Involvement of the Serum Response Factor Coactivator Megakaryoblastic Leukemia (MKL) in the Activin-regulated Dendritic Complexity of Rat Cortical Neurons

Abstract Megakaryoblastic leukemia 1 (MKL1), identified as part of the t(1;22) translocation specific to acute megakaryoblastic leukemia, is highly expressed in differentiated muscle cells and promotes muscle differentiation by activating serum response factor (SRF). Here we show that Mkl1 expression is up-regulated during murine megakaryocytic differentiation and that enforced overexpression of MKL1 enhances megakaryocytic differentiation. When the human erythroleukemia (HEL) cell line is induced to differentiate with 12-O-tetradecanoylphorbol 13-acetate, overexpression of MKL1 results in an increased number of megakaryocytes with a concurrent increase in ploidy. MKL1 overexpression also promotes megakaryocytic differentiation of primary human CD34+ cells cultured in the presence of thrombopoietin. The effect of MKL1 is abrogated when SRF is knocked down, suggesting that MKL1 acts through SRF. Consistent with these findings in human cells, knockout of Mkl1 in mice leads to reduced platelet counts in peripheral blood, and reduced ploidy in bone marrow megakaryocytes. In conclusion, MKL1 promotes physiologic maturation of human and murine megakaryocytes.

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MKL1 Promotes Megakaryocytic Differentiation Via Stimulation of Serum Response Factor Target Genes.

Blood ◽

10.1182/blood.v110.11.871.871 ◽

2007 ◽

Vol 110 (11) ◽

pp. 871-871 ◽

Cited By ~ 1

Author(s):

Ee-chun Cheng ◽

Matthew J. Renda ◽

Lin Wang ◽

Diane S. Krause

Keyword(s):

Target Genes ◽

Serum Response Factor ◽

Critical Role ◽

Cytoskeletal Proteins ◽

Chromosome 1 ◽

Platelet Glycoprotein ◽

Response Factor ◽

Megakaryoblastic Leukemia ◽

Average Percentage ◽

Serum Response

Abstract Our studies demonstrate a critical role for MKL1 (megakaryoblastic leukemia 1) in the molecular regulation of megakaryocytopoiesis. MKL1 is part of the fusion protein formed by the t (1; 22) translocation, which is found uniquely in Acute Megakaryoblastic Leukemia (AMKL). The translocation fuses the RBM15 (also known as OTT) gene on chromosome 1 with the MKL1 (also known as MAL) gene on chromosome 22. Previous studies in muscle cells show that MKL1 is a positive cofactor for the transcription factor serum response factor (SRF), and works via the Rho-A pathway to turn on immediate early genes and muscle specific genes. Using qRT-PCR we show that MKL1 mRNA is markedly up-regulated during megakaryocyte (MK) differentiation of primary murine bone marrow and fetal liver cells. When we overexpress MKL1 in the human erythroleukemia (HEL) cell line and differentiate the cells to become MK by phorbol ester (TPA), there is far greater MK differentiation than in control HEL cells. Via analysis of Wright-Geimsa stained cytospins, MKL1 overexpression increases the average percentage of mature MK from 26% to 48%. Using flow cytometry, platelet glycoprotein V positive cells increase from 14% to 43% on average. The percentage of cells with greater than 4N ploidy increases from 13% to 34%. In order to assess the mechanisms by which MKL1 promotes MK differentiation, we tested whether SRF expression was required for the effects of MKL1. The stimulatory effects of MKL1 are strongly abrogated when cells are transfected with siRNA against SRF, proving that MKL1 acts via SRF to stimulate MK differentiation. Interestingly, SRF siRNA also causes a statistically significant decrease in ploidy in control cells stimulated with TPA. By microarray analyses using both Affymetrix and Illumina platforms, enforced MKL1 expression upregulates many cytoskeletal genes and adhesion molecules, enhances the expression of platelet specific genes such as glycoprotein V (consistent with the FACS data), and accelerates the loss of expression of genes associated with erythropoiesis, such as erythrocyte membrane protein band 4.2. These data indicate that MKL1 enhances MK differentiation by promoting endoduplication as well as increasing expression of platelet-specific genes and of multiple cytoskeletal proteins required for proplatelet formation.

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Megakaryoblastic Leukemia 1, a Potent Transcriptional Coactivator for Serum Response Factor (SRF), Is Required for Serum Induction of SRF Target Genes

Molecular and Cellular Biology ◽

10.1128/mcb.23.18.6597-6608.2003 ◽

2003 ◽

Vol 23 (18) ◽

pp. 6597-6608 ◽

Cited By ~ 207

Author(s):

Bo Cen ◽

Ahalya Selvaraj ◽

Rebecca C. Burgess ◽

Johann K. Hitzler ◽

Zhigui Ma ◽

...

Keyword(s):

Target Genes ◽

Serum Response Factor ◽

Gene Activation ◽

Reporter Genes ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Related Factor ◽

Response Factor ◽

Megakaryoblastic Leukemia ◽

Serum Response

ABSTRACT Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related transcription factor that we found strongly activated serum response element (SRE)-dependent reporter genes through its direct binding to serum response factor (SRF). The c-fos SRE is regulated by mitogen-activated protein kinase phosphorylation of ternary complex factor (TCF) but is also regulated by a RhoA-dependent pathway. The mechanism of this pathway is unclear. Since MKL1 (also known as MAL, BSAC, and MRTF-A) is broadly expressed, we assessed its role in serum induction of c-fos and other SRE-regulated genes with a dominant negative MKL1 mutant (DN-MKL1) and RNA interference (RNAi). We found that DN-MKL1 and RNAi specifically blocked SRE-dependent reporter gene activation by serum and RhoA. Complete inhibition by RNAi required the additional inhibition of the related factor MKL2 (MRTF-B), showing the redundancy of these factors. DN-MKL1 reduced the late stage of serum induction of endogenous c-fos expression, suggesting that the TCF- and RhoA-dependent pathways contribute to temporally distinct phases of c-fos expression. Furthermore, serum induction of two TCF-independent SRE target genes, SRF and vinculin, was nearly completely blocked by DN-MKL1. Finally, the RBM15-MKL1 fusion protein formed by the t(1;22) translocation of acute megakaryoblastic leukemia had a markedly increased ability to activate SRE reporter genes, suggesting that its activation of SRF target genes may contribute to leukemogenesis.

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Serum response factor is an essential transcription factor in megakaryocytic maturation

Blood ◽

10.1182/blood-2010-01-261743 ◽

2010 ◽

Vol 116 (11) ◽

pp. 1942-1950 ◽

Cited By ~ 23

Author(s):

Stephanie Halene ◽

Yuan Gao ◽

Katherine Hahn ◽

Stephanie Massaro ◽

Joseph E. Italiano ◽

...

Keyword(s):

Transcription Factor ◽

Serum Response Factor ◽

Critical Role ◽

Response Factor ◽

Megakaryoblastic Leukemia ◽

Serum Response ◽

Essential Transcription Factor

Abstract Serum response factor (Srf) is a MADS–box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1, a cofactor of Srf, is part of the t(1;22) translocation in acute megakaryoblastic leukemia, and plays a critical role in megakaryopoiesis. To test the role of Srf in megakaryocyte development, we crossed Pf4-Cre mice, which express Cre recombinase in cells committed to the megakaryocytic lineage, to SrfF/F mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/SrfF/F knockout (KO) mice are born with normal Mendelian frequency, but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast, the BM has increased number and percentage of CD41+ megakaryocytes (WT: 0.41% ± 0.06%; KO: 1.92% ± 0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation, and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are down-regulated in KO megakaryocytes. Thus, Srf is required for normal megakaryocyte maturation and platelet production partly because of regulation of cytoskeletal genes.

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