Serum Response Factor Binding Sites Research Articles

Diaphanous-Related Formin Protein mDia2 Regulates Hematopoietic Stem and Progenitor Cell Functions through Srf-Integrins Pathway

Distinct mature cell types in mammalian blood system are derived from the Hematopoietic Stem/Progenitor cells (HSPCs), which are multipotent progenitor cells with self-renewing and engraftment ability. Various intrinsic and extrinsic molecules modulate HSPCs' fates to either undergo self-renewal or differentiation. The Diaphanous-related formin protein mDia2, is an effector of Rho GTPases. Previously, we reported that hematopoietic-specific mDia2 knock out mice developed anemia due to ineffective erythropoiesis caused by bi-nucleated erythroblasts. However, how mDia2 is involved in HSPC functions are unknown. Here we show that mDia2 expression is enriched in HSPCs. Although mDia2 was largely dispensable for HSPCs' function at steady-state, hematopoietic-specific inactivation of mDia2 severely impaired the competitive repopulation capacity of HSPCs. After primary bone marrow transplantation, the absolute number of bone marrow HSPCs from mDia2 knockout (KO) transplants was significantly decreased. mDia2 deficient LSK cells also showed skewed differentiation with increased percentage of more differentiated multi-potent progenitor cells (MPP), which was further supported by loss of stem cell quiescence. Under serial transplantation, mice transplanted with mDia2 knockout (KO) bone marrow showed rapid lethality. Importantly, these defects can be rescued by the constitutively active mDia2 mutant mDia2-ΔDAD. Furthermore, mDia2 KO HSPCs showed a marked reduction in the motility ability from bone marrow to the peripheral blood upon G-CSF stimulation. Loss of mDia2 also severely compromised the recovery of the mice from 5-FU-induced myelosuppression.mDia2 functions to nucleate linear actin filaments. We found that loss of mDia2 expression in HSPCs reduced the F-actin levels. This further attenuated the adhesion of HSPCs that was confirmed by adhesion assay and decreased protrusion formation in vitro . We demonstrated that the defect in F-actin formation caused by mDia2 deficiency repressed the transcriptional activity of serum response factor (SRF), which caused down-regulation of SRF target genes such as Acta2, Actb and SRF itself. Ectopic expression of SRF rescued HSPC phenotypes with loss of mDia2. We further identified that the expression levels of several integrins, including Itga2, Itgam (encodes CD11b) , Itagal (encodes CD11a)and Itgb2 (encodes CD18) , were significantly attenuated in mDia2 KO c-Kit+ HSPCs. Chromatin immunoprecipitation (ChIP) sequencing data further revealed intronic SRF binding sites in the genomic loci of Itgam and Itgb2, therefore suggesting Itgam and Itgb2 as new downstream targets of SRF pathway. Our data revealed a novel mDia2-SRF-integrin axis that regulates HSPCs adhesion and engraftment. DisclosuresNo relevant conflicts of interest to declare.

AR-mTOR-SRF Axis Regulates HMMR Expression in Human Prostate Cancer Cells.

The elevated expression of the hyaluronan-mediated motility receptor (HMMR) is known to be highly associated with tumor progression in prostate cancer, but the molecular mechanisms underlying the regulation of HMMR expression remain unclear. Here, we report that mammalian target of rapamycin (mTOR) is a key regulator of HMMR expression, for which its kinase activity is required. Pharmacological inhibitors of mTOR, such as rapamycin and Torin2, markedly suppressed the mRNA level as well as the protein level of HMMR in LNCaP and PC-3 cells. Our data demonstrate that such regulation occurs at the transcription level. HMMR promoter reporter assays revealed that the transcription factor SRF is responsible for the mTOR-mediated transcriptional regulation of HMMR gene. Consistently, the suppression of HMMR expression by Torin2 was noticeably reversed by the overexpression of SRF. Moreover, our findings suggest that the SRF binding sites responsible for the transcriptional regulation of HMMR through the mTOR-SRF axis are located in HMMR promoter sequences carrying the first intron, downstream of the translational start site. Furthermore, the upregulation of HMMR by DHT was abolished by stimulation with rapamycin, prior to DHT treatment, suggesting that mTOR activity is required for the induction of HMMR expression by androgen. Collectively, our study provides new mechanistic insights into the role of mTOR/SRF/AR signaling in HMMR regulation in prostate cancer cells.

NG2/CSPG4, CD146/MCAM and VAP1/AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

The present work addressed the hypothesis that NG2/CSPG4, CD146/MCAM, and VAP1/AOC3 are target genes of myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MKL1, MRTF-B/MKL2) and serum response factor (SRF). Using a bioinformatics approach, we found that CSPG4, MCAM, and AOC3 correlate with MYOCD, MRTF-A/MKL1, and SRF across human tissues. No other transcription factor correlated as strongly with these transcripts as SRF. Overexpression of MRTFs increased both mRNA and protein levels of CSPG4, MCAM, and AOC3 in cultured human smooth muscle cells (SMCs). Imaging confirmed increased staining for CSPG4, MCAM, and AOC3 in MRTF-A/MKL1-transduced cells. MRTFs exert their effects through SRF, and the MCAM and AOC3 gene loci contained binding sites for SRF. SRF silencing reduced the transcript levels of these genes, and time-courses of induction paralleled the direct target ACTA2. MRTF-A/MKL1 increased the activity of promoter reporters for MCAM and AOC3, and transcriptional activation further depended on the chromatin remodeling enzyme KDM3A. CSPG4, MCAM, and AOC3 responded to the MRTF-SRF inhibitor CCG-1423, to actin dynamics, and to ternary complex factors. Coincidental detection of these proteins should reflect MRTF-SRF activity, and beyond SMCs, we observed co-expression of CD146/MCAM, NG2/CSPG4, and VAP1/AOC3 in pericytes and endothelial cells in the human brain. This work identifies highly responsive vascular target genes of MRTF-SRF signaling that are regulated via a mechanism involving KDM3A.

Coronary Disease-Associated Gene TCF21 Inhibits Smooth Muscle Cell Differentiation by Blocking the Myocardin-Serum Response Factor Pathway.

The gene encoding TCF21 (transcription factor 21) has been linked to coronary artery disease risk by human genome-wide association studies in multiple racial ethnic groups. In murine models, Tcf21 is required for phenotypic modulation of smooth muscle cells (SMCs) in atherosclerotic tissues and promotes a fibroblast phenotype in these cells. In humans, TCF21 expression inhibits risk for coronary artery disease. The molecular mechanism by which TCF21 regulates SMC phenotype is not known. To better understand how TCF21 affects the SMC phenotype, we sought to investigate the possible mechanisms by which it regulates the lineage determining MYOCD (myocardin)-SRF (serum response factor) pathway. Modulation of TCF21 expression in human coronary artery SMC revealed that TCF21 suppresses a broad range of SMC markers, as well as key SMC transcription factors MYOCD and SRF, at the RNA and protein level. We conducted chromatin immunoprecipitation-sequencing to map SRF-binding sites in human coronary artery SMC, showing that binding is colocalized in the genome with TCF21, including at a novel enhancer in the SRF gene, and at the MYOCD gene promoter. In vitro genome editing indicated that the SRF enhancer CArG box regulates transcription of the SRF gene, and mutation of this conserved motif in the orthologous mouse SRF enhancer revealed decreased SRF expression in aorta and heart tissues. Direct TCF21 binding and transcriptional inhibition at colocalized sites were established by reporter gene transfection assays. Chromatin immunoprecipitation and protein coimmunoprecipitation studies provided evidence that TCF21 blocks MYOCD and SRF association by direct TCF21-MYOCD interaction. These data indicate that TCF21 antagonizes the MYOCD-SRF pathway through multiple mechanisms, further establishing a role for this coronary artery disease-associated gene in fundamental SMC processes and indicating the importance of smooth muscle response to vascular stress and phenotypic modulation of this cell type in coronary artery disease risk.

Involvement of SRF coactivator MKL2 in BDNF-mediated activation of the synaptic activity-responsive element in the Arc gene.

The expression of immediate early genes (IEGs) is thought to be an essential molecular basis of neuronal plasticity for higher brain function. Many IEGs contain serum response element in their transcriptional regulatory regions and their expression is controlled by serum response factor (SRF). SRF is known to play a role in concert with transcriptional cofactors. However, little is known about how SRF cofactors regulate IEG expression during the process of neuronal plasticity. We hypothesized that one of the SRF-regulated neuronal IEGs, activity-regulated cytoskeleton-associated protein (Arc; also termed Arg3.1), is regulated by an SRF coactivator, megakaryoblastic leukemia (MKL). To test this hypothesis, we initially investigated which binding site of the transcription factor or SRF cofactor contributes to brain-derived neurotrophic factor (BDNF)-induced Arc gene transcription in cultured cortical neurons using transfection and reporter assays. We found that BDNF caused robust induction of Arc gene transcription through a cAMP response element, binding site of myocyte enhancer factor 2, and binding site of SRF in an Arc enhancer, the synaptic activity-responsive element (SARE). Regardless of the requirement for the SRF-binding site, the binding site of a ternary complex factor, another SRF cofactor, did not affect BDNF-mediated Arc gene transcription. In contrast, chromatin immunoprecipitation revealed occupation of MKL at the SARE. Furthermore, knockdown of MKL2, but not MKL1, significantly decreased BDNF-mediated activation of the SARE. Taken together, these findings suggest a novel mechanism by which MKL2 controls the Arc SARE in response to BDNF stimulation.

Rnd3/RhoE expression is regulated by G-actin through MKL1-SRF signaling pathway

Rnd3/RhoE is an atypical member of the Rho family of small GTPases, devoid of intrinsic GTP hydrolytic activity and a general modulator of important cellular processes such as migration and proliferation. Here, we show that Rnd3 is a target of the transcription factor SRF and its co-activator MKL1. The MKL1-SRF pathway assures the translation of physical forces into a transcriptional response. Rho GTPases can modulate the activity of this mechanotransduction pathway through actin cytoskeleton regulation, and many MKL1-SRF targets are involved in the regulation of actin. We found that Rnd3 expression is altered by G-actin signaling and sensitive to actin-targeting drugs and MKL1 mutants. We further characterized a consensus SRF binding site in the Rnd3 promoter. We found that MKL1-SRF modulation regulates Rnd3 promoter activity and Rnd3 expression can affect MKL1-SRF pathway activity in return. We demonstrated that this novel MKL1-SRF target is required in mechanosensitive mechanisms such as cell spreading and spheroid formation. Thus, Rnd3 is a MKL1-SRF target that plays a key role in the feedback loop described between the MKL1-SRF pathway and the organization of the actin cytoskeleton.

Resveratrol stimulates c-Fos gene transcription via activation of ERK1/2 involving multiple genetic elements

The polyphenol resveratrol is found in many plant and fruits and is a constituent of our diet. Resveratrol has been proposed to have chemopreventive and anti-inflammatory activities. On the cellular level, resveratrol activates stimulus-regulated transcription factors. To identify resveratrol-responsive elements within a natural gene promoter, the molecular pathway leading to c-Fos gene expression by resveratrol was dissected. The c-Fos gene encodes a basic region leucine zipper transcription factor and is a prototype of an immediate-early gene that is regulated by a wide range of signaling molecules. We analyzed chromatin-integrated c-Fos promoter-luciferase reporter genes where transcription factor binding sites were destroyed by point mutations or deletion mutagenesis. The results show that mutation of the binding sites for serum response factor (SRF), activator protein-1 (AP-1) and cAMP response element binding protein (CREB) significantly reduced reporter gene transcription following stimulation of the cells with resveratrol. Inactivation of the binding sites for signal transducer and activator of transcription (STAT) or ternary complex factors did not influence resveratrol-regulated c-Fos promoter activity. Thus, the c-Fos promoter contains three resveratrol-responsive elements, the cAMP response element (CRE), and the binding sites for SRF and AP-1. Moreover, we show that the transcriptional activation potential of the c-Fos protein is increased in resveratrol-stimulated cells, indicating that the biological activity of c-Fos is elevated by resveratrol stimulation. Pharmacological and genetic experiments revealed that the protein kinase ERK1/2 is the signal transducer that connects resveratrol treatment with the c-Fos gene.

The cardiac calsequestrin gene transcription is modulated at the promoter by NFAT and MEF-2 transcription factors.

Calsequestrin-2 (CASQ2) is the main Ca2+-binding protein inside the sarcoplasmic reticulum of cardiomyocytes. Previously, we demonstrated that MEF-2 and SRF binding sites within the human CASQ2 gene (hCASQ2) promoter region are functional in neonatal cardiomyocytes. In this work, we investigated if the calcineurin/NFAT pathway regulates hCASQ2 expression in neonatal cardiomyocytes. The inhibition of NFAT dephosphorylation with CsA or INCA-6, reduced both the luciferase activity of hCASQ2 promoter constructs (-3102/+176 bp and -288/+176 bp) and the CASQ2 mRNA levels in neonatal rat cardiomyocytes. Additionally, NFATc1 and NFATc3 over-expressing neonatal cardiomyocytes showed a 2-3-fold increase in luciferase activity of both hCASQ2 promoter constructs, which was prevented by CsA treatment. Site-directed mutagenesis of the -133 bp MEF-2 binding site prevented trans-activation of hCASQ2 promoter constructs induced by NFAT overexpression. Chromatin Immunoprecipitation (ChIP) assays revealed NFAT and MEF-2 enrichment within the -288 bp to +76 bp of the hCASQ2 gene promoter. Besides, a direct interaction between NFAT and MEF-2 proteins was demonstrated by protein co-immunoprecipitation experiments. Taken together, these data demonstrate that NFAT interacts with MEF-2 bound to the -133 bp binding site at the hCASQ2 gene promoter. In conclusion, in this work, we demonstrate that the Ca2+-calcineurin/NFAT pathway modulates the transcription of the hCASQ2 gene in neonatal cardiomyocytes.

Histone acetyltransferase p300 promotes MRTF-A-mediates transactivation of VE-cadherin gene in human umbilical vein endothelial cells

Vascular endothelial cadherin (VE-cadherin) is the major determinant of endothelial cell contact integrity and is required in vascular development and angiogenesis. Serum response factor (SRF) plays essential roles in postnatal retinal angiogenesis and adult neovascularization. It is unclear whether transcription of VE-cadherin is mediated by a SRF co-activator, myocardin-related transcription factor-A (MRTF-A). Here we have demonstrated that MRTF-A is a key regulatory factor to activate the transcription of VE-cadherin in human umbilical vein endothelial cells (HUVECs). siRNA-mediated knockdown of MRTF-A decreased the level of VE-cadherin in HUVECs. Vascular endothelial growth factor (VEGF) induced MRTF-A binding to the SRF-binding site (CArG box) within VE-cadherin promoter. Histone acetyltransferase p300 and MRTF-A could synergistically augment the expression of VE-cadherin by enhancing acetylation of histone3K9 (H3K9Ac), histone3K14 (H3K14Ac) and histone4 at the SRF-binding site within VE-cadherin promoter. Taken together, these data identified a detailed regulatory mechanism of VE-cadherin gene expression.

Abstract 3197: RNase-L modulates transcriptional and post-transcriptional responses to mitogenic stimuli through serum response factor and tristetraprolin.

Abstract RNase-L is an endoribonuclease that functions in a range of important physiologic activities including the innate immune response and control of cell proliferation and is dysregulated in pathologic conditions including cancer. RNase-L is tightly regulated and requires the binding of 2’,5’-linked oligoadenylates (2-5A: px5′A(2′p5′A)n; x=1-2;n>2) to induce oligomerization and enzymatic activity. Activated RNase-L cleaves single-stranded regions of cellular and pathogen RNAs, and distinct profiles of RNA are targeted by RNase-L to mediate its biologic functions in different cellular contexts. Therefore an understanding of the mechanism(s) by which specific RNAs are targeted for RNase-L cleavage is essential to modulate its substrate selection and biologic activity for therapeutic applications. Towards this goal, we identified the RNA binding protein tristetraprolin (TTP) as a novel RNase-L interacting partner. TTP binds to A-U-rich elements (AREs) in the 3’-untranslated region (3’UTR) of target mRNAs, including its own mRNA, to promote their degradation. We hypothesize that TTP functions to direct RNase-L cleavage of a subset of ARE mRNAs. Consistent with this prediction, RNase-L interacted with TTP mRNA, and decreased steady-state levels and increased half-life of TTP mRNA in RNase-L-/- fibroblasts. In addition to this direct effect on basal TTP mRNA stability, RNase-L inhibited the transcriptional induction of TTP primary transcripts following serum stimulation. In this context, we determined that RNase-L targeted the mRNA encoding serum response factor (SRF), a transcription factor that induces the expression of proliferation and tumorigenesis-associated genes. The TTP promoter contains SRF binding sites suggesting that the RNase-L-mediated downregulation of SRF indirectly reduced TTP transcription; experimental validation of this regulation is in progress. Together these data support a model in which following mitogen stimulation, RNase-L constrains the transcriptional induction of proliferation-associated genes via its regulation of SRF and interacts with TTP to post-transcriptionally downregulate an overlapping set of mRNAs. The RNase-L-dependent regulation of TTP, which itself functions to limit the mitogenic response, suggests that RNase-L contributes to proliferative homeostasis through the regulation of both positive and negative effectors. These findings have implications for the therapeutic targeting of RNase-L-mediated cleavage in regenerative repair and tumorigenesis. Citation Format: Sarah E. Brennan-Laun, X-L Li, Heather J. Ezelle, Gerald M. Wilson, Bret A. Hassel. RNase-L modulates transcriptional and post-transcriptional responses to mitogenic stimuli through serum response factor and tristetraprolin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3197. doi:10.1158/1538-7445.AM2013-3197

Expression of Serum Response Factor in Gastric Carcinoma and Its Molecular Mechanisms Involved in the Regulation of the Invasion and Migration of SGC-7901 Cells

Serum response factor (SRF) is a transcription factor of the MADS box family. To date, DNA binding sites for SRF [serum response elements (SREs)] have been found in the promoters of approximately 50 different genes known to be involved in the regulation cell proliferation, differentiation, and apoptosis. Recent studies have indicated that SRF plays a role in the development of some tumors, including hepatocellular, thyroid, esophageal, and lung carcinomas. However, expression of SRF and its roles in gastric carcinoma are unclear. We found SRF to be highly expressed in human gastric carcinoma as well as ectopic or reduced expression for E-cadherin and β-catenin. Blockage of SRF expression was found to inhibit proliferation, invasion, and migration. We also found that an inhibitor (Y-27632) of Rho-associated coiled kinase (ROCK1), a regulator of actin cytoskeleton that regulates cell adhesion, migration, and motility, suppressed SRF expression as well. These results demonstrate that SRF is involved in the aggressive behavior of gastric carcinoma cells. We also found that the inhibition of ROCK1 by Y-27632 can inhibit the invasion and migration of gastric cells done at least, in part, by attenuating SRF expression.

Acetylation of Myocardin Is Required for the Activation of Cardiac and Smooth Muscle Genes

Myocardin belongs to the SAF-A/B, Acinus, PIAS (SAP) domain family of transcription factors and is specifically expressed in cardiac and smooth muscle. Myocardin functions as a transcriptional coactivator of SRF and is sufficient and necessary for smooth muscle gene expression. We have previously found that myocardin induces the acetylation of nucleosomal histones surrounding SRF-binding sites in the control regions of cardiac and smooth muscle genes through recruiting chromatin-modifying enzyme p300, yet no studies have determined whether myocardin itself is similarly modified. In this study, we show that myocardin is a direct target for p300-mediated acetylation. p300 acetylates lysine residues at the N terminus of the myocardin protein. Interestingly, a direct interaction between p300 and myocardin, which is mediated by the C terminus of myocardin, is required for the acetylation event. Acetylation of myocardin by p300 enhances the association of myocardin and SRF as well as the formation of the myocardin-SRF-CArG box ternary complex. Conversely, acetylation of myocardin decreases the binding of histone deacetylase 5 (HDAC5) to myocardin. Acetylation of myocardin is required for myocardin to activate smooth muscle genes. Our study demonstrates that acetylation plays a key role in modulating myocardin function in controlling cardiac and smooth muscle gene expression.

RhoA as a Mediator of Clinically Relevant Androgen Action in Prostate Cancer Cells

Recently, we have identified serum response factor (SRF) as a mediator of clinically relevant androgen receptor (AR) action in prostate cancer (PCa). Genes that rely on SRF for androgen responsiveness represent a small fraction of androgen-regulated genes, but distinguish benign from malignant prostate, correlate with aggressive disease, and are associated with biochemical recurrence. Thus, understanding the mechanism(s) by which SRF conveys androgen regulation to its target genes may provide novel opportunities to target clinically relevant androgen signaling. Here, we show that the small GTPase ras homolog family member A (RhoA) mediates androgen-responsiveness of more than half of SRF target genes. Interference with expression of RhoA, activity of the RhoA effector Rho-associated coiled-coil containing protein kinase 1 (ROCK), and actin polymerization necessary for nuclear translocation of the SRF cofactor megakaryocytic acute leukemia (MAL) prevented full androgen regulation of SRF target genes. Androgen treatment induced RhoA activation, increased the nuclear content of MAL, and led to MAL recruitment to the promoter of the SRF target gene FHL2. In clinical specimens RhoA expression was higher in PCa cells than benign prostate cells, and elevated RhoA expression levels were associated with aggressive disease features and decreased disease-free survival after radical prostatectomy. Overexpression of RhoA markedly increased the androgen-responsiveness of select SRF target genes, in a manner that depends on its GTPase activity. The use of isogenic cell lines and a xenograft model that mimics the transition from androgen-stimulated to castration-recurrent PCa indicated that RhoA levels are not altered during disease progression, suggesting that RhoA expression levels in the primary tumor determine disease aggressiveness. Androgen-responsiveness of SRF target genes in castration-recurrent PCa cells continued to rely on AR, RhoA, SRF, and MAL and the presence of intact SRF binding sites. Silencing of RhoA, use of Rho-associated coiled-coil containing protein kinase 1 inhibitors, or an inhibitor of SRF-MAL interaction attenuated (androgen-regulated) cell viability and blunted PCa cell migration. Taken together, these studies demonstrate that the RhoA signaling axis mediates clinically relevant AR action in PCa.

Actin-dependent activation of serum response factor in T cells by the viral oncoprotein tip

Serum response factor (SRF) acts as a multifunctional transcription factor regulated by mutually exclusive interactions with ternary complex factors (TCFs) or myocardin-related transcription factors (MRTFs). Binding of Rho- and actin-regulated MRTF:SRF complexes to target gene promoters requires an SRF-binding site only, whereas MAPK-regulated TCF:SRF complexes in addition rely on flanking sequences present in the serum response element (SRE). Here, we report on the activation of an SRE luciferase reporter by Tip, the viral oncoprotein essentially contributing to human T-cell transformation by Herpesvirus saimiri. SRE activation in Tip-expressing Jurkat T cells could not be attributed to triggering of the MAPK pathway. Therefore, we further analyzed the contribution of MRTF complexes. Indeed, Tip also activated a reporter construct responsive to MRTF:SRF. Activation of this reporter was abrogated by overexpression of a dominant negative mutant of the MRTF-family member MAL. Moreover, enrichment of monomeric actin suppressed the Tip-induced reporter activity. Further upstream, the Rho-family GTPase Rac, was found to be required for MRTF:SRF reporter activation by Tip. Initiation of this pathway was strictly dependent on Tip's ability to interact with Lck and on the activity of this Src-family kinase. Independent of Tip, T-cell stimulation orchestrates Src-family kinase, MAPK and actin pathways to induce SRF. These findings establish actin-regulated transcription in human T cells and suggest its role in viral oncogenesis.

The miR-143/145 Cluster Is a Novel Transcriptional Target of Jagged-1/Notch Signaling in Vascular Smooth Muscle Cells

Activation of Notch signaling by Jagged-1 (Jag-1) in vascular smooth muscle cells (VSMC) promotes a differentiated phenotype characterized by increased expression of contractile proteins. Recent studies show that microRNAs (miR)-143/145 regulates VSMC phenotype. The serum response factor (SRF)/myocardin complex binds to CArG sequences to activate miR-143/145 transcription, but no other regulators are known in VSMC. Using miR arrays, we found miR-143/145 induced following expression of a constitutively active Notch1 intracellular domain (N1ICD). We hypothesized that miR-143/145 is required for Jag-1/Notch-induced VSMC differentiation. Activation of Notch receptors by Jag-1 caused CBF1-dependent up-regulation of miR-143/145, increased differentiation, and decreased proliferation. Conversely, inhibiting basal Notch signaling decreased steady state levels of miR-143/145. Using SRF knockdown, we found that Jag-1/Notch induction of miR-143/145 is SRF independent, although full acquisition of contractile markers requires SRF. Using miR-143/145 promoter reporter constructs we show Jag-1/Notch increases promoter activity, and this is dependent on intact CBF1 consensus sites within the promoter. Chromatin immunoprecipitation (ChIP) assays revealed that N1ICD-containing complexes bind to CBF1 sites in the miR-143/145 promoter. We also identified N1ICD complex binding to CBF1 sites within the endogenous human miR-143/145 promoter. Using miR-143/145-interfering oligonucleotides, we demonstrate that Jag-1/Notch signaling requires induction of both miR-143 and miR-145 to promote the VSMC contractile phenotype. Thus, miR-143/145 is a novel transcriptional target of Jag-1/Notch signaling in VSMC. We propose miR-143/145 as activated independently by Jag-1/Notch and SRF in parallel pathways. Multiple pathways converging on miR-143/145 provides potential for fine-tuning or amplification of VSMC differentiation signals.

RTVP-1 expression is regulated by SRF downstream of protein kinase C and contributes to the effect of SRF on glioma cell migration

Gliomas are characterized by increased infiltration into the surrounding normal brain tissue. We recently reported that RTVP-1 is highly expressed in gliomas and plays a role in the migration of these cells, however the regulation of RTVP-1 expression in these cells is not yet described. In this study we examined the role of PKC in the regulation of RTVP-1 expression and found that PMA and overexpression of PKCα and PKCε increased the expression of RTVP-1, whereas PKCδ exerted an opposite effect. Using the MatInspector software, we identified a SRF binding site on the RTVP-1 promoter. Chromatin immunoprecipitation (ChIP) assay revealed that SRF binds to the RTVP-1 promoter in U87 cells, and that this binding was significantly increased in response to serum addition. Moreover, silencing of SRF blocked the induction of RTVP-1 expression in response to serum. We found that overexpression of PKCα and PKCε increased the activity of the RTVP-1 promoter and the binding of SRF to the promoter. In contrast, overexpression of PKCδ blocked the increase in RTVP-1 expression in response to serum and the inhibitory effect of PKCδ was abrogated in cells expressing a SRFT160A mutant. SRF regulated the migration of glioma cells and its effect was partially mediated by RTVP-1. We conclude that RTVP-1 is a PKC-regulated gene and that this regulation is at least partly mediated by SRF. Moreover, RTVP-1 plays a role in the effect of SRF on glioma cell migration.

Serum Response Factor Utilizes Distinct Promoter- and Enhancer-Based Mechanisms To Regulate Cytoskeletal Gene Expression in Macrophages

Cells of the monocyte/macrophage lineage play essential roles in tissue homeostasis and immune responses, but mechanisms underlying the coordinated expression of cytoskeletal genes required for specialized functions of these cells, such as directed migration and phagocytosis, remain unknown. Here, using genetic and genomic approaches, we provide evidence that serum response factor (SRF) regulates both general and cell type-restricted components of the cytoskeletal gene expression program in macrophages. Genome-wide location analysis of SRF in macrophages demonstrates enrichment of SRF binding at ubiquitously expressed target gene promoters, as expected, but also reveals that the majority of SRF binding sites associated with cell type-restricted target genes are at distal inter- and intragenic locations. Most of these distal SRF binding sites are established by the prior binding of the macrophage- and the B cell-specific transcription factor PU.1 and exhibit histone modifications characteristic of enhancers. Consistent with this, representative cytoskeletal target genes associated with these elements require both SRF and PU.1 for full expression. These findings suggest that SRF uses two distinct molecular strategies to regulate programs of cytoskeletal gene expression: a promoter-based strategy for ubiquitously expressed target genes and an enhancer-based strategy at target genes that exhibit cell type-restricted patterns of expression.

Identification of New SRF Binding Sites in Genes Modulated by SRF Over-Expression in Mouse Hearts

Background:To identify in vivo new cardiac binding sites of serum response factor (SRF) in genes and to study the response of these genes to mild over-expression of SRF, we employed a cardiac-specific, transgenic mouse model, with mild over-expression of SRF (Mild-O SRF Tg).Methodology:Microarray experiments were performed on hearts of Mild-O-SRF Tg at 6 months of age. We identified 207 genes that are important for cardiac function that were differentially expressed in vivo. Among them the promoter region of 192 genes had SRF binding motifs, the classic CArG or CArG-like (CArG-L) elements. Fifty-one of the 56 genes with classic SRF binding sites had not been previously reported. These SRF-modulated genes were grouped into 12 categories based on their function. It was observed that genes associated with cardiac energy metabolism shifted toward that of carbohydrate metabolism and away from that of fatty acid metabolism. The expression of genes that are involved in transcription and ion regulation were decreased, but expression of cytoskeletal genes was significantly increased. Using public databases of mouse models of hemodynamic stress (GEO database), we also found that similar altered expression of the SRF-modulated genes occurred in these hearts with cardiac ischemia or aortic constriction as well.Conclusion and significance:SRF-modulated genes are actively regulated under various physiological and pathological conditions. We have discovered that a large number of cardiac genes have classic SRF binding sites and were significantly modulated in the Mild-O-SRF Tg mouse hearts. Hence, the mild elevation of SRF protein in the heart that is observed during typical adult aging may have a major impact on many SRF-modulated genes, thereby affecting cardiac structure and performance. The results from our study could help to enhance our understanding of SRF regulation of cellular processes in the aged heart.

Myocardin-Related Transcription Factor A Is a Common Mediator of Mechanical Stress- and Neurohumoral Stimulation-Induced Cardiac Hypertrophic Signaling Leading to Activation of Brain Natriuretic Peptide Gene Expression

Subjecting cardiomyocytes to mechanical stress or neurohumoral stimulation causes cardiac hypertrophy characterized in part by reactivation of the fetal cardiac gene program. Here we demonstrate a new common mechanism by which these stimuli are transduced to a signal activating the hypertrophic gene program. Mechanically stretching cardiomyocytes induced nuclear accumulation of myocardin-related transcription factor A (MRTF-A), a coactivator of serum response factor (SRF), in a Rho- and actin dynamics-dependent manner. Expression of brain natriuretic peptide (BNP) and other SRF-dependent fetal cardiac genes in response to acute mechanical stress was blunted in mice lacking MRTF-A. Hypertrophic responses to chronic pressure overload were also significantly attenuated in mice lacking MRTF-A. Mutation of a newly identified, conserved and functional SRF-binding site within the BNP promoter, or knockdown of MRTF-A, reduced the responsiveness of the BNP promoter to mechanical stretch. Nuclear translocation of MRTF-A was also involved in endothelin-1- and angiotensin-II-induced activation of the BNP promoter. Moreover, mice lacking MRTF-A showed significantly weaker hypertrophic responses to chronic angiotensin II infusion than wild-type mice. Collectively, these findings point to nuclear translocation of MRTF-A as a novel signaling mechanism mediating both mechanical stretch- and neurohumoral stimulation-induced BNP gene expression and hypertrophic responses in cardiac myocytes.

Expression of Human Frataxin Is Regulated by Transcription Factors SRF and TFAP2

BackgroundFriedreich ataxia is an autosomal recessive neurodegenerative disease caused by reduced expression levels of the frataxin gene (FXN) due to expansion of triplet nucleotide GAA repeats in the first intron of FXN. Augmentation of frataxin expression levels in affected Friedreich ataxia patient tissues might substantially slow disease progression.Methodology/Principal FindingsWe utilized bioinformatic tools in conjunction with chromatin immunoprecipitation and electrophoretic mobility shift assays to identify transcription factors that influence transcription of the FXN gene. We found that the transcription factors SRF and TFAP2 bind directly to FXN promoter sequences. SRF and TFAP2 binding sequences in the FXN promoter enhanced transcription from luciferase constructs, while mutagenesis of the predicted SRF or TFAP2 binding sites significantly decreased FXN promoter activity. Further analysis demonstrated that robust SRF- and TFAP2-mediated transcriptional activity was dependent on a regulatory element, located immediately downstream of the first FXN exon. Finally, over-expression of either SRF or TFAP2 significantly increased frataxin mRNA and protein levels in HEK293 cells, and frataxin mRNA levels were also elevated in SH-SY5Y cells and in Friedreich ataxia patient lymphoblasts transfected with SRF or TFAP2.Conclusions/SignificanceWe identified two transcription factors, SRF and TFAP2, as well as an intronic element encompassing EGR3-like sequence, that work together to regulate expression of the FXN gene. By providing new mechanistic insights into the molecular factors influencing frataxin expression, our results should aid in the discovery of new therapeutic targets for the treatment of Friedreich ataxia.