Gene expression changes during mouse skeletal myoblast differentiation revealed by transcriptional profiling

Abstract

-Studies described here utilize high-density oligonucleotide arrays to characterize changes in global mRNA expression patterns during proliferation, cell cycle withdrawal, and terminal differentiation in mouse C2C12 myoblasts. Statistical analyses revealed 629 sequences differentially regulated between proliferating and differentiating myoblasts. These genes were clustered using self-organizing maps to identify sets of coregulated genes and were assigned to functional categories that were analyzed for distribution across expression clusters. Clusters were identified with statistically significant enrichment of functional categories including muscle contraction, cell adhesion, extracellular matrix function, cellular metabolism, mitochondrial transport, DNA replication, cell cycle control, mRNA transcription, and unexpectedly, immune regulation. In addition, functional category enrichment data can be used to predict gene function for numerous differentially regulated expressed sequence tags. The results provide new insight into how genes involved in these cellular processes may play a role in skeletal muscle growth and differentiation. C2C12 cells; oligonucleotide array; functional category enrichment SKELETAL MUSCLE DIFFERENTIATION is a highly ordered process requiring myocyte proliferation, expression of muscle-specific regulatory factors, cell cycle withdrawal, and the synthesis of muscle contractile proteins, resulting in the fusion of mononucleated myoblasts into terminally differentiated multinucleated myotubes (1). Myocyte differentiation is regulated by four myogenic regulatory factors (MRFs): MyoD, Myf5, myogenin, and MRF4 (Myf6) (28, 39). These musclespecific basic helix-loop-helix (bHLH) transcription factors cooperate with the MEF2 family of MADS box transcription factors to activate transcription of muscle structural genes through E-box and MEF2 promoter sites, respectively (5). Negative regulators of muscle gene transcription include the Id dominant-negative HLH proteins, which sequester bHLH proteins into complexes incapable of binding DNA, and the bHLH protein twist (reviewed in Ref. 2).The myogenic program consists of two temporally separated processes: myoblast proliferation and differentiation. Proliferating mononucleate myoblasts expressing MyoD and Myf5 are committed to the muscle lineage and will continue to proliferate in the presence of mitogens under high-serum conditions in vitro. Upon serum deprivation, myoblasts activate transcription of myogenin and undergo irreversible cell cycle arrest following transcription of the cyclin-dependent kinase (Cdk) inhibitor, p21, and dephosphorylation of pRb (1, 40). Skeletal muscle differentiation then proceeds through the induction of muscle-specific gene expression and fusion of myoblasts into myotubes (1,13,14,29,41).Here, we have used high-density oligonucleotide arrays to investigate transcriptional changes occurring during myoblast proliferation and differentiation in C2C12 cells, a well-characterized in vitro model of mouse skeletal muscle cell...

Mammalian Target of Rapamycin (mTOR) Signaling Is Required for a Late-stage Fusion Process during Skeletal Myotube Maturation

“…Our microarray data on RR/KI-mTOR C2C12 cells differentiating under normal conditions generally agreed with the previous report, even though a complete overlap was not expected because of the different experimental conditions and different coverage of the genome, our chip was a 15,000 cDNA array and theirs an unrelated 11,000 oligo array (32). Our microarray analysis was aimed at gathering more specific information: the comparison of RR/KI- mTOR cells differentiating in the absence and presence of rapamycin revealed potential targets of mTOR kinase-dependent signaling during myocyte differentiation and maturation, and furthermore, the data contributed to our general understanding of the gene expression programs specifically underlying myotube maturation.…”

“…Gene profiling comparing myoblast and myotube stages of C2C12 cells was reported previously (32). Our microarray data on RR/KI-mTOR C2C12 cells differentiating under normal conditions generally agreed with the previous report, even though a complete overlap was not expected because of the different experimental conditions and different coverage of the genome, our chip was a 15,000 cDNA array and theirs an unrelated 11,000 oligo array (32).…”

An initial blueprint for myogenic differentiation

“…We analyzed cells that were growing, in the process of differentiation (24 h in DM), or fully differentiated into myotubes. Globally, our results agree with data obtained previously (Moran et al 2002).…”

“…Beyond these first steps, our knowledge is somewhat fragmentary: Relatively few physiological targets of MRFs and MEF2 have been identified, and the number of genes known to be regulated by these factors is considerably smaller than the number of genes induced upon myogenic differentiation (Moran et al 2002). In addition, the role of MyoD in myogenic differentiation appears to be considerably more complex, since recent studies point to a role for this factor in transcriptional repression as well as activation (Mal and Harter 2003).…”

Alpha-mangostin promotes myoblast differentiation by modulating the gene-expression profile in C2C12 cells

“…5) Shen et al have focused on the events taking place during C2C12 myoblast cell cycle withdrawal. 6) Tomczak et al reported the analysis of 24,290 murine probe sets in a 12-d time course of differentiating C2C12 myoblasts.…”