The Dynamics of Myogenin Site-specific Demethylation Is Strongly Correlated with Its Expression and with Muscle Differentiation

Abstract

The molecular mechanisms underlying the activation of tissue-specific genes have not yet been fully clarified. We analyzed the methylation status of specific CCGG sites in the 5-flanking region and exon 1 of myogenin gene, a very important myogenic differentiation factor. We demonstrated a loss of methylation, at the onset of C2C12 muscle cell line differentiation, limited to the CCGG site of myogenin 5-flanking region, which was strongly correlated with the transcriptional activation of this gene and with myogenic differentiation. The same CCGG site was also found to be hypomethylated, in vivo, in embryonic mouse muscle (a myogenin-expressing tissue), as opposed to nonmuscle (nonexpressing) tissues that had a fully methylated site. In a C2C12-derived clone with enhanced myogenic ability, demethylation occurred within 2 h of induction of differentiation, suggesting the involvement of some active demethylation mechanism(s) that occur in the absence of DNA replication. Exposure to drugs that inhibit DNA methylation by acting on the S-adenosylmethionine metabolism produced a further reduction, to a few minutes, in the duration of the demethylation dynamics. These effects suggest that the final site-specific DNA methylation pattern of tissue-specific genes is defined through a continuous, relatively fast interplay between active DNA demethylation and re-methylation mechanisms.Cytosine methylation is, in eukaryotic nuclear DNA, a well established epigenetic mechanism that controls the expression of housekeeping and possibly also tissue-specific genes (1-3), as well as several important cellular functions such as X chromosome inactivation and genomic imprinting (4 -6), mutagenesis and tumorigenesis (7-9), senescence, and virus latency (10 -12).Developmental changes in the methylation pattern are particularly evident (13-16). In fact, during early embryogenesis, the original gamete methylation pattern is erased, and most of the DNA in the blastocyst becomes demethylated. After implantation, a de novo methylation activity produces in the gastrula a methylation pattern characteristic of the adult animal. During the subsequent development, tissue-specific genes undergo specific demethylation events required for their transcriptional activation, according to the general paradigm of an inverse correlation between DNA methylation and gene expression. Knock-out experiments have highlighted the lethality of even modest abnormal methylation patterns in the embryo (17). There are, in addition, several lines of evidence indicating that endogenous genes can be activated by demethylating agents and that exogenous methylated genes are not expressed when transfected into cells but that their expression is reactivated by demethylating agents (18 -21).Despite many years of intense studies on DNA methylation, neither the mechanism that regulates this process nor its exact functional role in the activation of genes has been fully clarified. Two steps need to be considered as follows: 1) the creation of a methylation pattern in the DNA co...

High-phosphate-induced calcification is related to SM22α promoter methylation in vascular smooth muscle cells

“…S-Adenosyl methionine has been used previously to induce methylation of myogenic genes in myoblasts. (30,31) Similar results were obtained using a small interfering RNA to knock down SM22a expression. A discrete fall in mRNA expression of SM22a (similar to that observed with phosphate treatment) was enough to enhance of Cbfa1 expression in shortterm cultures.…”

The methyl-CpG-binding protein CIBZ suppresses myogenic differentiation by directly inhibiting myogenin expression

“…Since the 51-kb region between Myog and its upstream gene lacks CpG islands, DNA methylation in the vicinity of the promoter is probably responsible for Myog silencing. Consistent with this, Lucarelli et al reported that the methylation status of a single CCGG site at 340 bp upstream from the transcriptional start site (TSS) affects Myog transcription in mouse tissues and in C2C12, a skeletal muscle satellite-derived myoblast cell line [17]. This HpaII recognition site (CCGG), which is in the region proximal to the Myog promoter, was identified using the methylationsensitive endonuclease HpaII-PCR assay.…”

“…Treatment of 10T1/2 fibroblasts with the DNA demethylating reagent 5-aza-dC, or expression of antisense Dnmt1, results in myotube formation, suggesting that global demethylation plays a role in myogenic differentiation [14,15]. Similarly, 5-aza-dC treatment induces Myog expression in myoblast cells, implying that DNA methylation is also involved in the suppression of Myog transcription [16,17]. Since the 51-kb region between Myog and its upstream gene lacks CpG islands, DNA methylation in the vicinity of the promoter is probably responsible for Myog silencing.…”

The social environment and the epigenome

“…Active demethylation was reported for genomic DNA upon induction of Epstein Barr virus lytic cycle [Szyf et al, 1985] for the myosin gene in differentiating myoblast cells [Lucarelli et al, 2001], for Il2 gene upon T cell activation [Bruniquel and Schwartz, 2003] and the INTERFERON GAMMA gene upon antigen exposure of memory CD8 T cells [Kersh et al, 2006]. However, if DNA methylation is plastic in mature postmitotic tissue and responsive to the environment throughout the life of an organism, mechanisms must exist to enable both introduction of new DNA methylation sites by de novo methylation and mechanisms for removal of DNA methylation by demethylation.…”