Specific and redundant roles of TEAD transcription factors in C2C12 cell and primary myoblast differentiation (ChIP-Seq)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE82190 | GEO | 2017/02/17
SECONDARY ACCESSION(S): PRJNA324278
REPOSITORIES: GEO
Similar Datasets
Project description:Defining the function of TEAD transcription factors in myogenic differentiation has proved elusive due to overlapping expression and functional redundancy. Here, we show that siRNA silencing of either Tead1, Tead2 or Tead4 did not effect differentiation of primary myoblasts (PMs) while their simultaneous knockdown strongly impaired differentiation. In contrast in C2C12 cells, silencing of Tead1 or Tead4 impaired differentiation showing a differential requirement for these factors in PMs and C2C12 cells that involved both differential regulation of their expression and intracellular localisation. Through integration of Tead1 and Tead4 ChIP-seq with chromatin modifications, we identify active enhancers associated with genes activated during C2C12 cell differentiation that are bound by combinations of Tead4, Myod1 or Myog and show a signature of frequently co-occuring motifs. We show that distinct but overlapping sets of genes are deregulated by Tead silencing in C2C12 cells and PMs therefore describing for the first time in a comprehensive manner the specific and redundant regulatory roles of Tead factors in myogenic differentiation. We also performed ChIP-seq from mouse muscle in vivo identifying a set of highly transcribed muscle cell-identity genes and revealing that Tead4 binds a distinct repertoire of sites in C2C12 cells and muscle.
2017-02-17 | GSE82192 | GEO
Project description:The TEAD (1-4) transcription factors comprise the conserved TEA/ATTS DNA binding domain recognising the MCAT element in the promoters of muscle-specific genes. Despite extensive genetic analysis, the function of TEAD factors in muscle differentiation has proved elusive due to redundancy amongst the family members. Expression of the TEA/ATTS DNA binding domain that acts a dominant negative repressor of TEAD factors in C2C12 myoblasts inhibits their differentiation, while selective shRNA knockdown of TEAD4 results in abnormal differentiation characterised by the formation of shortened myotubes. Chromatin immunoprecipitation coupled to array hybridisation (ChIP-chip) shows that TEAD4 occupies 867 promoters including those of myogenic miRNAs. We show that TEAD factors cooperate with MYOD1 to directly induce Myogenin, CDKN1A and Caveolin 3 expression to promote myoblast differentiation and fusion. RNA-seq identifies a novel set of TEAD4 target genes encoding muscle structural and regulatory proteins and those required for the unfolded protein response. In contrast, TEAD4 represses expression of the growth factor CTGF and Cyclin D1 to promote differentiation. Together these results show that TEAD factor activity is essential for C2C12 cell differentiation and define a novel and nonredundant role for TEAD4 in regulating the unfolded protein response. C2C12 cells were infected with retrotiviral vector expressing Flag-HA-Tagged TEAD4 or with empty control vector and selected in the continouos presence of puromycin. Infected cell populations were then differentiated for 5 days in DMEM medium with 2% horse serum and fixed in 0.4% formaldehyde.
2011-08-21 | E-GEOD-29208 | biostudies-arrayexpress
Project description:The TEAD (1-4) transcription factors comprise the conserved TEA/ATTS DNA binding domain recognising the MCAT element in the promoters of muscle-specific genes. Despite extensive genetic analysis, the function of TEAD factors in muscle differentiation has proved elusive due to redundancy amongst the family members. Expression of the TEA/ATTS DNA binding domain that acts a dominant negative repressor of TEAD factors in C2C12 myoblasts inhibits their differentiation, while selective shRNA knockdown of TEAD4 results in abnormal differentiation characterised by the formation of shortened myotubes. Chromatin immunoprecipitation coupled to array hybridisation (ChIP-chip) shows that TEAD4 occupies 867 promoters including those of myogenic miRNAs. We show that TEAD factors cooperate with MYOD1 to directly induce Myogenin, CDKN1A and Caveolin 3 expression to promote myoblast differentiation and fusion. RNA-seq identifies a novel set of TEAD4 target genes encoding muscle structural and regulatory proteins and those required for the unfolded protein response. In contrast, TEAD4 represses expression of the growth factor CTGF and Cyclin D1 to promote differentiation. Together these results show that TEAD factor activity is essential for C2C12 cell differentiation and define a novel and nonredundant role for TEAD4 in regulating the unfolded protein response.
2011-08-22 | GSE29208 | GEO
Project description:The TEAD (1-4) transcription factors comprise the conserved TEA/ATTS DNA binding domain recognising the MCAT element in the promoters of muscle-specific genes. Despite extensive genetic analysis, the function of TEAD factors in muscle differentiation has proved elusive due to redundancy amongst the family members. Expression of the TEA/ATTS DNA binding domain that acts a dominant negative repressor of TEAD factors in C2C12 myoblasts inhibits their differentiation, while selective shRNA knockdown of TEAD4 results in abnormal differentiation characterised by the formation of shortened myotubes. RNA-seq identifies a novel set of TEAD4 target genes encoding muscle structural and regulatory proteins and those required for the unfolded protein response. In contrast, TEAD4 represses expression of the growth factor CTGF and Cyclin D1 to promote differentiation. Together these results show that TEAD factor activity is essential for normal C2C12 cell differentiation and define a novel and non-redundant role for TEAD4 in regulating expression of the unfolded protein response genes.
2011-08-22 | GSE27845 | GEO
Project description:Abstract Hippo pathway downstream effectors Yap and Taz play key roles in cell proliferation and regeneration, regulating gene expression especially via interaction with Tead transcription factors. To investigate their role in skeletal muscle stem cells, we analysed Taz in vivo and ex vivo in comparison to Yap. Taz was expressed in activated satellite cells. siRNA knockdown or constitutive expression of wildtype or constitutively active TAZ mutants showed that TAZ promoted proliferation, a function that was shared with YAP. However, at later stages of myogenesis, TAZ also enhanced myogenic differentiation of myoblasts, whereas YAP inhibits such differentiation. Functionally, while muscle growth was mildly affected in Taz (gene symbol Wwtr1-/-) knockout mice, there were no overt effect on regeneration. However, conditional knockout of Yap in satellite cells of Pax7Cre-ERT2/+ : Yapflox/flox : Rosa26Lacz mice produced a marked regeneration deficit. To identify potential mechanisms, microarray analysis showed many common Taz/Yap targets, but Taz also regulates some genes independently of Yap, including myogenic genes such as Pax7, Myf5 and Myod1. Proteomic analysis of Yap/Taz revealed many common binding partners, but Taz also interacts with proteins distinct from Yap, that are mainly involved in myogenesis and aspects of cytoskeleton organization. Neither TAZ nor YAP bind members of the Wnt destruction complex but both extensively changed expression of Wnt and Wnt-cross talking genes with known roles in myogenesis. Finally, TAZ operates through Tead4 to enhance myogenic differentiation. In summary, Taz and Yap have overlapping functions in promoting myoblast proliferation but Taz then switches to promote myogenic differentiation.
2017-06-13 | PXD005751 | Pride
Project description:KSRP knock-down and BMP2 treatment produce a largely overlapping reshape of the transcriptome in C2C12 cells. microRNAs (miRNAs) are essential regulators of development, physiology, and evolution with miRNA biogenesis being strictly controlled at multiple levels. Regulatory proteins, such as KH-type splicing regulatory protein (KSRP), modulate rates and timing of the enzymatic reactions responsible for maturation of select miRNAs from their primary transcripts in response to specific stimuli. Induction of myogenic miRNAs (myomiRs) is essential for muscle differentiation with KSRP phosphorylation being required to convey myogenic signals to enhanced myomiR maturation. Here we show that either KSRP silencing or Bone Morphogenetic Protein (BMP)2-signaling activation in mesenchimal C2C12 cells prevented myogenic differentiation while induced osteoblastic differentiation as revealed by the reshaping of the whole transcriptome analyzed by RNA deep-sequencing. The most striking feature common to both BMP2 signaling activation and KSRP silencing was a blockade of myomiR maturation. Our results demonstrate that phosphorylated SMAD proteins, the transducers of BMP signaling, associate with KSRP and block its interaction with primary-myomiRs. This, in turn, abrogates KSRP-dependent myomiR maturation with the knock-down of SMAD4, 5, and 9 being able to rescue KSRP function. SMAD-induced blockade of KSRP-dependent myomiR maturation, in parallel to the well known SMAD function on gene transcription, inhibits C2C12 cell differentiation into myofibers and contributes to orient cells towards osteoblast lineage. We propose that remodeling of co-regulatory complexes affecting primary-miRNA processing is a mechanism well suited to guide cell fate determination in eukaryotes. Total RNA was prepared from 1. untreated mock-transfected C2C12 cells; 2. BMP2-treated mock-transfected C2C12 cells; 3. untreated shKSRP-transfected C2C12 cells and analyzed by RNA-seq
2013-06-25 | E-GEOD-38907 | biostudies-arrayexpress
Project description:Regulation of organ size is important for development and tissue homeostasis. In Drosophila, Hippo signaling controls organ size by regulating the activity of a TEAD transcription factor, Scalloped, through modulation of its coactivator protein Yki. The role of mammalian Tead proteins in growth regulation, however, remains unknown. Here we examined the role of mouse Tead proteins in growth regulation. In NIH3T3 cells, cell density and Hippo signaling regulated the activity of Tead proteins by modulating nuclear localization of a Yki homologue, Yap, and the resulting change in Tead activity altered cell proliferation. Tead2-VP16 mimicked Yap overexpression, including increased cell proliferation, reduced cell death, promotion of EMT, lack of cell contact inhibition, and promotion of tumor formation. Growth promoting activities of various Yap mutants correlated with their Tead-coactivator activities. Tead2-VP16 and Yap regulated largely overlapping sets of genes. However, only a few of the Tead/Yapregulated genes in NIH3T3 cells were affected in Tead1-/-;Tead2-/- or Yap-/- embryos. Most of the previously identified Yap-regulated genes were not affected in NIH3T3 cells or mutant mice. In embryos, levels of nuclear Yap and Tead1 varied depending on cell types. Strong nuclear accumulation of Yap and Tead1 were seen in myocardium, correlating with requirements of Tead1 for proliferation. However, their distribution did not always correlate with proliferation. Taken together, mammalian Tead proteins regulate cell proliferation and contact inhibition as a transcriptional mediator of Hippo signaling, but the mechanisms by which Tead/Yap regulate cell proliferation differ depending on cell types, and Tead, Yap and Hippo signaling may play multiple roles in mouse embryos. We used microarrays to know the gene expression profiles regurated by Tead2-VP16, Tead2-EnR, Yap, and cell density in NIH3T3 cells. Keywords: Cell density, genetic modification Tead2-VP16-, Tead2-EnR-, Yap- and control vector-expressing cells were cultured at low or high density for RNA extraction and hybridization on Affymetrix microarrays.
2008-11-20 | E-GEOD-12498 | biostudies-arrayexpress
Project description:Gene expression profiling was performed to identify Sfmbt1-dependent regulation in myogenic programs. To establish the magnitude of the Sfmbt1 effect on muscle cells, we have compared gene expression profiles of C2C12 cells transduced with lentiviruses expressing scramble shRNA control or shSfmbt1. Our analysis suggested that Sfmbt1 critically confers transcriptional silencing of muscle genes in myogenic progenitor cells. Two biological replicates for each sample group: C2C12 cells stably transduced with pLKO.1 expressing scramble shRNA control and C2C12 cells transduced with pLKO.1 shSFMBT1. Samples were independently cultured and RNAs were then harvested for microarray analysis.
2013-01-28 | E-GEOD-24346 | biostudies-arrayexpress
Project description:Skeletal muscle differentiation (myogenesis) is a complex and highly coordinated biological process regulated by a series of myogenic marker genes. Chromatin interactions between gene’s promoters and their enhancers have an important role in transcriptional control. However, the high-resolution chromatin interactions of myogenic genes and their functional enhancers during myogenesis remain largely unclear. Here, we used circularized chromosome conformation capture coupled with next-generation sequencing (4C-seq) to investigate eight myogenic marker genes in C2C12 myoblasts (C2C12-MBs) and C2C12 myotubes (C2C12-MTs). We revealed dynamic chromatin interactions of these marker genes during differentiation, and identified 163 and 314 significant interaction sites (SISs) in C2C12-MBs and C2C12-MTs, respectively. The interacting genes of SISs in C2C12-MTs were mainly involved in muscle development, and histone modifications of the SISs changed during differentiation. Through functional genomic screening, we also identified 25 and 41 putative active enhancers in C2C12-MBs and C2C12-MTs, respectively. Using luciferase reporter assays for putative enhancers of Myog and Myh3, we identified eight activating enhancers. Furthermore, dCas9-KRAB epigenome editing and RNA-seq revealed a role for Myog enhancers in the regulation of Myog expression and myogenic differentiation in the native genomic context. Taken together, this study lays the groundwork for understanding 3D chromatin interaction changes of myogenic genes during myogenesis and provides insights that contribute to our understanding of the role of enhancers in regulating myogenesis.
2022-06-10 | GSE201138 | GEO
Project description:KSRP knock-down and BMP2 treatment produce a largely overlapping reshape of the transcriptome in C2C12 cells. microRNAs (miRNAs) are essential regulators of development, physiology, and evolution with miRNA biogenesis being strictly controlled at multiple levels. Regulatory proteins, such as KH-type splicing regulatory protein (KSRP), modulate rates and timing of the enzymatic reactions responsible for maturation of select miRNAs from their primary transcripts in response to specific stimuli. Induction of myogenic miRNAs (myomiRs) is essential for muscle differentiation with KSRP phosphorylation being required to convey myogenic signals to enhanced myomiR maturation. Here we show that either KSRP silencing or Bone Morphogenetic Protein (BMP)2-signaling activation in mesenchimal C2C12 cells prevented myogenic differentiation while induced osteoblastic differentiation as revealed by the reshaping of the whole transcriptome analyzed by RNA deep-sequencing. The most striking feature common to both BMP2 signaling activation and KSRP silencing was a blockade of myomiR maturation. Our results demonstrate that phosphorylated SMAD proteins, the transducers of BMP signaling, associate with KSRP and block its interaction with primary-myomiRs. This, in turn, abrogates KSRP-dependent myomiR maturation with the knock-down of SMAD4, 5, and 9 being able to rescue KSRP function. SMAD-induced blockade of KSRP-dependent myomiR maturation, in parallel to the well known SMAD function on gene transcription, inhibits C2C12 cell differentiation into myofibers and contributes to orient cells towards osteoblast lineage. We propose that remodeling of co-regulatory complexes affecting primary-miRNA processing is a mechanism well suited to guide cell fate determination in eukaryotes.
2013-06-25 | GSE38907 | GEO