Transcription factor Runx1 cooperates with MyoD and c-Jun to regulate the balance of myoblast proliferation/differentiation [ATAC-Seq]
ABSTRACT: Runx1 is expressed in regenrating muscle, specifically in the muscle adult stem cells- the satellite cells. Its exact role and target genes were yet to be identified. We report here the genome wide open chromatin paatern, as established by ATAC-seq Examination ofgenome wide pattern naked and therefore trhansposase accecible genomic DNA in primary myoblasts (PM)
Project description:Runx1 is expressed in regenrating muscle, specifically in the muscle adult stem cells- the satellite cells. Its exact role and target genes were yet to be identified. We report here the genome wide histone modification using an active enhancer pattern of H3K4 monomethylation (H3K4me1) and H3K27 acetylation (H3K27ac). Examination of genome wide chromatin binding of several TFs in primary myoblasts (PM)
Project description:Runx1 is expressed in regenerating muscle, specifically in the muscle adult stem cells- the satellite cells. Its exact role and target genes were yet to be identified. We report here Runx1 and potential TF counterparts bind important muscle differentiation related genes Examination of genome-wide chromatin binding of several TFs in primary myoblasts (PM)
Project description:Runx1 is expressed in regenrating muscle, specifically in the muscle adult stem cells- the satellite cells. Its exact role and target genes were yet to be identified This experiment aimed at determining the Runx1 regulated genes in muscle regeneration, specifically in Satellite cells To elucidate the Runx1-mediated transcriptional program involved in early stages of muscle regeneration, we compared gene expression profiles in WT and Runx1f/f primary myobalsts (PM). PM were purified from both genotypes , Total RNA was isolated, reverse-transcribed, fragmented, labeled and hybridized to Affymetrix MoGene 1.0 ST DNA array.
Project description:To understand the mechanism underlying the transcriptional regulation by Sox2, we analyzed genome-wide binding sites of Sox2, Tfap2c, and Cdx2 in trophoblast stem (TS) cells by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). ZHBTc4- and embryo-derived TS cell lines were maintained in the presence of FGF4 and mouse embryonic fibroblasts (MEFs).
Project description:While the core members of the Polycomb family of proteins (PRC2, PRC1, PR-DUB) are well-characterized, little is known about the specific composition of and protein-protein interactions within these complexes in different cell types. We performed quantitative interaction proteomics and cross-linking mass spectrometry on core Polycomb complex members to identify novel interactors, the relative abundance (stoichiometry) of subunits, and the architecture of these complexes in mouse embryonic stem cells (mESCs) and neural progenitor cells (NPCs). Differentiation to NPCs resulted in dramatic binding changes for several substoichiometric interactors of PRC2 and PRC1. ChIP-seq of core PRC2 and PRC1 subunits in mESCs and NPCs also identified dynamic changes in the genomic localization of these complexes. We observed a loss of PRC2 from most H3K27me3 sites during differentiation, whereas PRC1 is retained at these sites. Additionally, we found PRC1 at enhancers and promoters of active genes independent of PRC2 binding. Overexpression studies using NPC-specific PRC1 interactors demonstrated that the subunit switching observed during differentiation can change PRC1 target site binding. Altogether, these findings extend our understanding of Polycomb family composition, architecture, and genome-wide localization. ChIP-seq samples for Suz12, Ezh2, Ring1b, Pcgf2, and inputs from mouse embryonic stems cells (mES) and neural progenitor cells (NPC) as well as NPC histone H3K4me1 ChIP-seq.
Project description:We use ChIP-seq targeting histone 3 lysine 4 mono-methylation (H3K4me1) to identify putative enhancer sites genome-wide, in the retrosplenial cortex of adult prairie vole males. ChIP samples were generated by targeting a known enhancer mark (H3K4me1) in chromatin extracted from the retrosplenial cortex of 8 males. Illumina libraries were prepared from ChIP and INPUT DNA and sequenced on Illimuna HiSeq 2500 platform.
Project description:A number of microRNAs have been shown to regulate skeletal muscle development and differentiation. MicroRNA-222 is downregulated during myogenic differentiation and its overexpression leads to alteration of muscle differentiation process and specialized structures. By using RNA induced silencing complex (RISC) pulldown followed by RNA sequencing, combined with in silico microRNA target prediction, we have identified two new targets of microRNA-222 involved in the regulation of myogenic differentiation, Ahnak and Rbm24. Specifically, the RNA binding protein Rbm24 is a major regulator of muscle specific alternative splicing and its downregulation by microRNA-222 results in defective exon inclusion impairing the production of muscle-specific isoforms of Coro6, Fxr1 and NACA transcripts. Reconstitution of normal levels of Rbm24 in cells overexpressing microRNA-222 rescues muscle-specific splicing. In conclusion, we have identified a new function of microRNA-222 leading to alteration of myogenic differentiation at the level of alternative splicing, and we provide evidence that this effect is mediated by Rbm24 protein. We built linear models using 2 different experiments and two conditions (miR222 over expression (n=1) and control siRNA(n=2)) with the linear formula (~condition + experiment).
Project description:Clarification of the mechanisms underlying osteoclast differentiation enable us to understand the physiology of bone metabolism as well as the pathophysiology of bone diseases, such as osteoporosis. Recently, it has been reported that epigenetics can determine the cell fate and regulate cell type specific gene expression. However, little is known about epigenetics during osteoclastogenesis. To reveal a part of epigenetics, especially focused on chromatin dynamics, during early osteoclastogenesis and identify novel transcription factors involved in osteoclastogenesis, we investigated genome-wide analysis of open chromatin during receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis using DNase I hypersensitive sites sequencing (DNase-seq). DNase-seq was performed using the extracted nuclei obtained from RAW264 cells treated with or without RANKL for 24 hours, followed by several bioinformatic analyses. DNase I hypersensitive sites (DHSs) during RANKL-induced osteoclastogenesis were dynamically changed and accumulated in promoter regions, although the distributions of DHSs among cis-regulatory DNA regions were identical regardless of RANKL stimulation. Motif discoveries from DHSs successfully identified well-known osteoclastogenic transcription factors such as Jun, CREB1, FOS, ATF2 and ATF4, but also novel transcription factors for osteoclastogenesis such as Zscan10, Atf1 Nrf1 and Srebf2. siRNA knockdown of these identified novel transcription factors impaired osteoclastogenesis. Taken together, DNase-seq can be a useful tool for comprehension of epigenetics, especially chromatin dynamics during osteoclastogenesis and for identification of novel transcription factors involved in osteoclastogenesis. This study may reveal underlying mechanisms that determine cell-type specific differentiation of bone cells and may lead to investigate novel therapeutic targets for osteoporosis. Examination of genome-wide DNase Hypersensitive Sites in differentiated and undifferentiated RAW264 cells.
Project description:The Polycomb repressive complexes PRC1 and PRC2 are key mediators of heritable gene silencing in multicellular organisms. Here we characterize AEBP2, a known PRC2 cofactor which, in vitro, has been shown to stimulate PRC2 activity. We show that AEBP2 localises specifically to PRC2 target loci, including the inactive X chromosome. Proteomic analysis confirms that AEBP2 associates exclusively with PRC2 complexes. However, analysis of embryos homozygous for a targeted mutation of Aebp2 unexpectedly revealed a Trithorax phenotype, normally linked to antagonism of Polycomb function. Consistent with this we observe elevated levels of PRC2 mediated histone H3K27 methylation at target loci in Aebp2 mutant embryonic stem cells. We further demonstrate that mutant ES cells assemble atypical hybrid PRC2 sub-complexes, potentially accounting for enhancement of Polycomb activity, and suggesting that AEBP2 normally plays a role in defining the mutually exclusive composition of PRC2 sub-complexes. H3K27me3, SUZ12, and AEBP2 ChIP-Seq in wild-type and AEBP2 KO mouse ESCs, biological replicates, pre-cleared chromatin as input, additionally FS2 ChIP-Seq in cells with FS2 tagged AEBP2, HiSeq2000
Project description:We identified target genes for NHR-25 by ChIP-seq at L1 stage of C. elegans. Transcription factor genes were tagged with GFP and their expression examined at L1 stage. Since there are no direct target genes known for NHR-25 that can be used for assessment of enrichment efficiency by quantitative PCR (qPCR), we chose to repeat ChIP-seq experiment of another GFP tagged transcription factor, PHA-4 for which the ChIP-seq was performed during a pilot experiment of modENCODE project using the same transgenic strain and antibody (a gift from Tony Hyman lab). pha-4 and nhr-25 transgenic worm were studied in Fed L1 stage.