Structure-function analysis of the role of megakaryoblastic leukemia 1 in megakaryocyte polyploidization

Megakaryoblastic leukemia 1 (MKL1; also known as MRTFA, MAL, or BSAC) is a coactivator of serum response factor (SRF). SRF is a transcription factor that participates in the activation of immediate-early genes as well as genes associated with the cytoskeleton, proliferation, and apoptosis by binding promoter sequences known as serum response elements (SRE). 1 The MKL1/SRF complex plays a critical role in megakaryocyte maturation.2,3 As megakaryocytes mature, they undergo successive rounds of endomitosis to become highly polyploid; Mkl1 knockout (KO) murine megakaryocytes exhibit reduced polyploidization, a phenotype that is more severe in double KO (dKO) megakaryocytes lacking both Mkl1 and the closely related Megakaryoblastic leukemia 2 (Mkl2, or Mrtfb). 3,4 Additionally, MKL1 is part of the recurrent t(1;22) chromosomal translocation found in acute megakaryoblastic leukemia, which results in a fusion protein known as RBM15-MKL1.5 MKL1 and MKL2 each contain five highly conserved domains which have been investigated in the context of SRF coactivation using luciferase reporter assays.1,3,6,7 However, their functional effects in megakaryocyte polyploidization remain unknown, so structure-function analysis of these domains in primary megakaryocyte polyploidization is needed.7,8 Furthermore, the pro-tumorigenic mechanism of RBM15-MKL1 remains unclear, warranting further investigation of the individual proteins’ roles in both normal and pathological megakaryocyte development – particularly considering that all functional domains of MKL1 are retained in the fusion protein. In order to define which domains of MKL1 are necessary for polyploidization, we transduced wild-type (WT), Mkl1 KO and dKO murine bone marrow with domain-specific deletion constructs of MKL1, and analyzed subsequent megakaryocyte polyploidization. Published data have established that Mkl1 KO and dKO megakaryocytes have reduced polyploidization relative to WT cells in vivo. We expanded upon this by demonstrating reduced polyploidization of megakaryocytes differentiated in culture from hematopoietic stem and progenitor cells (HSPC). 2,3,7 Murine bone marrow-derived HSPC were cultured for 48 hours in expansion medium (50 ng/mL murine Flt3, 20 ng/mL murine interleukin-3, 100 ng/mL murine stem cell factor, 50 ng/mL murine thrombopoietin in StemSpan [Stemcell Technologies]), then switched into megakaryocyte maturation medium (50 ng/mL murine thrombopoietin) for 4 days. Polyploidization was analyzed via flow cytometry using propidium iodide to visualize DNA content, and anti-CD41 to identify megakaryocytes. In order to capture the full spectrum of polyploidization, the mean fluorescence intensity (MFI) of propidium iodide was determined for each population. MFI was normalized to the WT value within each experiment to account for variations in fluorescence intensity between experiments and cytometers. Mkl1 KO and dKO megakaryocytes showed decreased MFI relative to WT, indicating a lower degree of polyploidization.2,3,7 This decrease was statistically significant for dKO cells (P=0.01, n=4). Mkl1 KO megakaryocytes exhibited a directional decrease in MFI which did not reach statistical significance (P=0.05, n=6) (Figure 1A). To test whether restoration of Mkl1 expression restores polyploidization, we overexpressed full-length (FL) MKL1 in WT, Mkl1 KO, and dKO megakaryocytes. We cloned FL MKL1 into a pMSCV-GW-RfA-PGK-EGFP retroviral backbone using vectors VB160922-1087ttu and VB1609221086xsa (VectorBuilder). Constructs were validated in HEL cells via western blot (Online Supplementary Figure S1A). Murine HSPC were cultured according to the above protocol, and transduced after 24 hours in expansion medium with 8 μg/mL polybrene via spinfection for 1 hour at 800 x g. The transduced megakaryocyte population was gated based on CD41/GFP fluorescence. Representative ploidy histograms are shown in Figure 1C and indicate that overexpression of FL MKL1 increases the number of high ploidy cells. Overexpression of MKL1 resulted in a statistically significant increase in polyploidization (as represented by the MFI of propidium iodide) of cells from each genotype (WT n=5, Mkl1 KO n=6, and dKO n=4), compared to empty vector controls (Figure 1B). Previous work has defined the roles of each domain in MKL1-mediated regulation of SRF in luciferase reporter assays.9 Two canonical N-terminal RPEL domains serve as negative regulators of MKL1/SRF by binding to G-actin, which promotes nuclear export;8,9,10 the Basic domain contains a nuclear localization signal and promotes dimerization of MKL1 with SRF;9,11 the LZ domain permits MKL1 heteroand homodimerization;1 and the C-terminal TAD domain is required for transcriptional activation of MKL1/SRF target genes.1 The SAP domain does not affect MKL1-induced luciferase reporter activity and has no clear role in MKL1 function.11 While SRF-independent functions have been reported for the SAP domain of MKL1, these findings have not been reproduced.12 We therefore tested