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

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 871-871 ◽  
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.

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

2003 ◽  
Vol 23 (18) ◽  
pp. 6597-6608 ◽  
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.

scholarly journals Serum response factor is an essential transcription factor in megakaryocytic maturation

Blood ◽  
2010 ◽  
Vol 116 (11) ◽  
pp. 1942-1950 ◽  
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.

scholarly journals Critical Role of Serum Response Factor in Pulmonary Myofibroblast Differentiation Induced by TGF-β

2009 ◽  
Vol 41 (3) ◽  
pp. 332-338 ◽  
Author(s):  
Nathan Sandbo ◽  
Steven Kregel ◽  
Sebastien Taurin ◽  
Sangeeta Bhorade ◽  
Nickolai O. Dulin
Keyword(s):  
Serum Response Factor ◽  
Critical Role ◽  
Myofibroblast Differentiation ◽  
Response Factor ◽  
Serum Response

scholarly journals The Cytoskeleton-associated PDZ-LIM Protein, ALP, Acts on Serum Response Factor Activity to Regulate Muscle Differentiation

2007 ◽  
Vol 18 (5) ◽  
pp. 1723-1733 ◽  
Author(s):  
Pascal Pomiès ◽  
Mohammad Pashmforoush ◽  
Cristina Vegezzi ◽  
Kenneth R. Chien ◽  
Charles Auffray ◽  
...  
Keyword(s):  
Serum Response Factor ◽  
Critical Role ◽  
Muscle Differentiation ◽  
Actin Dynamics ◽  
Response Factor ◽  
Cytoskeletal Regulation ◽  
Expression Construct ◽  
Filament Bundles ◽  
Serum Response

In this report, an antisense RNA strategy has allowed us to show that disruption of ALP expression affects the expression of the muscle transcription factors myogenin and MyoD, resulting in the inhibition of muscle differentiation. Introduction of a MyoD expression construct into ALP-antisense cells is sufficient to restore the capacity of the cells to differentiate, illustrating that ALP function occurs upstream of MyoD. It is known that MyoD is under the control of serum response factor (SRF), a transcriptional regulator whose activity is modulated by actin dynamics. A dramatic reduction of actin filament bundles is observed in ALP-antisense cells and treatment of these cells with the actin-stabilizing drug jasplakinolide stimulates SRF activity and restores the capacity of the cells to differentiate. Furthermore, we show that modulation of ALP expression influences SRF activity, the level of its coactivator, MAL, and muscle differentiation. Collectively, these results suggest a critical role of ALP on muscle differentiation, likely via cytoskeletal regulation of SRF.

scholarly journals RAGE‐DIAPH1 modulates hyperglycemia driven induction of Serum response factor target genes in cardiac cells

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Gopalkrishna Sreejit ◽  
Nosirudeen Quadri ◽  
Radha Ananthakrishnan ◽  
Ann Marie Schmidt ◽  
Ravichandran Ramasamy
Keyword(s):  
Target Genes ◽  
Serum Response Factor ◽  
Cardiac Cells ◽  
Response Factor ◽  
Serum Response

2007 Russell Ross Memorial Lectureship in Vascular Biology—Epigenetic Control of Vascular Smooth Muscle Differentiation in Development and Disease

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Gary K. Owens
Keyword(s):  
Smooth Muscle ◽  
Vascular Injury ◽  
Serum Response Factor ◽  
Critical Role ◽  
Conditional Knockout ◽  
Phenotypic Switching ◽  
Marker Genes ◽  
List Type ◽  
Response Factor ◽  
Serum Response

There is clear evidence that alterations in the differentiated state of the smooth muscle cell (SMC) play a key role in the pathogenesis of a number of major human diseases, including atherosclerosis and postan-gioplasty restenosis. This process is referred to as “phenotypic switching” and likely evolved to promote repair of vascular injury. However, the mechanisms controlling phenotypic switching as well as normal differentiation of SMCs in vivo are poorly understood. This talk will provide an overview of molecular mechanisms that control differentiation of SMCs during vascular development. A particular focus will be to consider the role of CArG elements found within the promoters of many SMC differentiation marker genes, as well as regulation of their activity by serum response factor and the potent SMC-selective serum response factor coactivator myocardin. In addition, I will summarize recent work in our laboratory showing that SMC- and gene-locus–selective changes in chromatin structure play a critical role both in normal control of SMC differentiation and in phenotypic switching in response to vascular injury. Finally, I will present evidence based on conditional knockout experiments in mice showing that krupple-like factor 4 is induced in SMCs after vascular injury and regulates SMC phenotypic switching and growth through: binding to G/C repressor elements located in close proximity of CArG elements within the promoters of many SMC marker genes, suppressing expression of myocardin, and inducing epigenetic modifications of SMC marker gene loci associated with chromatin condensation and transcriptional silencing. Supported by NIH grants P01 HL19242, R37 HL57353, and R01 HL 38854.

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