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

Abstract

Postnatal growth and regeneration of skeletal muscle are carried out mainly by satellite cells, which, upon stimulation, begin to express myogenin (Myog), the critical determinant of myogenic differentiation. DNA methylation status has been associated with the expression of Myog, but the causative mechanism remains almost unknown. Here, we report that the level of CIBZ, a methyl-CpG-binding protein, decreases upon myogenic differentiation of satellitederived C2C12 cells, and during skeletal muscle regeneration in mice. We present data showing that the loss of CIBZ promotes myogenic differentiation, whereas exogenous expression of CIBZ impairs it, in cultured cells. CIBZ binds to a Myog promoter-proximal region and inhibits Myog transcription in a methylation-dependent manner. These data suggest that the suppression of myogenic differentiation by CIBZ is dependent, at least in part, on the regulation of Myog. Our data show that the methylation status of this proximal Myog promoter inversely correlates with Myog transcription in cells and tissues, and during postnatal growth of skeletal muscle. Notably, induction of Myog transcription by CIBZ suppression is independent of the demethylation of CpG sites in the Myog promoter. These observations provide the first reported molecular mechanism illustrating how Myog transcription is coordinately regulated by a methyl-CpG-binding protein and the methylation status of the proximal Myog promoter.

Cullin E3 Ligase Activity Is Required for Myoblast Differentiation

“…We analyzed ZBTB38 protein levels in differentiating C2C12 cells in presence of MLN4924, and in combination with Aza (Figure 8A). While ZBTB38 protein levels decrease significantly during differentiation, a finding that has been reported previously [37, 43], treatment with MLN4924 was unable to block its active degradation. Intriguingly however, the additional incubation with Aza was consistently able to restore ZBTB38 protein levels comparable to those in proliferative C2C12 cells.…”

“…Recently, a zinc finger and BTB-domain containing protein, ZBTB38 (CIBZ), was shown to be important for the initiation of the myogenic program [37]. ZBTB38 acts as a transcriptional repressor for myogenin expression in proliferative C2C12 myoblasts.…”

“…Its repressor function can be overcome by treating C2C12 cells with 5-aza-dC (Aza), which is a DNA-methylation inhibitor. DNA-methylation is required for ZBTB38 binding to the myogenin promoter, and repression of myogenin expression [37]. We therefore wondered if additional treatment of C2C12 with Aza may overcome some of the inhibitory effects of MLN4924 on terminal muscle cell differentiation.…”

“…Among them, ZBTB38 (CIBZ), Bhlhe41 and Id1 represent potential CRL substrates, because they either contain a BTB domain that indicates their interaction with cullin-3 [38, 39], or due to the regulation of their degradation [40], or because of their known interaction with a cullin substrate adaptor [41, 42]. While ZBTB38 protein levels are downregulated during differentiation, its mRNA levels remain unchanged throughout differentiation ([37, 43] and Supplemental Figure S2A). In addition, the differentiation-dependent loss of ZBTB38 on the protein level can be blocked by the proteasome inhibitor MG132, indicating that the ubiquitin-proteasome pathway is actively regulating ZBTB38 protein levels [43].…”

Putting muscle in DNA methylation

“…Recently, Oikawa et al [11] advanced our understanding of a less wellstudied methyl-CpG-binding protein, CIBZ, by studying its role in muscle differentiation through regulation of Myogenin, a gene encoding a basic HLH protein that is an essential musclespecific transcription factor [12]. CIBZ (CtBP-interacting BTB zinc finger protein, ZBTB38) possesses a modified zinc finger domain capable of binding methylated DNA in vitro and can act as a transcriptional repressor of methylated reporter genes [5].…”