Megakaryoblastic Leukemia-1/2, a Transcriptional Co-activator of Serum Response Factor, Is Required for Skeletal Myogenic Differentiation

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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Lysine demethylase, KDM2B, demethylates serum response factor K165 to inhibit myogenic differentiation

Proceedings for Annual Meeting of The Japanese Pharmacological Society ◽

10.1254/jpssuppl.92.0_jkp-06 ◽

2019 ◽

Vol 92 (0) ◽

pp. JKP-06

Author(s):

Hosouk Joung ◽

Ju Young Kang ◽

Sang Beom Seo ◽

Hyun Kook

Keyword(s):

Serum Response Factor ◽

Myogenic Differentiation ◽

Response Factor ◽

Lysine Demethylase ◽

Serum Response

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Serum response factor p67SRF is expressed and required during myogenic differentiation of both mouse C2 and rat L6 muscle cell lines.

The Journal of Cell Biology ◽

10.1083/jcb.118.6.1489 ◽

1992 ◽

Vol 118 (6) ◽

pp. 1489-1500 ◽

Cited By ~ 64

Author(s):

M Vandromme ◽

C Gauthier-Rouvière ◽

G Carnac ◽

N Lamb ◽

A Fernandez

Keyword(s):

Skeletal Muscle ◽

Cell Lines ◽

Muscle Cell ◽

Serum Response Factor ◽

Troponin T ◽

Myogenic Differentiation ◽

Muscle Differentiation ◽

Response Factor ◽

Serum Response

The 67-kD serum response factor (p67SRF) is a ubiquitous nuclear transcription factor that acts by direct binding to a consensus DNA sequence, the serum response element (SRE), present in the promoter region of numerous genes. Although p67SRF was initially implicated in the activation of mitogen-stimulated genes, the identification of a sequence similar to SRE, the CArG box motif, competent to interact with SRE binding factors in many muscle-specific genes, has led to speculation that, in addition to its function in cell proliferation, p67SRF may play a role in muscle differentiation. Indirect immunofluorescence using affinity-purified antibodies specifically directed against p67SRF reveals that this factor is constitutively expressed and localized in the nucleus of two skeletal muscle cell lines: rat L6 and mouse C2 myogenic cells during myogenic differentiation. This result was further confirmed through immunoblotting and Northern blot analysis. Furthermore, specific inhibition of p67SRF in vivo through microinjection of purified p67SRF antibodies prevented the myoblast-myotube transition and the expression of muscle-specific genes such as the protein troponin T. We further showed that anti-p67SRF injection also inhibited the expression of the myogenic factor myogenin, implying an early requirement for p67SRF in muscle differentiation. These results demonstrate that p67SRF is involved in the process of skeletal muscle differentiation. The potential action of p67SRF via CArG sequences is discussed.

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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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Growth and differentiation of C2 myogenic cells are dependent on serum response factor.

Molecular and Cellular Biology ◽

10.1128/mcb.16.11.6065 ◽

1996 ◽

Vol 16 (11) ◽

pp. 6065-6074 ◽

Cited By ~ 69

Author(s):

M Soulez ◽

C G Rouviere ◽

P Chafey ◽

D Hentzen ◽

M Vandromme ◽

...

Keyword(s):

Myosin Heavy Chain ◽

Heavy Chain ◽

Antisense Rna ◽

Serum Response Factor ◽

Myogenic Differentiation ◽

Receptor Expression ◽

Rna Expression ◽

Response Factor ◽

Myogenic Cells ◽

Serum Response

In order to study to what extent and at which stage serum response factor (SRF) is indispensable for myogenesis, we stably transfected C2 myogenic cells with, successively, a glucocorticoid receptor expression vector and a construct allowing for the expression of an SRF antisense RNA under the direction of the mouse mammary tumor virus long terminal repeat. In the clones obtained, SRF synthesis is reversibly down-regulated by induction of SRF antisense RNA expression by dexamethasone, whose effect is antagonized by the anti-hormone RU486. Two kinds of proliferation and differentiation patterns have been obtained in the resulting clones. Some clones with a high level of constitutive SRF antisense RNA expression are unable to differentiate into myotubes; their growth can be blocked by further induction of SRF antisense RNA expression by dexamethasone. Other clones are able to differentiate and are able to synthesize SRF, MyoD, myogenin, and myosin heavy chain at confluency. When SRF antisense RNA expression is induced in proliferating myoblasts by dexamethasone treatment, cell growth is blocked and cyclin A concentration drops. When SRF antisense RNA synthesis is induced in arrested confluent myoblasts cultured in a differentiation medium, cell fusion is blocked and synthesis of not only SRF but also MyoD, myogenin, and myosin heavy chain is inhibited. Our results show, therefore, that SRF synthesis is indispensable for both myoblast proliferation and myogenic differentiation.

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