Abstract
Pediatric non-Down Syndrome acute megakaryoblastic leukemia (AMKL), a disease where megakaryocyte (MK) maturation is blocked, has a very poor prognosis. In 13% of AMKL cases of this type, the transcriptional co-factor MRTFA is expressed as part of a fusion protein. Normally, MRTFA levels increase in hematopoietic cells during megakaryopoiesis, making it likely that expression of MRTFA as part of a fusion protein leads to aberrant gene regulation, which results in leukemia. However, the mechanism by which megakaryopoiesis is blocked in AMKL is unknown. Therefore, we sought to parse out the role of MRTFA in normal megakaryopoiesis, so that we could better understand the cause for maturation block in leukemia.
MRTFA is a co-factor of serum response factor (SRF). Knockout of either SRF or MRTFA in mice decreases MK maturation causing thrombocytopenia; and MRTFA overexpression (MRTFAOE) promotes MK maturation of primary human bone marrow (BM) cells in vitro. Our novel study shows that genomic regulation by MRTFA promotes MK maturation. In the human erythroleukemia (HEL) cell line, MRTFAOE enhances phorbol ester (TPA)-induced megakaryopoiesis, mimicking the effects of MRTFA on primary MK maturation. TPA-induced HEL cells with MRTFAOE achieve significantly higher 8N and 16N ploidy (p < 0.001, N = 4), compared to those without MRTFAOE. To identify the mechanisms underlying megakaryocytic differentiation, we bioinformatically analyzed anti-SRF chromatin immunoprecipitation (ChIP)-sequencing [approx. 15 million reads/sample] and RNA-sequencing data [approx. 20 million reads/sample], from non-induced and TPA-induced HEL cells, with and without MRTFAOE (N = 2/condition). We identified SRF peaks that change during TPA-induction, with and without MRTFAOE, and analyzed the strength of their binding and motif status. MRTFOE not only increased SRF binding to the genome during MK maturation (p ≤ 10-8), but preferentially retained binding at genomic CArG (CC[W]6GG) motifs, where SRF binds to in association with either MRTFA or specific ETS proteins (ELK1, ELK4). We then analyzed upregulated and downregulated genes during TPA-induction, with and without MRTFAOE, and identified those that had associated SRF peaks. As expected, TPA-induction upregulated megakaryocytic and cytoskeletal genes (VWF, ACTN1, CORO1A) and downregulated erythroid genes (KLF1, GYPB, GYPE). Interestingly, TPA-induction with MRTFAOE increased the number of upregulated genes by 27% and the number of downregulated genes by 10%. In TPA-induced cells, MRTFAOE increased the percentage of differentially expressed genes that had SRF peaks (25% versus 11% for upregulated genes, and 9% versus 4% for downregulated genes), further highlighting the direct role that MRTFA plays in MK maturation. Also, genes upregulated by TPA-induction alone have both ETS and CArG motifs, whereas those upregulated by TPA-induction with MRTFAOE lack ETS binding motifs, suggesting that MRTFAOE skews SRF binding toward CArG motifs. With anti-MRTFA ChIP-PCR in HEL cells, we confirmed the novel finding that along with SRF, MRTFA binds to regions associated with megakaryocytic and cytoskeletal genes (XRK6, CORO1A). Therefore, SRF and MRTFA together regulate expression of genes that are important for normal megakaryopoiesis, which explains why lack of these proteins adversely affects megakaryopoiesis in mice.
We asked whether our findings in HEL cells were applicable to the more clinically relevant primary human BM cells. Anti-SRF and anti-MRTFA ChIP-PCR on day 0 primary human cells (CD34+ cells) and day 8 differentiated cell subpopulations (CD41+CD42-, CD41+CD42+) confirmed that both SRF and MRTFA have increased binding during megakaryopoiesis at target sites associated with upregulated genes, such as CORO1A, TNS1, and XRK6 (p < 0.001). Therefore, we illustrated that transcriptional regulation by SRF/MRTFA function similarly in human BM cells undergoing megakaryopoiesis.
We show for the first time that MRTFA increases both the genomic association and activity of SRF, and upregulates genes that enhance primary human megakaryopoiesis. These findings suggest that aberrant expression of MRTFA as a fusion protein in AMKL may disrupt essential transcriptional regulation via the SRF/MRTFA axis, resulting in blocked MK maturation. This forms a crucial reference point for future studies to understand the altered SRF/MRTFA function in AMKL.
Disclosures
No relevant conflicts of interest to declare.
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