Definition of the fibroblast SRF-mediated immediate-early transcriptional response
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE45888 | GEO | 2014/04/24
SECONDARY ACCESSION(S): PRJNA196820
REPOSITORIES: GEO
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Project description:To control transcription, SRF recruits signal-regulated co-activators, the Ternary Complex Factors (TCFs) and the Myocardin-related Transcription Factors (MRTFs), which compete for a common site on its DNA-binding domain. The TCFs - SAP-1, Elk-1 and Net - are Ets proteins that link SRF activity to Ras-ERK signalling. In contrast, the two MRTFs, MRTF-A and MRTF-B, link SRF activity to Rho-actin signalling. In this novel signalling pathway, the actin-binding MRTF RPEL domain acts as a G-actin sensor, controlling MRTF nuclear accumulation in response to signal-induced depletion of the G-actin pool. Previous studies have suggested that the Ras-ERK signalling and Rho-actin pathways control specific subsets of SRF target genes. We used ChIP-seq and RNA-seq to analyse the immediate-early transcriptional response in NIH3T3 fibroblasts, using pathway-specific inhibitors to identify the contributions of Ras-ERK and Rho-actin signalling Chromatin immunoprecipitation and sequencing (ChIP-seq) in NIH3T3 fibroblast after serum stimulation in presence or absence of LatrunculinB or U0126 drugs and using antibodies against SRF, MRTF-A, MRTF-B, SAP1, ELK1, NET, Pol II, PolII S5P, PolII S2P and total H3. Validation by ChIP-PCR. Strand specific total-RNA-seq following DSN normalisation and validation by qRT-PCR from NIH3T3 stimulated by serum or Cytochalasin D in presence or absence of LatrunculinB and/or U0126 drugs.
2014-04-24 | E-GEOD-45888 | biostudies-arrayexpress
Project description:The transcription factor SRF is a master regulator of growth factor inducible and cytoskeletal gene expression. Two transcription cofactor families, the TCFs & the MRTFs, are responsible for SRF's activity to direct transcriptional programme in response to extracellular signalling through the Ras-ERK and Rho-actin pathways respectively. We are investigating the role of the SRF network in the response to infection, using the Listeria monocytogenes model. We find that inactivation of the network impairs the ability of CD8 T cells to proliferate in response to listeria and to generate memory cells. Surprisingly, the defect reflects impaired MRTF-SRF signalling rather than the TCF-SRF signalling pathway usually associated with proliferation. Moreover, the defec doesn'tt lie downstream of the initial activation of the TCR, appearing instead to arise from an inability of CD8 T cells to present IL-2 to each other in paracrine fashion. Consistent with these observations, we find that while cultured CD8 MRTF-null T cells can be effectively activated by TCR crosslinking invitro, their ability to form clusters, which is IL-2-dependent, is impaired. Moreover, as expected, pilot experiments indicate that these cells exhibit deficits both in the basal levels of MRTF-SRF target gene expression, and in their ability to induce these genes in response to IL-2 stimulation:
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Project description:Myocardin-related transcription factors (MRTFs) play a central role in the regulation of actin expression and cytoskeletal dynamics. Stimuli that promote actin polymerization allow for shuttling of MRTFs to the nucleus where they activate serum response factor (SRF), a regulator of actin and other cytoskeletal protein genes. SRF is an essential regulator of skeletal muscle differentiation and numerous components of the muscle sarcomere, but the potential involvement of MRTFs in skeletal muscle development has not been examined. We explored the role of MRTFs in muscle development in vivo by generating mutant mice harboring a skeletal muscle-specific deletion of MRTF-B and a global deletion of MRTF-A. These double knockout (dKO) mice were able to form sarcomeres during embryogenesis. However, the sarcomeres were abnormally small and disorganized, causing skeletal muscle hypoplasia and perinatal lethality. Transcriptome analysis demonstrated dramatic dysregulation of actin genes in MRTF dKO mice, highlighting the importance of MRTFs in actin cycling and myofibrillogenesis. MRTFs were also necessary for the survival of skeletal myoblasts and for the efficient formation of intact myotubes. Our findings reveal a central role for MRTFs in sarcomere formation during skeletal muscle development and point to the potential involvement of these transcriptional coactivators in skeletal myopathies. Gene expression profile was generated comparing wild type (WT) and HSA-Cre, MRTF-A/B double knockout mice, by deep seqencing, with three biological replicates, using Illumina HiSeq 2500.
2016-08-02 | E-GEOD-84063 | biostudies-arrayexpress
Project description:Myocardin-related transcription factors (MRTFs) play a central role in the regulation of actin expression and cytoskeletal dynamics. Stimuli that promote actin polymerization allow for shuttling of MRTFs to the nucleus where they activate serum response factor (SRF), a regulator of actin and other cytoskeletal protein genes. SRF is an essential regulator of skeletal muscle differentiation and numerous components of the muscle sarcomere, but the potential involvement of MRTFs in skeletal muscle development has not been examined. We explored the role of MRTFs in muscle development in vivo by generating mutant mice harboring a skeletal muscle-specific deletion of MRTF-B and a global deletion of MRTF-A. These double knockout (dKO) mice were able to form sarcomeres during embryogenesis. However, the sarcomeres were abnormally small and disorganized, causing skeletal muscle hypoplasia and perinatal lethality. Transcriptome analysis demonstrated dramatic dysregulation of actin genes in MRTF dKO mice, highlighting the importance of MRTFs in actin cycling and myofibrillogenesis. MRTFs were also necessary for the survival of skeletal myoblasts and for the efficient formation of intact myotubes. Our findings reveal a central role for MRTFs in sarcomere formation during skeletal muscle development and point to the potential involvement of these transcriptional coactivators in skeletal myopathies.
2016-08-02 | GSE84063 | GEO
Project description:Specific spectra of chromatin modifications are associated with different transcriptional states and gene regulatory elements, but relatively little is known about how such chromatin modifications are established. In particular the relationship of chromatin modifications to the activity of specific transcription factors and the signalling pathways that control them remain largely unclear. We investigated the relationship between chromatin modifications and transcription in response to Ras/ERK activationby stimulating cells with TPA (12-O-tetradecanoyl phorbol-13-acetate) for 30 minutes. Oncogenic active Ras is known to mediate the cellular response to multiple growth factors leading to the activation of immediate-early genes (IE). A key player in IE gene response is the SRF transcriptional network that we identified being crucial to mediate allegedly the entire transcriptional response downstream of Ras/ERK. The Serum response factor (SRF) is a transcription factor with low intrinsic transcriptional activity and requires the recruitment of one of two families of co-activators: the MRTFs (myocardin-related transcription factors) and the TCFs (ternary complex factors). In particular the TCF, member of the wide family of ETS transcription factors, are specifically activated by MAPK signalling downstream of Ras. Cells lacking all three TCFs are defective in responding to Ras/MAPK signalling. We analysed changes in 5 main histone modifications (H3K9acS10ph, H4K16ac, H3K27ac, H3K9acK14ac and H3K4me3) associated with active transcription by ChIP-seq in Mouse Embryonic Fibroblasts (MEFs) derived from wild type mice (MEF WT) or animals lacking all three TCFs (TCF KO). In addition the distribution of the 5 modifications have been assessed in TCF KO cells reconstituted with Elk-1 (one of the member of the TCF factos) variants.
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Project description:Ras plays roles across the spectrum of proliferation, invasion and metastasis. The activity of Ras genes, usually K-Ras, is altered by mutations in around 20% of human cancers, and activation of the Ras-ERK pathway is a critical event in melanomas, colorectal and renal cancers but the degree to which Ras- or Raf-driven transformation is dependent on the activity of the SRF network remains largely obscure. Oncogenic active Ras is known to mediate the cellular response to multiple growth factors leading to the activation of immediate-early genes (IE). A key player in IE gene response is the SRF transcriptional network that we identified being crucial to mediate allegedly the entire transcriptional response downstream of Ras/ERK. The Serum response factor (SRF) is a transcription factor with low intrinsic transcriptional activity and requires the recruitment of one of two families of co-activators: the MRTFs (myocardin-related transcription factors) and the TCFs (ternary complex factors, Elk-1, SAP-1, Net). In particular the TCF, member of the wide family of ETS transcription factors, are specifically activated by MAPK signalling downstream of Ras. We characterised the transcriptional program downstream of Ras/ERK signalling using short TPA stimulations (12-O-tetradecanoyl phorbol-13-acetate, compunt known to induced Ras/ERK activation) by RNA-seq. The investigation of the transcriptional response was performed using Mouse Embryonic Fibroblasts (MEFs) derived from different animals: (i) wild type, (ii) TKO (MEFs derived from mice lacking all three TCFs), (iiI) TKO recon pMY Empty (TKO reconstituted with empty vectors by retrovirus infection), (iv) TKO recon Elk-1 wt (TKO reconstituted with retrovirus vectors expressing Elk-1 wild type by retrovirus infection), (v) TKO recon Elk-1 FW (TKO reconstituted with retrovirus vectors expressing Elk-1 mutant lacking the FW residues in its activation domain), (vi) TKO recon Elk-1 nonA (TKO reconstituted with retrovirus vectors expressing Elk-1 mutant where all phosphor-acceptor residues in its activation domain have been mutagenized to alanines). In order to map SRF and Elk-1 binding sites we performed ChIP-seq of SRF in wild type MEFs and TKO and ChIP-seq of Elk-1 in TKO reconstituted cell lines.
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Project description:Myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF), and thereby regulate cytoskeletal gene expression in response to actin dynamics. MRTFs have also been implicated in heat shock protein (hsp) transcription in fly ovaries, but the mechanisms remain unclear. Here we demonstrate that in mammalian cells, MRTFs are dispensable for hsp gene induction. However, the widely used small molecule inhibitors of MRTF/SRF transcription pathway, derived from CCG-1423, efficiently inhibit hsp gene transcription in both fly and mammalian cells also in absence of MRTFs. Quantifying RNA synthesis and RNA polymerase distribution demonstrates that CCG-1423-derived compounds have a genome-wide effect on transcription. Indeed, tracking nascent transcription at nucleotide resolution reveals that CCG-1423-derived compounds reduce RNA polymerase II elongation, and severely dampen the transcriptional response to heat shock. The effects of CCG-1423-derived compounds therefore extend beyond the MRTF/SRF pathway into nascent transcription, opening novel opportunities for their use in transcription research.
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Project description:Myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF), and thereby regulate cytoskeletal gene expression in response to actin dynamics. MRTFs have also been implicated in heat shock protein (hsp) transcription in fly ovaries, but the mechanisms remain unclear. Here we demonstrate that in mammalian cells, MRTFs are dispensable for hsp gene induction. However, the widely used small molecule inhibitors of MRTF/SRF transcription pathway, derived from CCG-1423, efficiently inhibit hsp gene transcription in both fly and mammalian cells also in absence of MRTFs. Quantifying RNA synthesis and RNA polymerase distribution demonstrates that CCG-1423-derived compounds have a genome-wide effect on transcription. Indeed, tracking nascent transcription at nucleotide resolution reveals that CCG-1423-derived compounds reduce RNA polymerase II elongation, and severely dampen the transcriptional response to heat shock. The effects of CCG-1423-derived compounds therefore extend beyond the MRTF/SRF pathway into nascent transcription, opening novel opportunities for their use in transcription research.
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Project description:Chemokine signaling is important for the seeding of different sites by hematopoietic stem cells during development. Serum Response Factor (SRF) controls multiple genes governing adhesion and migration, mainly by recruiting members of the Myocardin-Related Transcription Factor (MRTF) family of G-actin regulated cofactors. We used vav-iCre to inactivate MRTF-SRF signaling early during hematopoietic development. In both Srf- and Mrtf-deleted animals, hematopoiesis in fetal liver and spleen is intact, but does not become established in fetal bone marrow. Srf-null HSC/Ps (hematopoietic stem/progenitor cells) fail to effectively engraft in transplantation experiments, exhibiting normal proximal signaling responses to SDF-1, but reduced adhesiveness, F-actin assembly, and reduced motility. Srf-null HSC/Ps fail to polarise in response to SDF-1, and cannot migrate through restrictive membrane pores to SDF-1 or Scf in vitro. Mrtf-null HSC/Ps were also defective in chemotactic responses to SDF-1. MRTF-SRF signaling is thus critical for the response to chemokine signaling during hematopoietic development. Strand specific RNA sequencing (RNA-seq) in sorted WT and SRF deleted LSK cells with or without a 30 minute SDF stimulation and validation by qRT-PCR
2015-03-04 | E-GEOD-63820 | biostudies-arrayexpress