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PubMed2006

Cellular toxicity induced by SRF-mediated transcriptional squelching.

The transcriptional activator serum response factor (SRF) is a member of the immediate early gene family known to promote embryonic development, cell growth, and myogenesis through interaction with multiple nuclear protein factors. Previous studies have shown that SRF possesses potent transcriptional activation domains that can interfere with gene expression at artificially high expression levels through "transcriptional squelching." The current work sought to characterize toxicological aspects of SRF-mediated transcriptional squelching. An adenoviral expression system driven by the potent cytomegalovirus promoter was used to achieve up to a 50-fold increase in SRF protein levels. The overexpressed SRF is nuclear localized and interferes with gene expression independent of specific promoter interaction as expected for transcriptional squelching. SRF-mediated squelching elicits robust cell killing affecting multiple cell types including normal and abnormal proliferating cells as well as postmitotic cells such as cardiomyocytes in culture, and the cell killing is more pronounced than that mediated by the tumor suppressor protein p53. Although both the DNA-binding and transcriptional activation domains of SRF are normally required for the physiological roles of SRF, only the transcriptional activation domain is required for cell killing. Unlike c-myc-induced cell killing, squelching-induced cell death does not require serum withdrawal and cannot be effectively attenuated by blocking the caspase and calpain proteolytic pathways or by overexpression of the antiapoptotic gene bcl-xL. These findings suggest transcriptional squelching may be engineered for killing cancer cells, and the SRF gene may represent a novel molecular target for cancer therapeutics.

Adenoviridae↗

PubMed1994

Serum-regulated transcription by serum response factor (SRF): a novel role for the DNA binding domain.

The transcription factors Serum Response Factor (SRF) and Ternary Complex Factor (TCF) form a ternary complex at the c-fos Serum Response Element (SRE). We show that in NIH3T3 cells TCF binding is required for regulated transcription in response to stimulation by phorbol myristate acetate (PMA), but not by whole serum. We constructed a novel transcriptionally inactive SRE variant whose serum-regulated activity can be partially restored by overexpression of SRF in the absence of bound TCF. Activation by SRF does not require the SRF N-terminal phosphorylation sites, but is potentiated 2- to 3-fold by the SRF C-terminal activation domain. Mutations in the SRF DNA binding domain, which do not affect the ability of SRF to bind DNA, abolish its ability to mediate TCF-independent serum-regulated activation and reduce activation by the SRF/TCF(Elk-1) ternary complex. Efficient activation requires that SRF be targeted to DNA via its own DNA binding domain.

3T3 Cells↗

PubMed1999

Expression of the SRF gene occurs through a Ras/Sp/SRF-mediated-mechanism in response to serum growth signals.

Serum Response Factor (SRF) plays a central role in the transcriptional response of mammalian cells to a variety of extracellular signals. It is a key regulator of many cellular early response genes which are believed to be involved in cell growth, differentiation, and development. The mechanism by which SRF activates transcription in response to mitogenic agents has been extensively studied, however, less is known about regulation of the SRF gene itself. Previously, we identified distinct regulatory elements in the SRF promoter that play a role in activation, including an ETS domain binding site, an overlapping Sp1/Egr-1 binding site, and two SRF binding sites. We further showed that serum induces the SRF gene by a mechanism that requires an intact SRF binding site, also termed a CArG box. In the present study we demonstrate that in response to stimulation by cells by lysophosphatidic acid (LPA) or whole serum, the SRF promoter is upregulated by a bipartite pathway that requires both an Sp1 factor binding site and the CArG motifs for maximal stimulation. The CArG box-dependent component of this pathway is targeted by Rho mediated signals, and the Sp1 binding site dependent component is targeted by Ras mediated signals.

3T3 Cells↗

PubMed2004

The ETS domain transcription factor Elk-1 regulates the expression of its partner protein, SRF.

The ternary complex factors (TCF) are a subfamily of ETS domain transcription factors that bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). Here, we have identified the SRF gene as a target for the TCFs, thereby providing a positive feedback loop whereby TCF activation leads to the enhancement of the expression of its partner protein SRF. The binding of the TCF Elk-1 to the SRF promoter and subsequent regulation of SRF expression occurs in a ternary complex-dependent manner. Our data therefore reveal that SRF is an important target for the ERK and Rho signaling pathways that converge on a ternary TCF-SRF complex at the SRE on the SRF promoter.

Base Sequence↗

PubMed1996

Elk-1 can recruit SRF to form a ternary complex upon the serum response element.

The initial genomic response to serum growth factors is the transcriptional activation of a set of immediate-early genes. Serum-induced transcriptional activation of several of these genes involves the formation of a ternary complex that includes the serum response factor (SRF), a 62 kDa ternary complex factor (TCF) and a serum response element (SRE). TCF alone does not bind the SRE of the protooncogene c-fos, but requires the prior assembly of the SRF-SRE binary complex for it to be recruited into a ternary complex. Here we show that this SRF-SRE binary complex is not an obligatory prerequisite for the formation of a serum responsive ternary complex. We demonstrate that Elk-1, which has properties of TCF can recruit SRF into a ternary complex on elements that do not support formation of the SRF-DNA binary complex. We also show that for two immediate-early genes, pip92 and nur77, formation of the ternary complex may occur without the prior assembly of SRF-DNA binary complex. Finally, we show that the ability of different sequences to support formation of Elk-l-SRF-DNA ternary complex in vitro correlates with their ability to respond to serum growth factors in vivo. Our results suggest that a much broader range of DNA sequences than the consensus SRF and TCF binding sites can support ternary complex formation, and by inference, serum induction. Possible implications of these results are discussed.

3T3 Cells↗

PubMed1991

Human SRF-related proteins: DNA-binding properties and potential regulatory targets.

Serum response factor (SRF) is a transcription factor that binds the sequence CC(A/T)6GG found in a number of growth factor-inducible and muscle-specific promoters. We describe the isolation and characterization of cDNA clones encoding a family of three human SRF-related DNA-binding proteins. Each of these RSRF (related to SRF) proteins contains an 86-amino-acid amino-terminal region related to the SRF DNA-binding domain: In RSRFC4 and RSRFC9, this region is identical, whereas that present in RSRFR2 differs by seven conservative amino acid substitutions. The DNA-binding specificity of the RSRF proteins, which recognize the consensus sequence CTA(A/T)4TAG, is distinct from that of SRF. The entire RSRF common region is required for DNA binding, and the differential sequence specificity of the RSRFs and SRF is the result of differences in the basic amino-terminal part of this domain. The RSRF proteins bind DNA as dimers and can dimerize with one another but not with SRF. Although the RSRF mRNAs are expressed in many cell types, RSRFR2 mRNA is expressed at elevated levels in several B-cell lines. Consistent with this, extracts from many cell types form CTA(A/T)4TAG-binding complexes that contain RSRF proteins, and oligonucleotides containing RSRF-binding sites function as promoter elements in transfection assays. Like SRF-binding sites, RSRF-binding sites are found in the regulatory sequences of a number of growth factor-inducible and muscle-specific genes, and we show that RSRF polypeptides are components of previously characterized binding activities that interact with these elements. We discuss the potential role of RSRF proteins in the regulation of these genes.

3T3 Cells↗

PubMed2004

Regulation of Egr-1, SRF, and Sp1 mRNA expression in contracting skeletal muscle cells.

The early cellular signals associated with contractile activity initiate the activation and induction of transcription factors that regulate changes in skeletal muscle phenotype. The transcription factors Egr-1, Sp1, and serum response factor (SRF) are potentially important mediators of mitochondrial biogenesis based on the prevalence of binding sites for them in the promoter regions of genes encoding mitochondrial proteins, including PGC-1 alpha, the important regulator of mitochondrial biogenesis. Thus, to further define a role for transcription factors at the onset of contractile activity, we examined the time-dependent alterations in Egr-1, Sp1, and SRF mRNA and the levels in electrically stimulated mouse C(2)C(12) skeletal muscle cells. Early transient increases in Egr-1 mRNA levels within 30 min (P < 0.05) of contractile activity led to threefold increases (P < 0.05) in Egr-1 protein by 60 min. The increase in Egr-1 mRNA was not because of increased stability, as Egr-1 mRNA half-life after 30 min of stimulation showed only a 58% decline. Stimulation of muscle cells had no effect on Sp1 mRNA but led to progressive increases (P < 0.05) in SRF mRNA by 30 and 60 min. This was not matched by increases in SRF protein but occurred coincident with increases (P < 0.05) in SRF-serum response element DNA binding at 30 and 60 min as a result of SRF phosphorylation on serine-103. To assess the importance of the recovery period, 12 h of continuous contractile activity was compared with four successive 3-h bouts, with an intervening 21-h recovery period after each bout. Continuous contractile activity led to a twofold increase (P < 0.05) in Egr-1 mRNA, no change in SRF mRNA, and a 43% decrease in Sp1 mRNA expression. The recovery period prevented the decline in Sp1 mRNA, produced a decrease in Egr-1 mRNA, and had no effect on SRF mRNA. Thus continuous and intermittent contractile activity evoked different specific transcription factor expression patterns, which may ultimately contribute to divergent qualitative, or temporal patterns of, phenotypic adaptation in muscle.

Animals↗

PubMed2006

Control of SRF binding to CArG box chromatin regulates smooth muscle gene expression in vivo.

Precise control of SMC transcription plays a major role in vascular development and pathophysiology. Serum response factor (SRF) controls SMC gene transcription via binding to CArG box DNA sequences found within genes that exhibit SMC-restricted expression. However, the mechanisms that regulate SRF association with CArG box DNA within native chromatin of these genes are unknown. Here we report that SMC-restricted binding of SRF to murine SMC gene CArG box chromatin is associated with patterns of posttranslational histone modifications within this chromatin that are specific to the SMC lineage in culture and in vivo, including methylation and acetylation to histone H3 and H4 residues. We found that the promyogenic SRF coactivator myocardin increased SRF association with methylated histones and CArG box chromatin during activation of SMC gene expression. In contrast, the myogenic repressor Kruppel-like factor 4 recruited histone H4 deacetylase activity to SMC genes and blocked SRF association with methylated histones and CArG box chromatin during repression of SMC gene expression. Finally, we observed deacetylation of histone H4 coupled with loss of SRF binding during suppression of SMC differentiation in response to vascular injury. Taken together, these findings provide novel evidence that SMC-selective epigenetic control of SRF binding to chromatin plays a key role in regulation of SMC gene expression in response to pathophysiological stimuli in vivo.

Animals↗

PubMed1991

A protein domain conserved between yeast MCM1 and human SRF directs ternary complex formation.

MCM1 and SRF bind to the same DNA sequence and form ternary complexes with STE12 and p62TCF, respectively. We show that in gel retardation assays, MCM1 recruits both ternary complex factors whereas SRF interacts only with p62TCF. A protein domain of 90 amino acids, shared by MCM1 and SRF, was found to be sufficient for ternary complex formation. The domain is also required for dimerization and DNA binding. Similar regions are found in other proteins, such as ARG80, Deficiens and Agamous. ARG80 and Agamous exhibit similar DNA binding specificities but do not interact with either STE12 or p62TCF. By exchanging three residues of ARG80 with those of corresponding positions in SRF (residues 198, 200 and 203), the ARG80 protein acquires the ability to recruit p62TCF into a ternary complex. Likewise, the substitution of four SRF amino acids by MCM1-derived residues (amino acids 73, 75, 77 and 78) confers on SRF the ability to interact with STE12. Thus, we have identified specific amino acids in MCM1 and SRF that are critical for ternary complex formation and which map to equivalent positions within the shared domains. Therefore, the structural basis for specific protein-protein interaction appears to be conserved in evolution between a class of transcription factors.

Amino Acid Sequence↗

PubMed2004

Myocardin/MKL family of SRF coactivators: key regulators of immediate early and muscle specific gene expression.

Myocardin, megakaryoblastic leukemia-1 (MKL1), and MKL2 belong to a newly defined family of transcriptional coactivators. All three family members bind to serum response factor (SRF) and strongly activate transcription from promoters with SRF binding sites. SRF is required for the serum induction of immediate early genes such as c-fos and for the expression of many muscle specific genes. Consistent with a role in muscle specific gene expression, myocardin is specifically expressed in cardiac and smooth muscle cells while MKL1 and 2 are broadly expressed. Myocardin has particularly been shown to be required for smooth muscle development while MKL1/2 are required for the RhoA signaling pathway for induction of immediate early genes. SRF can be activated by at least two families of coactivators, p62TCF and myocardin/MKL. These factors bind to the same region of SRF such that their binding is mutually exclusive. This provides one mechanism of regulation of SRF target genes by pathways that differentially activate the coactivators. The RhoA pathway appears to activate MKL1 by altering MKL1's binding to actin and causing MKL1's translocation from the cytoplasm to the nucleus. However, this mechanism of activation of the myocardin/MKL family has not been observed in all cell types such that other regulatory mechanism(s) likely exist. In particular, rapid serum inducible phosphorylation of MKL1 was observed. The regulation of this coactivator family is key to understanding how SRF target genes are activated during muscle cell differentiation or growth factor induced cell proliferation.

Cell Differentiation↗

PubMed2001

Activation of skeletal muscle afferents evokes release of glutamate in the subretrofacial nucleus (SRF) of cats.

The subretrofacial nucleus (SRF) is a region of the rostral ventrolateral medulla known to play a crucial role in sympathoexcitation. SRF neurons send direct projections to the intermediolateral cell columns of the spinal cord where they form synaptic contact with preganglionic sympathetic motor neurons. Activation of this neural pathway increases sympathetic outflow to the heart and blood vessels affecting cardiac function and vasomotor tone. Previous studies utilizing electrophysiological recording techniques and c-Fos expression have established that the activity of SRF neurons is increased during skeletal muscle contraction. However, the excitatory neurotransmitter mediating this increased activity remains in question. In the present study, static contraction of the triceps surae was induced by electrical stimulation of L7 and S1 ventral roots in anesthetized cats (n=12). Endogenous release of glutamate (Glu) from the SRF was recovered by microdialysis and measured by HPLC. Static muscle contraction for 4 min increased mean arterial pressure (MAP) 38+/-4 mmHg from a control level of 102+/-12 mmHg (P< 0.05). During muscle contraction the extracellular concentration of Glu recovered from the SRF increased from 623+/-117 to 1078+/-187 nM (P<0.05). To determine the effect of muscle contraction on Glu release in the absence of synaptic input from other reflexogenic areas, contraction was repeated following acute sinoaortic denervation and vagotomy. Following this denervation, muscle contraction increased MAP 41+/- 4 mmHg (P < 0.05) and Glu concentration from 635+/-246 to 1106+/-389 nM (P < 0.05). Muscle paralysis prevented the increases in MAP and Glu concentration during ventral root stimulation. These results suggest that: (i) Glu is released in the SRF during activation of contraction-sensitive skeletal muscle afferent fibers in the cat; and (ii) synaptic input from other reflexogenic areas appears to be ineffective in modulating the release of Glu in the SRF during static muscle contraction.

Animals↗

PubMed2002

Effect of nitric oxide on release of glutamate in the subretrofacial nucleus (SRF) during the exercise pressor reflex in cats.

The subretrofacial nucleus (SRF) has been known to play a crucial role in the expression of the exercise pressor reflex. Previously, we have reported that the release of glutamate (Glu) in the SRF was increased during muscle contraction in anesthetized cats. In this study, static muscle contraction of the triceps surae for 4 min was induced by electrical stimulation of L7 and S1 ventral roots. Endogenous release of Glu and citrulline (Cit) from the SRF was recovered by microdialysis and measured by HPLC. The microdialysis probes were also used to deliver L-arginine and L-NAME to test the effect of nitric oxide (NO) on release of Glu in the SRF and on the cardiovascular responses during muscle contraction. During control, muscle contraction significantly increased mean arterial pressure (MAP) from 98+/-8 to 151+/-9 mmHg, and the extracellular concentration of Glu from 610+/-120 to 1280+/-290 nM. Dialyzing 2 mM L-arginine into the SRF increased basal Cit concentration from 260+/-50 to 760+/-210 nM (P<0.05). During contraction after L-arginine, the increases in MAP and Glu concentration were significantly attenuated (86+/-3-124+/-6 mmHg and 300+/-60-460+/-100 nM, respectively). Dialysis of 0.5 mM L-NAME into the SRF decreased Cit concentration from 340+/-40 to 180+/-20 nM (P<0.05). During contraction after dialyzing L-NAME, the increases in MAP and Glu concentration were significantly potentiated (93+/-6-154+/-9 mmHg and 520+/-80-1290+/-380 nM, respectively). These results suggest that endogenous NO modulates the cardiovascular responses to static muscle contraction by affecting the release of Glu in the SRF.

Animals↗

PubMed2003

Actin dynamics control SRF activity by regulation of its coactivator MAL.

Rho GTPases regulate the transcription factor SRF via their ability to induce actin polymerization. SRF activity responds to G actin, but the mechanism of this has remained unclear. We show that Rho-actin signaling regulates the subcellular localization of the myocardin-related SRF coactivator MAL, rearranged in t(1;22)(p13;q13) AML. The MAL-SRF interaction displays the predicted properties of a Rho-regulated SRF cofactor. MAL is predominantly cytoplasmic in serum-starved cells, but accumulates in the nucleus following serum stimulation. Activation of the Rho-actin signaling pathway is necessary and sufficient to promote MAL nuclear accumulation. MAL N-terminal sequences, including two RPEL motifs, are required for the response to signaling, while other regions mediate its nuclear export (or cytoplasmic retention) and nuclear import. MAL associates with unpolymerized actin through its RPEL motifs. Constitutively cytoplasmic MAL derivatives interfere with MAL redistribution and Rho-actin signaling to SRF. MAL associates with several SRF target promoters regulated via the Rho-actin pathway.

3T3 Cells↗

PubMed1997

A competitive mechanism of CArG element regulation by YY1 and SRF: implications for assessment of Phox1/MHox transcription factor interactions at CArG elements.

In the promoters of many immediate early genes, including c-fos, CArG DNA regulatory elements mediate basal constituitive expression and rapid and transient serum induction. CArG boxes also occur in the promoters of muscle-specific genes, including skeletal alpha-actin, where it confers muscle-specific expression. These elements are regulated, at least in part, by the ubiquitous transcription factors serum response factor (SRF) and YY1. The homeobox transcription factor Phox1/MHox has also been implicated in regulation of the c-fos CArG element and is thought to function by facilitating SRF binding to DNA. Here, we provide in vitro and in vivo evidence that the mechanism of YY1 repression of CArG elements results from competition with SRF for overlapping binding sites. We describe in detail the binding sites of YY1 and SRF through serial point mutations of the skeletal alpha-actin proximal CArG element and identify a mutation that dramatically reduces YY1 binding but retains normal SRF binding. YY1 competes with SRF for binding to wild-type CArG elements, but not to this point mutant in vitro. This mutant is sufficient for muscle-specific expression in vivo but is much less sensitive to repression by YY1 overexpression. We utilized the YY1/SRF competition to address the role of Phox1 at these elements. Phox1 overexpression did not diminish YY1-mediated repression, suggesting that transcriptional activation by Phox1 does not result from enhanced SRF binding to these elements. These methods may prove to be useful for assessing interactions between other CArG element regulatory factors.

Actins↗

PubMed2000

The mechanism of complex formation between Fli-1 and SRF transcription factors.

The mechanisms of multicomponent transcription factor complex assembly are currently poorly defined. A paradigm for this type of complex is the ETS-domain transcription factor Elk-1 and the MADS-box transcription factor SRF which form a ternary complex with the c- fos serum response element (SRE). In this study we have analysed how a different ETS-domain transcription factor Fli-1 interacts with SRF to form ternary complexes with this element. Two regions of Fli-1 that are required for ternary complex formation have been identified. These SRF binding motifs are located on either side of the ETS DNA-binding domain. Hydrophobic amino acids within these motifs have been identified that play important roles in binding to SRF and ternary complex formation. By using Fli-1 derivatives with mutations in the N-terminal SRF binding motif, the significance of Fli-1-SRF interactions in recruitment of Fli-1 to the c- fos SRE in vivo has been demonstrated. Collectively our data provide a model of how Fli-1 interacts with SRF that differs significantly from the mechanism used by a different ETS-domain protein, Elk-1.

3T3 Cells↗

PubMed2005

Temporally controlled onset of dilated cardiomyopathy through disruption of the SRF gene in adult heart.

BACKGROUND: Serum response factor (SRF) is a cardiac transcription factor involved in cell growth and differentiation. We have shown, using the Cre/loxP system, that cardiac-specific disruption of SRF gene in the embryonic heart results in lethal cardiac defects. The role of SRF in adult heart is unknown. METHODS AND RESULTS: We disrupted SRF in the adult heart using a heart-specific tamoxifen-inducible Cre recombinase. This disruption led to impaired left ventricular function with reduced contractility, subsequently progressing to dilated cardiomyopathy, as demonstrated by serial echocardiography, including tissue Doppler imaging. The cytoarchitecture of cardiomyocytes was altered in the intercalated disks. All mutant mice died from heart failure 10 weeks after treatment. These functional and structural defects were preceded by early alterations in the cardiac gene expression program: major decreases in mRNA levels for cardiac alpha-actin, muscle creatine kinase, and calcium-handling genes. CONCLUSIONS: SRF is crucial for adult cardiac function and integrity. We suggest that the rapid progression to heart failure in SRF mutant mice results primarily from decreased expression of proteins involved in force generation and transmission, low levels of polymerized actin, and changes in cytoarchitecture, without hypertrophic compensation. These cardiac-specific SRF-deficient mice have the morphological and clinical features of acquired dilated cardiomyopathy in humans and may therefore be used as an inducible model of this disorder.

Animals↗

PubMed2003

Interaction of Smad3 and SRF-associated complex mediates TGF-beta1 signals to regulate SM22 transcription during myofibroblast differentiation.

Myofibroblasts play important roles in a variety of developmental and pathological processes, such as vascular remodeling, atherosclerosis and wound healing. In this study, we used the TGF-beta1-treated 10T1/2 cells as an in vitro model to understand how Smad-mediated TGF-beta1 signals regulate SM22 promoter transcription during myofibroblast differentiation. We found that TGF-beta1 transiently induces SRF and SM22 transcription, and that this process is accompanied by transient increases of SRF and Smad3 binding to the SM22 promoter. Interestingly, Smad3, not Smad2, is the primary mediator for TGF-beta1-induced transactivation of the SM22 promoter, while Smad6 and Smad7 repress such a transactivation. Smad3 can bind to a Smad-binding element (SBE) in the first exon of SM22, and directly associate with the SRF complex in response to TGF-beta1 treatment. Moreover, Smad3 and I-Smads regulate the SM22 promoter through CArG box-dependent transcription using dominant-negative SRF mutants and SRF-VP16. Although SBE as well as CArG boxes and TGF-beta control element are all important for the SM22 promoter activities, the promoters with mutations at either one or all of them still respond to TGF-beta1 treatment. Consistently, TGF-beta1 stimulates SM22 transcription in Smad3 null mouse embryonic fibroblasts. These findings provide the first evidence that Smad3 directly links TGF-beta1 signaling to an SRF-associated regulatory network in controlling SM22 transcription; it also implies that TGF-beta1 regulates the SM22 promoter via Smad3-dependent and Smad3-independent pathways.

Animals↗

PubMed1995

The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF.

The c-fos serum response element (SRE) forms a ternary complex with the transcription factors SRF (serum response factor) and TCF (ternary complex factor). By itself, SRF can mediate transcriptional activation induced by serum, lysophosphatidic acid, or intracellular activation of heterotrimeric G proteins. Activated forms of the Rho family GTPases RhoA, Rac1, and CDC42Hs also activate transcription via SRF and act synergistically at the SRE with signals that activate TCF. Functional Rho is required for signaling to SRF by several stimuli, but not by activated CDC42Hs or Rac1. Activation of the SRF-linked signaling pathway does not correlate with activation of the MAP kinases ERK, SAPK/JNK, or MPK2/p38. Functional Rho is required for regulated activity of the c-fos promoter. These results establish SRF as a nuclear target of a novel Rho-mediated signaling pathway.

3T3 Cells↗