpains 1 and 2, as well as by treatment of cells with the calpain inhibitors calpeptin and Calpain Inhibitor III. All three approaches blocked cellular enlargement, CD41 upregulation, and polyploidization, indicating a critical role for calpain activity in early steps of megakaryocytic differentiation. We next addressed the hypothesis that calpain contributed to megakaryocytic differentiation through positive regulation of P-TEFb activity. In support of this hypothesis, calpain inhibition prevented the P-TEFb-driven processes of RNAP II hyperphosphorylation and HEXIM1 upregulation, both normally seen in megakaryocytic differentiation. In addition, calpain inhibition blocked the transcriptional cooperation of RUNX1 and GATA-1, which we have previously shown to be dependent on P-TEFb activity. How calpain activity regulates P-TEFb remains unclear, but in vitro and in vivo assays identified cyclin T1 and RNAP II as highly sensitive targets of calpain 2/S1 protease activity. Because P-TEFb remodeling in megakaryopoiesis requires Cdk9 kinase activity, we examined the possibility that calpain itself might be regulated by Cdk9, a notion supported by multiple experiments. In particular, Cdk9 inhibition by shRNA kd or flavopiridol treatment prevented the calpain-dependent cleavage of cyclin T1 and RNAP II normally seen in megakaryocytic differentiation. Furthermore, using purified, recombinant factors, in vitro calpain 2 cleavage of the RNAP II carboxy terminal domain (CTD) showed an absolute requirement for active P-TEFb complex. We thus postulate the existence of a novel regulatory circuit in which P-TEFb and calpain control the activity of one another during megakaryocytic differentiation. The participation of RUNX1 and GATA-1 in this circuit is suggested by the requirements for P-TEFb and calpain activity for their transcriptional cooperation. In addition, a murine strain with megakaryocytic GATA-1 deficiency, the GATA-1Lo strain, showed drops in platelet counts when treated with the calpain inhibitor E64, in contrast to to wild type counterparts, which responded with increased platelet counts. This novel regulatory circuit most likely has clinical relevance for at least two reasons: 1) P-TEFb inhibition in GATA-1Lo mice has been shown to elicit a disorder resembling the DS-TMD, and 2) megakaryocytes expressing GATA-1s display defective upregulation of calpains 2 and S1.