Serum Response Factor Is An Essential Transcription Factor in Megakaryocytic Maturation.

Abstract

Abstract 3652Poster Board III-588SRF is a MADS-box transcription factor first identified as an inducer of immediate-early gene expression, such as c-fos, Junb, Fosb and Egr1, in response to cytokines. It plays a critical role in cardiac and smooth muscle development and differentiation by regulating cell cycle, apoptosis, cell growth, and differentiation. SRF regulates expression of contractile and cytoskeletal genes. Its function in hematopoiesis has not yet been revealed. While no hematopoietic disease specific mutations of SRF have been identified, the region on chromosome 6 where the SRF gene is located, is a site of frequent chromosomal aberrations in MDS. MKL1, a target of the t(1;22) translocation in acute megakaryoblastic leukemia, is a cofactor for SRF mediated gene activation. In published work (Cheng EC et al., Blood 2009;113:2826), we showed that MKL1 expression increases with normal megakaryocytic (Mk) differentiation, that it promotes Mk differentiation by increasing the expression of Mk specific genes such as CD42b, and that mice lacking MKL1 have decreased platelet counts while showing increased numbers of low ploidy Mk in the BM. In addition, we showed that the effects of MKL1 on Mk differentiation require SRF. In order to test the role of SRF in Mk development, we crossed PF4-Cre mice (Tiedt R et al., Blood 2007;109:1503), which express Cre recombinase in cells committed to the Mk lineage, to SRFF/F mice (Miano JM et al., PNAS 2004;101:17132) in which functional SRF is no longer expressed after Cre-mediated excision. Our findings are quite surprising, as knockout of SRF in the Mk lineage leads to a far more profound phenotype than MKL1 KO. PF4-Cre x SRFF/F (PF4-Cre/SRF) mice are born with a normal mendelian frequency, but have significant macrothrombocytopenia. The platelet counts in WT and KO mice are 703 ± 33 × 103/μl vs 460 ± 23 × 103/μl, respectively. Despite the decreased platelet number, the BM has increased numbers and percentages of CD41+ Mk (WT: 0.41 ± 0.06% and KO: 1.92 ± 0.12%) with a predominance of Mk with hypolobated nuclei. Spleens of PF4-Cre/SRF mice show significantly increased numbers of Mk (mean of 4 and 15 Mk per high power field for WT and KO spleens, respectively). Ploidy as an assay of Mk maturation in the BM is significantly reduced in SRF −/− Mk. The percentage of CD41+ cells in SRF KO BM that is 2N is nearly 2-fold higher, and the percentage with greater than 8N ploidy is decreased by 70%. Hematopoietic stem and progenitor populations in PF4-Cre/SRF BM are unaffected, as expected due to stage and lineage specific knockout of SRF. In contrast, there is an increase in Mk progenitors in vivo in PF4-Cre/SRF mice, reflected both by FACS analysis and CFU-Mk in vitro analysis. The mechanism by which SRF disrupts Mk maturation was studied by assessment of Mk morphology, and gene expression analysis. Mk lacking SRF expression have abnormal stress fiber formation based on phalloidin staining, a phenotype parallel to that observed in mice lacking myosin heavy chain 9 (MYH9) expression, and MYH9 expression was decreased in SRF KO Mk. In summary, the data show that SRF is critical for normal Mk maturation including polyploidization and platelet production, and the mechanism by which loss of SRF disrupts megakaryocytopoiesis is, at least in part, by loss of normal expression of known targets of SRF, such as cytoskeletal genes including MYH9. Disclosures:No relevant conflicts of interest to declare.