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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Definition of the fibroblast SRF-mediated immediate-early transcriptional response

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Aengus Stewart

PROVIDER: E-GEOD-45888 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Definition of the fibroblast SRF-mediated immediate-early transcriptional response

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

2014-04-24 | GSE45888 | GEO

MRTF-SRF signaling is required for seeding of HSC/Ps in bone marrow during development

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

MRTF-SRF signalling in the T cell IL2 response

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:

2023-10-23 | GSE241689 | GEO

Regulation of nuclear actin levels and MRTF/SRF target gene expression during PC6.3 cell differentiation

Project description:Actin has important functions in both cytoplasm and nucleus of the cell, with active nuclear transport mechanisms maintaining the cellular actin balance. Nuclear actin levels are subject to regulation during many cellular processes from cell differentiation to cancer. Here we show that nuclear actin levels increase upon differentiation of PC6.3 cells towards neuron-like cells. Photobleaching experiments demonstrate that this increase is due to decreased nuclear export of actin during cell differentiation. Increased nuclear actin levels lead to decreased nuclear localization of MRTF-A, a well-established transcription cofactor of SRF. In line with MRTF-A localization, transcriptomics analysis reveals that MRTF/SRF target gene expression is first transiently activated, but then substantially downregulated during PC6.3 cell differentiation. This study therefore describes a novel cellular context, where regulation of nuclear actin is utilized to tune MRTF/SRF target gene expression during cell differentiation.

2022-06-27 | GSE206781 | GEO

MRTF-SRF signaling is required for seeding of HSC/Ps in bone marrow during development

2015-03-04 | GSE63820 | GEO

The CAP1 represses MRTF-SRF–dependent gene expression in the mouse cerebral cortex by an actin-dependent mechanism

Project description:Serum response factor (SRF) is a ubiquitously expressed transcription factor that is essential for brain development and function. SRF activity is controlled by two competing classes of coactivators, myocardin-related transcription factors (MRTF) and ternary complex factors, which introduce specificity into gene expression programs. Here, we explored the MRTF-mediated regulatory mechanism in mouse cortical neurons. Using gene-reporter assays and pharmacological and genetic approaches in isolated mouse cortical neurons, we found that cyclase-associated protein 1 (CAP1) repressed neuronal MRTF-SRF activity. CAP1 promoted cytosolic retention of MRTF by controlling cytosolic G-actin levels that required its helical folded domain and its CARP domain. This function of CAP1 was not redundant with that of its homolog CAP2 and was independent of cofilin1 and actin-depolymerizing factor. Deep RNA sequencing and mass spectrometry in cerebral cortex lysates from CAP1 knockout (CAP1-KO) mice supported the in vivo relevance for the CAP1-actin-MRTF-SRF signaling axis. Our study identified CAP1 as a repressor of neuronal gene expression and led to the identification of likely MRTF-SRF target genes in the developing cerebral cortex, whose dysregulation may contribute to impaired formation of neuronal networks in CAP1-KO mice. Together with our previous studies that implicated CAP1 in actin dynamics in axonal growth cones or excitatory synapses, we established CAP1 as a crucial actin regulator in neurons.

2024-03-22 | GSE262154 | GEO

Lap2α is required for MRTF-A-SRF transcriptional activity [ChIP-seq]

Project description:Myocardin-related transcription factor A (MRTF-A) in complex with the serum response factor (SRF) regulates the expression of cytoskeletal genes in response to cytoplasmic and nuclear actin dynamics. Different components of nucleoskeleton, besides nuclear actin, also regulate the activity of MRTF-A. Here we extend these studies by showing that lamina-associated polypeptide 2α (Lap2α), the nucleoplasmic isoform of Lap2, is a novel regulator, and a direct binding partner of MRTF-A. Lap2α is a nucleoplasmic protein that interacts with A-type lamins and the retinoblastoma protein (pRb) and contributes to chromatin organization. Unlike other known MRTF-A regulators, Lap2α is not required for MRTF-A nucleo-cytoplasmic shuttling; it functions within the nucleus where it binds MRTF-A directly via its unique C-terminal domain prior to MRTF-A forming the complex with chromatin and SRF. Such interaction affects MRTF-A transcriptional activity since genome-wide analysis revealed reduced binding of MRTF-A to SRF target genes in Lap2α knockout cells compared to control cells that consequently led to impaired expression of target genes. Our studies therefore add another regulatory layer to the control MRTF-A-SRF-mediated gene expression, and broaden the role of Lap2α in transcriptional regulation.

2020-12-15 | GSE159370 | GEO

Lap2α is required for MRTF-A-SRF transcriptional activity [RNA-seq]

2020-12-15 | GSE159369 | GEO

Treatment of melanoma cell lines with CCG-222740

Project description:The Ras/MEK/ERK pathway has been the primary focus of targeted therapies in melanoma, given that it is aberrantly activated in almost 80% of human cutaneous melanomas (~50% BRAFV600 mutations and ~30% NRAS mutations). While targeted therapies have yielded success in BRAFV600 mutant melanoma patients, such therapies have been ineffective in NRAS mutant melanomas in part due to their cytostatic effects and primary resistance in this patient population. Here, we demonstrate that increased Rho/MRTF-pathway activation correlates with high intrinsic resistance to trametinib, a MEK inhibitor, in a panel of NRAS mutant melanoma cell lines. Combination of trametinib with the Rho/MRTF-pathway inhibitor, CCG-222740, synergistically reduced cell viability in NRAS mutant melanoma cell lines in vitro. Furthermore, the combination of CCG-222740 with trametinib induced apoptosis and reduced clonogenicity in the highly trametinib-resistant SK-Mel-147 cells. These findings suggest a role of the Rho/MRTF-pathway in high intrinsic trametinib resistance to a subset of NRAS mutant melanoma cell lines and highlights the potential of concurrently targeting the Rho/MRTF-pathway and MEK in NRAS mutant melanomas.

2018-12-30 | GSE115938 | GEO

Treatment of melanoma cell lines with CCG-222740

2019-07-16 | GSE134320 | GEO

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