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.

We previously identified and characterized a murine BTB domain-containing protein, CIBZ (ZBTB38 in human), that interacts with CtBP and binds to methylated CpGs. However, its physiological function remained unknown. As CtBP is reportedly involved in p53-independent programmed cell death, we examine here whether CIBZ is associated with apoptosis. We found that CIBZ was highly expressed in proliferating C2C12 cells but that its expression levels decreased upon induction of apoptosis by serum starvation. Knockdown of CIBZ by small interfering RNA in C2C12 cells induced apoptosis, as determined by an increase of annexin V/propidium iodide labeling, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. CIBZ inhibition also activated caspase-7 and caspase-9, suggesting that CIBZ-associated apoptosis occurs through the mitochondrial pathway. Notably, knockdown of CIBZ in p53 ؊/؊ mouse embryonic fibroblast cells also activated caspase-3 and cleavage of poly(ADP-ribose) polymerase, indicating that CIBZassociated apoptosis is mediated by a p53-independent pathway; however, because both common and distinct targets are regulated by CIBZ-and CtBP-associated apoptosis, we conclude that more than one pathway is involved. Finally, using mutagenesis and an in vitro caspase cleavage assay, we show that CIBZ is a novel substrate of caspase-3 and identify two caspase-3 recognition sites. These findings indicate, collectively, that CIBZ plays an important role by participating in the negative regulation of apoptosis in murine cells.Apoptosis is a genetically controlled form of cell death that plays critical roles during development and tissue homeostasis by ensuring the removal of damaged or unnecessary cells (1). Activation of proteolytic enzymes called caspases is a key step in the apoptotic program. Once the initiator caspase, the best characterized of which is caspase-9, is activated by cellular stress signals, it processes and activates downstream effector caspases such as caspase-3 and caspase-7. For caspase-3, the 32-kDa inactive proenzyme is cleaved to 17-and 12-kDa fragments to form an active heterotetramer. This active form can specifically cleave its substrates at a DXXD motif to induce DNA fragmentation and morphological changes typical of cells undergoing apoptosis (1, 2). One of the major substrates of caspase-3 is poly(ADP-ribose) polymerase (PARP), 3 and cleaved PARP and cleaved caspase-3 itself are regarded as signature markers of apoptosis (2, 3).Murine C2C12 cells are a well established in vitro model system to study apoptosis as well as myogenesis in developing muscle because a significant fraction of C2C12 cells undergo apoptosis, cell cycle withdrawal, and differentiation when cultured with 2% horse serum-containing medium (referred to as differentiation medium (DM)) (4 -7). Growing evidence indicates that the expression of many regulatory proteins is stimulated or suppressed during apoptosis induced by DM. The upregulated proteins include the cyclin-dependent kinase inhibitors p21 and p27 (4, 8...

Background: G1/S transition is important for embryonic stem cell (ESC) proliferation; however, the transcriptional factor that regulates this remains largely unknown. Results: CIBZ, a transcription factor, regulates ESC proliferation and G1/S transition. Conclusion: CIBZ-associated ESC proliferation and G1/S transition is dependent on Nanog expression. Significance: This study provides insights into the mechanism underlying the rapid proliferation and G1/S transition of ESCs.

Background: Glypican-3 (GPC3) is expressed in most of hepatocellular carcinoma (HCC) and GPC3 immunohistochemical staining is widely used in the clinical setting, but it has not been recommended as a blood biomarker, mainly due to its heterogeneous nature and the lack of established assay system. Here, we developed and evaluated the basic performance of fully automated GPC3 immunoassay kits which detect the full-length or the N-terminal fragments. We analyzed the molecular forms of GPC3 in HCC serum and evaluated the diagnostic performance of GPC3 and other biomarkers. Methods: We examined the analytical performance of the GPC3 kits. Then, the automated GPC3 assays were compared with an established ELISA kit. Afterwards, we determined the clinical cutoff of GPC3 and compared its diagnostic performance to alpha-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist-II (PIVKA-II) using 180 serum samples from clinically diagnosed patients. Results: GPC3 assays showed good analytical performance. The level of GPC3 in HCC was higher than recorded in healthy or other liver diseases' sera. The AUC of GPC3 was 0.90, whereas the AUCs of AFP and PIVKA-II were 0.89 and 0.76, respectively. Conclusion: Automated GPC3 assays with stable performance against GPC3 in screening HCC have been established and the diagnostic accuracy of GPC3 was as good as AFP.