Genome-wide maps of H3K9Me3, H3K27Me and Brg1 distribution between Wild Type (Wt) and Actb-/- (KNO) Mouse Embryonic Fibroblast
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE100096 | GEO | 2017/07/01
SECONDARY ACCESSION(S): PRJNA390667
REPOSITORIES: GEO
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Project description:β-actin is a crucial component of several chromatin remodeling complexes which control chromatin structure and accessibility. The mammalian Brahma-associated factor (BAF) is one such complex that plays essential roles in development and differentiation by regulating the chromatin state of critical genes and opposing the repressive activity of polycomb repressive complexes. While previous work has shown that β-actin loss can lead to extensive changes in gene expression and heterochromatin organization, it is not known if changes in β-actin levels can directly influence chromatin remodeling activities of BAF and polycomb proteins. Here we conduct a comprehensive genomic analysis of β-actin knockout mouse embryonic fibroblasts (MEFs) using ATAC-Seq, HiC-seq, RNA-Seq and ChIP-Seq of various epigenetic marks. We demonstrate that β-actin levels can affect the complex interplay between chromatin remodelers such as BAF/BRG1 and EZH2 in a dosage-dependent manner. Our results show that changes in β-actin levels and associated chromatin remodeling activities can not only impact local chromatin accessibility but also induce reversable changes in 3D genome architecture. Our findings support a novel role for β-actin levels in shaping the chromatin landscape during development and differentiation.
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Project description:Using Chromatin-immunoprecipitation and NGS, we investigated the binding of TFAM protein at mitochondrial genome between Actb+/+ Wild Type (WT) and Actb-/- (KO) Mouse Embryonic Fibroblast
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Project description:β-actin is a crucial component of several chromatin remodeling complexes which control chromatin structure and accessibility. The mammalian Brahma-associated factor (BAF) is one such complex that plays essential roles in development and differentiation by regulating the chromatin state of critical genes and opposing the repressive activity of polycomb repressive complexes. While previous work has shown that β-actin loss can lead to extensive changes in gene expression and heterochromatin organization, it is not known if changes in β-actin levels can directly influence chromatin remodeling activities of BAF and polycomb proteins. Here we conduct a comprehensive genomic analysis of β-actin knockout mouse embryonic fibroblasts (MEFs) using ATAC-Seq, HiC-seq, RNA-Seq and ChIP-Seq of various epigenetic marks. We demonstrate that β-actin levels can affect the complex interplay between chromatin remodelers such as BAF/BRG1 and EZH2 in a dosage-dependent manner. Our results show that changes in β-actin levels and associated chromatin remodeling activities can not only impact local chromatin accessibility but also induce reversable changes in 3D genome architecture. Our findings support a novel role for β-actin levels in shaping the chromatin landscape during development and differentiation.
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Project description:Genome-wide ChIP-Seq maps of EZH2 distribution between Wild Type (WT), Actb+/- (HET), and Actb-/- (KO) Mouse Embryonic Fibroblast
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Project description:Genome-wide maps of H3K9Me3, H3K27Me and Brg1 distribution between Wild Type (Wt) and Actb-/- (KNO) Mouse Embryonic Fibroblast
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Project description:The SWI/SNF complex remodels chromatin in an ATP-dependent manner through the ATPase subunits BRG1 and BRM. Chromatin remodeling alters nucleosome structure to change gene expression, however aberrant remodeling and gene expression can result in cancer. The function and localization on chromatin of the SWI/SNF complex depends on the protein makeup of the complex. Here we report the protein-protein interactions of wild-type BRG1 or mutant BRG1 in which the HSA domain has been deleted (BRG1-HSA). We demonstrate the interaction of BRG1 with most SWI/SNF complex members and a failure of a number of these members to interact with BRG1-HSA. These results demonstrate that the HSA domain of BRG1 is a critical interaction platform for the correct formation of SWI/SNF remodeling complexes.
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Project description:ACTB is a cytoskeletal protein involved in intracellular trafficking. In recent years, it has become evident that, in addition to its established roles in these compartments, ACTB also participates in the regulation of transcription. However, the molecular mechanisms underlying this function remain poorly understood. The methyltransferase SETD3 has previously been shown to methylate ACTB at H73, thereby regulating ACTB polymerization and smooth muscle contraction. Here, we show that the genomic distribution of ACTB is SETD3-dependent and that this regulation modulates the transcription of genes involved in cell adhesion and mRNA translation in colorectal cancer cells. Proteomic analyses reveal that ACTB and SETD3 interact with multiple large protein complexes, including complexes associated with transcriptional regulation. Specifically, we demonstrate that SETD3-mediated ACTB methylation is required for the co-localization of SMARCA4, a subunit of the SWI/SNF BAF complex, at specific genomic loci. Genomic analyses further show that this co-localization enables the coordinated occupancy of SMARCA4 and H73-methylated ACTB at genes involved in cell adhesion and mRNA translation. Finally, phenotypic assays confirm these regulatory effects. Together, these findings uncover a new mechanistic layer of selective transcriptional regulation mediated by an ACTB–SETD3–SMARCA4 axis in colorectal cancer cells.
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Project description:ACTB is a cytoskeletal protein involved in intracellular trafficking. In recent years, it has become evident that, in addition to its established roles in these compartments, ACTB also participates in the regulation of transcription. However, the molecular mechanisms underlying this function remain poorly understood. The methyltransferase SETD3 has previously been shown to methylate ACTB at H73, thereby regulating ACTB polymerization and smooth muscle contraction. Here, we show that the genomic distribution of ACTB is SETD3-dependent and that this regulation modulates the transcription of genes involved in cell adhesion and mRNA translation in colorectal cancer cells. Proteomic analyses reveal that ACTB and SETD3 interact with multiple large protein complexes, including complexes associated with transcriptional regulation. Specifically, we demonstrate that SETD3-mediated ACTB methylation is required for the co-localization of SMARCA4, a subunit of the SWI/SNF BAF complex, at specific genomic loci. Genomic analyses further show that this co-localization enables the coordinated occupancy of SMARCA4 and H73-methylated ACTB at genes involved in cell adhesion and mRNA translation. Finally, phenotypic assays confirm these regulatory effects. Together, these findings uncover a new mechanistic layer of selective transcriptional regulation mediated by an ACTB–SETD3–SMARCA4 axis in colorectal cancer cells.
2026-01-29 | GSE308376 | GEO
Project description:The cytoplasmic actin proteins, beta- and gamma-actin, are 99% identical but perform non-redundant functions. Genes encoding the cytoplasmic actins, Actb and Actg1, respectively, are less similar but still share 89% of their nucleotide sequences. Knockout (KO) of Actb by deletion of first coding exons 2 and 3 in mice is embryonic lethal while KO embryonic fibroblasts (MEFs) fail to proliferate. In contrast, KO of Actg1 is viable but mice lacking Actg1 present with increased perinatal lethality and Actg1 KO MEFs present with a much milder defect in cell proliferation. Recent studies have identified important protein-independent functions for both Actb and Actg1 and demonstrate that deletions within the Actb nucleotide sequence, and not loss of the beta-actin protein, cause the most severe phenotypes in KO mice and cells. Here, we use a multi-omics approach to better understand what drives the phenotypes of Actb KO MEFs. RNA-sequencing and mass spectrometry of Actb KO MEFs reveal largescale changes to the transcriptome, proteome, and phosphoproteome. Pathway analysis of genes and proteins differentially expressed upon Actb KO shows widespread dysregulation of genes involved in the cell cycle. Together, these data suggest novel, protein-independent roles for Actb in regulating gene expression associated with control of cell proliferation.
2024-02-19 | PXD039786 | Pride
Project description:Purpose: The goals of this study are to use NGS to perform transcriptome profiling (RNA-seq) to find the difference of the transcriptomes of chemically induced neurons from wild type (WT), Actb+/- (HET) and Actb-/- (KO) mouse embryonic fibroblasts. Methods: MEFs were reprogramed into neurons by small chemical cocktails. Small chemical molecules were dissolved and diluted in DMSO and used at the following final concentrations: ISX9: 20 mM; Forskolin: 50 mM; CHIR99021: 20 mM; and I-BET151: 2 mM. In addition to the small molecules, the neuron induction medium (Neurobasal Medium) contains the following supplements: N2 (1X) and B27 (2X) supplements, GlutaMAX (1X), penicillin-streptomycin (100 µg/ml), bFGF (20 ng/ml), 100 µM cAMP, Non-essential Amino Acid (1X) and Trace element B (1X). MEFs were seeded to Matrigel-coated plate (1:30 dilution in pre-cold PBS and coat overnight at 4 oC, at a density of 200,000 cells per well in 6-well plate and 10,000 cells/well in 96 well plate. The MEFs were cultured in DMEM until confluent. When the cells are confluent, the DMEM was replaced with neuron induction medium with 4 small molecules. The induction medium was refreshed every two days for the first week and every 3 days for the remaining induction period until day 20. 3 biological replicates of total RNA were used for RNA-seq library preparation. Results: Using an optimized data analysis workflow, we identified neuronal gene programs induced during direct reprograming. The induction of neuronal programs were impaired in neurons from Actb-/- mouse embryonic fibroblasts. Conclusions: Our study shows that neuronal program induction was impaired in chemically induced neurons from Actb-/- mouse embryonic fibroblasts.
2018-04-27 | GSE113733 | GEO