Adult stem cell quiescence is critical to ensure regeneration while minimizing tumorigenesis. Epigenetic regulation contributes to cell cycle control and differentiation, but few regulators of the chromatin state in quiescent cells are known. Here we report that the tumor suppressor PRDM2/RIZ, an H3K9 methyltransferase, is enriched in quiescent muscle stem cells in vivo and controls reversible quiescence in cultured myoblasts. We find that PRDM2 associates with >4400 promoters in G0 myoblasts, 5 ...[more]
Project description:Knockdown of PRDM2 led to precocious differentiation. To understand the molecular basis for this phenotype, we performed microaary analysis of growing myoblasts. Genes differentially regulated by PRDM2 knock down were reveraled by microarray analysis using NIA15K mouse chips. Control and knock down cells were grown in proliferating conditions
Project description:Using cDNA arrays, we compared G0 myoblasts (S48) with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture which show important similarities with muscle satellite cells, and establish that distinct gene expression profiles characterize irreversible and reversible arrest Cell were synchronized in G0 by resuspending them in semi-solid media for 48 hours and harvested for RNA isolation
Project description:Knockdown of MLL5 led to deregulation of S phase. To understand the molecular basis for this phenotype, we performed microarray analysis of S phase synchronized myoblasts. Genes differentially regulated by MLL5 knock down were revealed by microarray analysis using NIA15K mouse chips. Control and knock down cells were synchronized at G0 by suspension culture and reactivated to enter S phase by replating for 24hrs in growth medium.
Project description:Knockdown of PRDM2 led to precocious differentiation. To understand the molecular basis for this phenotype, we performed microaary analysis of quiescent myoblasts. Genes differentially regulated by PRDM2 knock down were reveraled by microarray analysis using affymetrix mouse chips. Control and knock down cells were synchronized at G0 by suspension culture and total RNA was used to perform microarray analysis
Project description:High-fat diet or exposure to saturated fatty acids affects skeletal muscle growth and function. The aim of the present study was to investigate the effect of palmitate on the transcriptomic profile of mouse C2C12 myoblasts. Global gene expression was evaluated using whole mouse genome oligonucleotide microarrays, and the results were validated through real-time PCR. A total of 4047genes were identified as differentially expressed, including 3492 down-regulated and 555 up-regulated genes during 48-h-exposure to palmitate (0.1 mmol/l). Functional classification showed the involvement of PA-regulated genes in several processes which regulate cell growth. In conclusion, palmitate modifies the expression of genes associated with: i) myoblast responsiveness to hormones and growth factors, ii) cytokine and growth factor expression, and iii) regulation of cell-cell cell-matrix communication. Such alterations can affect myoblast growth and differentiation, however further studies in this field are required. To whom the correspondence should be addressed: Dr K. Grzelkowska-Kowalczyk; e-mail: email@example.com, tel/fax: (48 22) 847 24 52 After scanning of hybridized microarrays, quantitation of slide images was performed using Feature Extraction Software (Agilent) using default parameters and the raw data were exported to GeneSpring GX 12 (Agilent, Santa Clara, CA) and log2 transformed. For identification of genes significantly altered in cell compared with the control normal gene set. Total detected entities were filtered by flags (detected, non detected) and error (coefficient of variation: CV < 50.0 percent) to remove very low signal entities and to select reproducible signal values of entities among the replicated experiments, respectively. In statistical analysis, separated for experiment with myoblasts treated with palmitate acid was used t-test unpaired (p < 0.05) with multiple testing correction: Benjamini-Hochberg <0.05, all significant changes over fold change 1.6 were selected.
Project description:Knockdown of PRDM2 led to precocious differentiation. To understand the molecular basis for this phenotype, we performed microaary analysis of 28hr differentiated myoblasts. Genes differentially regulated by PRDM2 knock down were reveraled by microarray analysis using affymetrix mouse chips. Control and knock down cells were grown in differentiating conditions for 28hrs and total RNA was used to perform microarray analysis
Project description:PRDM2 directly associates with the Myogenin promoter and repress its transcription. This led to the hypothesis that PRDM2 could potentially associate directly with other promoters and regulate their expression.To gain further insight into the pathways and genes controlled by PRDM2, ChIP-on-Chip analysis was performed using mouse 44K promoter array. Since PRDM2 represses transcription by H3K9 dimethylation, we were interested in determining which targets were occupied by PRDM2 as well as showed enrichment for H3K9me2. Hence ChIP-on-Chip analysis for H3K9me2 was performed to find the overlap between PRDM2 and H3K9me2 marked promoters. Agilent two-color ChIP-on-Chip experiment, Organism: Mus musculus ,Genotypic Technology designed Custom Mouse Promoter 244k ChIP-on-chip Array (AMADID-019046)
Project description:APC is a key regulator of canonical Wnt signalling since it participates to beta-catenin targeting to proteasomal degradation when the pathway is inactive. Moreover, independently of Wnt signaling, APC regulates several cellular functions such as mycrotubule dynamics, chromosome segregation, cell adhesion. Although APC has been widely studied for its implication in initation and progression of several cancers, its role in satellite cells (skeletal muscle stem cells) has never been investigated. Here we used microarrays and to clarify APC functions and we identified several pathways and cellular processes to be affected following APC silencing. Overall design: We cultured satellite cells-derived primary myoblasts in proliferating conditions and we transfected them either by a siRNA targeting APC or by a control siRNA. 48 hours after transfection, cells were processed for RNA extraction.
Project description:We use tag-based next generation sequencing analysis to quantify gene expression levels during myogenic differentiation and focused on the identifcation of novel 3' ends with DeepSAGE and novel 5' ends with DeepCAGE. Illumina DeepCAGE and DeepSAGE with C2C12 cells at time points T0 (proliferating) and T9 (differentiated) days of differentiation.