scholarly journals Sarcomeres regulate murine cardiomyocyte maturation through MRTF-SRF signaling

2020 ◽  
Vol 118 (2) ◽  
pp. e2008861118
Author(s):  
Yuxuan Guo ◽  
Yangpo Cao ◽  
Blake D. Jardin ◽  
Isha Sethi ◽  
Qing Ma ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Serum Response Factor ◽  
Mitochondrial Genes ◽  
Dominant Negative ◽  
Response Factor ◽  
Strongly Correlated ◽  
Transcriptional Dysregulation ◽  
Transcriptional Changes ◽  
Major Bottleneck ◽  
Serum Response

The paucity of knowledge about cardiomyocyte maturation is a major bottleneck in cardiac regenerative medicine. In development, cardiomyocyte maturation is characterized by orchestrated structural, transcriptional, and functional specializations that occur mainly at the perinatal stage. Sarcomeres are the key cytoskeletal structures that regulate the ultrastructural maturation of other organelles, but whether sarcomeres modulate the signal transduction pathways that are essential for cardiomyocyte maturation remains unclear. To address this question, here we generated mice with cardiomyocyte-specific, mosaic, and hypomorphic mutations of α-actinin-2 (Actn2) to study the cell-autonomous roles of sarcomeres in postnatal cardiomyocyte maturation. Actn2 mutation resulted in defective structural maturation of transverse-tubules and mitochondria. In addition, Actn2 mutation triggered transcriptional dysregulation, including abnormal expression of key sarcomeric and mitochondrial genes, and profound impairment of the normal progression of maturational gene expression. Mechanistically, the transcriptional changes in Actn2 mutant cardiomyocytes strongly correlated with those in cardiomyocytes deleted of serum response factor (SRF), a critical transcription factor that regulates cardiomyocyte maturation. Actn2 mutation increased the monomeric form of cardiac α-actin, which interacted with the SRF cofactor MRTFA and perturbed its nuclear localization. Overexpression of a dominant-negative MRTFA mutant was sufficient to recapitulate the morphological and transcriptional defects in Actn2 and Srf mutant cardiomyocytes. Together, these data indicate that Actn2-based sarcomere organization regulates structural and transcriptional maturation of cardiomyocytes through MRTF-SRF signaling.

Nuclear import of serum response factor (SRF) requires a short amino-terminal nuclear localization sequence and is independent of the casein kinase II phosphorylation site

1994 ◽  
Vol 107 (11) ◽  
pp. 3029-3036
Author(s):  
J. Rech ◽  
I. Barlat ◽  
J.L. Veyrune ◽  
A. Vie ◽  
J.M. Blanchard
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Serum Response Factor ◽  
Phosphorylation Site ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Resting Cells ◽  
Response Factor ◽  
Amino Terminal ◽  
Serum Response

Serum stimulation of resting cells is mediated at least in part at the transcriptional level by the activation of numerous genes among which c-fos constitutes a model. Serum response factor (SRF) forms a ternary complex at the c-fos serum response element (SRE) with an accessory protein p62TCF/Elk-1. Both proteins are the targets of multiple phosphorylation events and their role is still unknown in the amino terminus of SRF. While the transcriptional activation domain has been mapped between amino acids 339 and 508, the DNA-binding and the dimerization domains have been mapped to between amino acids 133–235 and 168–235, respectively, no role has been proposed for the amino-terminal portion of the molecule. We demonstrate in the present work that amino acids 95 to 100 contain a stretch of basic amino acids that are sufficient to target a reporter protein to the nucleus. Moreover, this sequence appears to be the only nuclear localization signal operating in SRF. Finally, whereas the global structure around this putative nuclear location signal is reminiscent of what is found in the SV40 T antigen, the casein kinase II phosphorylation site does not determine the rate of cyto-nuclear protein transport of this protein.

scholarly journals Four isoforms of serum response factor that increase or inhibit smooth-muscle-specific promoter activity

2000 ◽  
Vol 345 (3) ◽  
pp. 445-451 ◽  
Author(s):  
Paul R. KEMP ◽  
James C. METCALFE
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Serum Response Factor ◽  
Muscle Cells ◽  
Dominant Negative ◽  
C2c12 Cells ◽  
Specific Gene ◽  
Response Factor ◽  
Transactivation Domain ◽  
Serum Response

Serum response factor (SRF) is a key transcriptional activator of the c-fos gene and of muscle-specific gene expression. We have identified four forms of the SRF coding sequence, SRF-L (the previously identified form), SRF-M, SRF-S and SRF-I, that are produced by alternative splicing. The new forms of SRF lack regions of the C-terminal transactivation domain by splicing out of exon 5 (SRF-M), exons 4 and 5 (SRF-S) and exons 3, 4 and 5 (SRF-I). SRF-M is expressed at similar levels to SRF-L in differentiated vascular smooth-muscle cells and skeletal-muscle cells, whereas SRF-L is the predominant form in many other tissues. SRF-S expression is restricted to vascular smooth muscle and SRF-I expression is restricted to the embryo. Transfection of SRF-L and SRF-M into C2C12 cells showed that both forms are transactivators of the promoter of the smooth-muscle-specific gene SM22α, whereas SRF-I acted as a dominant negative form of SRF.

scholarly journals Dominant Negative Murine Serum Response Factor: Alternative Splicing within the Activation Domain Inhibits Transactivation of Serum Response Factor Binding Targets

1999 ◽  
Vol 19 (7) ◽  
pp. 4582-4591 ◽  
Author(s):  
Narasimhaswamy S. Belaguli ◽  
Wei Zhou ◽  
Thuy-Hanh T. Trinh ◽  
Mark W. Majesky ◽  
Robert J. Schwartz
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Smooth Muscles ◽  
Dominant Negative ◽  
C2c12 Cells ◽  
Luciferase Reporter ◽  
Response Factor ◽  
Activation Domain ◽  
Alternatively Spliced ◽  
Serum Response

ABSTRACT Primary transcripts encoding the MADS box superfamily of proteins, such as MEF2 in animals and ZEMa in plants, are alternatively spliced, producing several isoformic species. We show here that murine serum response factor (SRF) primary RNA transcripts are alternatively spliced at the fifth exon, deleting approximately one-third of the C-terminal activation domain. Among the different muscle types examined, visceral smooth muscles have a very low ratio of SRFΔ5 to SRF. Increased levels of SRFΔ5 correlates well with reduced smooth muscle contractile gene activity within the elastic aortic arch, suggesting important biological roles for differential expression of SRFΔ5 variant relative to wild-type SRF. SRFΔ5 forms DNA binding-competent homodimers and heterodimers. SRFΔ5 acts as a naturally occurring dominant negative regulatory mutant that blocks SRF-dependent skeletal α-actin, cardiac α-actin, smooth α-actin, SM22α, and SRF promoter-luciferase reporter activities. Expression of SRFΔ5 interferes with differentiation of myogenic C2C12 cells and the appearance of skeletal α-actin and myogenin mRNAs. SRFΔ5 repressed the serum-induced activity of the c-fos serum response element. SRFΔ5 fused to the yeast Gal4 DNA binding domain displayed low transcriptional activity, which was complemented by overexpression of the coactivator ATF6. These results indicate that the absence of exon 5 might be bypassed through recruitment of transcription factors that interact with extra-exon 5 regions in the transcriptional activating domain. The novel alternatively spliced isoform of SRF, SRFΔ5, may play an important regulatory role in modulating SRF-dependent gene expression.

scholarly journals Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha-actin gene transcription.

1996 ◽  
Vol 16 (11) ◽  
pp. 6372-6384 ◽  
Author(s):  
C Y Chen ◽  
R J Schwartz
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Binding Activity ◽  
Dominant Negative ◽  
Actin Gene ◽  
Response Factor ◽  
Dna Binding Activity ◽  
Actin Promoter ◽  
Alpha Actin ◽  
Serum Response

We recently showed that the cardiogenic homeodomain factor Nkx-2.5 served as a positive acting accessory factor for serum response factor (SRF) and that together they provided strong transcriptional activation of the cardiac alpha-actin promoter, depending upon intact serum response elements (SREs) (C. Y. Chen, J. Croissant, M. Majesky, S. Topouz, T. McQuinn, M. J. Frankovsky, and R. J. Schwartz, Dev. Genet. 19:119-130, 1996). As shown here, Nkx-2.5 and SRF collaborated to activate the endogenous murine cardiac alpha-actin gene in 10T1/2 fibroblasts by a mechanism in which SRF recruited Nkx-2.5 to the alpha-actin promoter. Activation of a truncated promoter consisting of the proximal alpha-actin SRE1 occurred even when Nkx-2.5 DNA-binding activity was blocked by a point mutation in the third helix of its homeodomain. Investigation of protein-protein interactions showed that Nkx-2.5 was bound to SRF in the absence of DNA in soluble protein complexes retrieved from cardiac myocyte nuclei but could also be detected in coassociated binding complexes on the proximal SRE1. Recruitment of Nkx-2.5 to an SRE depended upon SRF DNA-binding activity and was blocked by the dominant negative SRFpm1 mutant, which allowed for dimerization of SRF monomers but prevented DNA binding. Interactive regions shared by Nkx-2.5 and SRF were mapped to N-terminal/helix I and helix II/helix III regions of the Nkx-2.5 homeodomain and to the N-terminal extension of the MADS box. Our study suggests that physical association between Nkx-2.5 and SRF is one way that cardiac specified genes are activated in cardiac cell lineages.

scholarly journals The RhoA/Rho Kinase Pathway Regulates Nuclear Localization of Serum Response Factor

2003 ◽  
Vol 29 (1) ◽  
pp. 39-47 ◽  
Author(s):  
Hong Wei Liu ◽  
Andrew J. Halayko ◽  
Darren J. Fernandes ◽  
Gregory S. Harmon ◽  
Joel A. McCauley ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Serum Response Factor ◽  
Rho Kinase ◽  
Response Factor ◽  
Serum Response ◽  
Kinase Pathway

scholarly journals RhoA GTPase and Serum Response Factor Control Selectively the Expression of MyoD without Affecting Myf5 in Mouse Myoblasts

1998 ◽  
Vol 9 (7) ◽  
pp. 1891-1902 ◽  
Author(s):  
Gilles Carnac ◽  
Michael Primig ◽  
Magali Kitzmann ◽  
Philippe Chafey ◽  
David Tuil ◽  
...  
Keyword(s):  
Gene Expression ◽  
G Proteins ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Muscle Differentiation ◽  
Dominant Negative ◽  
Response Factor ◽  
Rhoa Protein ◽  
Myod Gene ◽  
Serum Response

MyoD and Myf5 belong to the family of basic helix-loop-helix transcription factors that are key operators in skeletal muscle differentiation. MyoD and Myf5 genes are selectively activated during development in a time and region-specific manner and in response to different stimuli. However, molecules that specifically regulate the expression of these two genes and the pathways involved remain to be determined. We have recently shown that the serum response factor (SRF), a transcription factor involved in activation of both mitogenic response and muscle differentiation, is required for MyoD gene expression. We have investigated here whether SRF is also involved in the control of Myf5 gene expression, and the potential role of upstream regulators of SRF activity, the Rho family G-proteins including Rho, Rac, and CDC42, in the regulation of MyoD and Myf5. We show that inactivation of SRF does not alter Myf5 gene expression, whereas it causes a rapid extinction of MyoD gene expression. Furthermore, we show that RhoA, but not Rac or CDC42, is also required for the expression of MyoD. Indeed, blocking the activity of G-proteins using the general inhibitor lovastatin, or more specific antagonists of Rho proteins such as C3-transferase or dominant negative RhoA protein, resulted in a dramatic decrease of MyoD protein levels and promoter activity without any effects on Myf5 expression. We further show that RhoA-dependent transcriptional activation required functional SRF in C2 muscle cells. These data illustrate that MyoD and Myf5 are regulated by different upstream activation pathways in which MyoD expression is specifically modulated by a RhoA/SRF signaling cascade. In addition, our results establish the first link between RhoA protein activity and the expression of a key muscle regulator.

scholarly journals Increased Myosin Light Chain Kinase Expression in Hypertension: Regulation by Serum Response Factor via an Insertion Mutation in the Promoter

2006 ◽  
Vol 17 (9) ◽  
pp. 4039-4050 ◽  
Author(s):  
Yoo-Jeong Han ◽  
Wen-Yang Hu ◽  
Olga Chernaya ◽  
Nenad Antic ◽  
Lianzhi Gu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Myosin Light Chain Kinase ◽  
Promoter Activity ◽  
Myosin Light Chain ◽  
Serum Response Factor ◽  
Vascular Diseases ◽  
Dominant Negative ◽  
Response Factor ◽  
Carg Box ◽  
Serum Response

Regulation of gene transcription in vascular smooth muscle cells (VSMCs) by serum response factor (SRF) plays a crucial role in vascular development and in the pathophysiology of vascular diseases. Nevertheless, the regulation of specific genes by SRF in vascular diseases is poorly understood. Therefore, we investigated the regulation of smooth muscle myosin light chain kinase (smMLCK) by using spontaneously hypertensive rats (SHR) as an experimental model. We found that smMLCK expression in blood vessels increases during the development of hypertension and is always greater in blood vessels from SHR compared with normotensive rats. Analysis of the DNA sequences of the promoters isolated from SHR and normotensive rats revealed that SHR contain a 12-base pair insertion adjacent to the CArG box. This insertion increases SRF binding to the CArG box and positively regulates SRF-dependent promoter activity. The increase in smMLCK expression was blocked by dominant-negative SRF, dominant-negative Ras, or antisense oligonucleotides to ERK. In vivo, inhibiting MEK decreased smMLCK expression and blood pressure in SHR partly by decreasing SRF binding to the smMLCK promoter. These data provide novel insight into the regulation of smMLCK expression at the molecular level and demonstrate the importance of SRF in regulating smMLCK promoter activity in SHR.

scholarly journals Serum-Induced Phosphorylation of the Serum Response Factor Coactivator MKL1 by the Extracellular Signal-Regulated Kinase 1/2 Pathway Inhibits Its Nuclear Localization

2008 ◽  
Vol 28 (20) ◽  
pp. 6302-6313 ◽  
Author(s):  
Susanne Muehlich ◽  
Ruigong Wang ◽  
Seung-Min Lee ◽  
Thera C. Lewis ◽  
Chao Dai ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Export ◽  
Serum Response Factor ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Actin Binding ◽  
Response Factor ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Serum Response

ABSTRACT Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related coactivator of the serum response factor (SRF) transcription factor, which has an integral role in differentiation, migration, and proliferation. Serum induces RhoA-dependent translocation of MKL1 from the cytoplasm to the nucleus and also causes a rapid increase in MKL1 phosphorylation. We have mapped a serum-inducible phosphorylation site and found, surprisingly, that its mutation causes constitutive localization to the nucleus, suggesting that phosphorylation of MKL1 inhibits its serum-induced nuclear localization. The key site, serine 454, resembles a mitogen-activated protein kinase phosphorylation site, and its modification was blocked by the MEK1 inhibitor U0126, implying that extracellular signal-regulated kinase 1/2 (ERK1/2) is the serum-inducible kinase that phosphorylates MKL1. Previous results indicated that G-actin binding to MKL1 promotes its nuclear export, and we found that MKL1 phosphorylation is required for its binding to actin, explaining its effect on localization. We propose a model in which serum induction initially stimulates MKL1 nuclear localization due to a decrease in G-actin levels, but MKL1 is then downregulated by nuclear export due to ERK1/2 phosphorylation.

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.

Sign in / Sign up

Export Citation Format

Share Document