miR-29 in Myoblast Conversion to Myofibroblast Figure 3. TGF-b inhibits miR-29 during myogenic and fibrogenic differentiation of C2C12 cells. (A) IF staining of MyoD (red) and a-SMA (green) in C2C12 cells treated with or without TGF-b. Photos were taken by confocal scanning microscope. Dashed oval is used to circle a-SMA+/ MyoD2 cells; arrow, a-SMA+/MyoD+ cell. Scale bar = 50 mm. (B) miR-29 expressions in C2C12 incubated without (2) or with (+) TGF-b for 0, 3 or 6 days in DM. (C) miR-29 promoter luciferease reporter activities in C2C12 incubated with indicated doses of TGF-b for 48 hrs in DM. (D) C2C12 cells stably expressing miR-29 or vector control (NC) were treated with TGF-b in DM for 0, 2, 4, or 6 days at which times MyHC, Troponin, a-Actin were probed by Western blotting. (E) Cells were photographed under phase contrast or immunostained for MyHC and visualized by confocal scanning microscope at DM day 4. Positively stained cells were quantified. Data are plotted as mean 6 S.D. Scale bars = 50 mm. (F) NC or miR-29 stable cells were treated without or with TGF-b for 48 hrs at which time Col 1, a-SMA and Lims1 were probed by Western blotting with a-Tubulin as a loading control. (G) Col 1, a-SMA, VIM and Lims1 were stained by IF and visualized by confocal scanning microscope. Scale bars = 50 mm. (H) mRNA expressions of Col 1A1, Col 3A1, a-SMA and Lims1. Data are plotted as mean 6 S.D. *p,0.05. doi:10.1371/journal.pone.0033766.g003 abolished the inhibition of TGF-b on both miR-29 expression and miR-29 promoter activity. In contrast, knockdown of Smad7, an inhibitor of Smad3 activation, enhanced the inhibition of TGF-b on miR-29 expression and promoter activity. To substantiate this finding, primary myoblasts were isolated from tibialis anterior muscles of wild type (Smad3+/+), Smad3 heterozygous (Smad3+/2) or knockout (Smad32/2) mice and examined for miR-29 expression. In agreement, miR-29 expression levels were signifi- cantly elevated in Smad32/2 myoblasts (Figure 4D, 7.360.1 fold, p,0.0001) compared to Smad3+/+ cells. Only mild increase (1.9 fold, p,0.01) was detected in Smad3+/2 cells, suggesting a Smad3 dose-dependent regulation on miR-29 expression. On the contrary, primary myoblasts isolated from Smad72/2 mice displayed a significant reduction on miR-29 level (Figure 4E, 0.1160.2 fold, p,0.001). Additionally, when injected with Cardiotoxin, a snake venom that induces extensive muscle necrotic injury and subsequent regeneration, a steady increase of miR-29 levels were observed during the course of degeneration and regeneration (day 1, 2, 4, 6, 9) in Smad7+/+ muscles while Smad72/2 mice displayed much lower levels of miR-29 expression at all time points examined (Fig. 4F). These results reaffirm that Smad3 and Smad7 are critical mediators of TGF-b inhibition on miR-29. Interestingly, Smad3 protein was inhibited by miR-29 over- expression but increased upon miR-29 knock-down in C2C12 cells (Fig. S4), suggesting miR-29 regulates Smad3 expression although it is not predicted to be a direct target of miR-29. This is in line with a recent report showing miR-29 suppresses basal Smad3 expression possibly through inhibiting TGF-b3 [13]. Interestingly, most studies on Smads have documented their role as transcriptional activators, although TGF-b signaling often results in down-regulation of gene expression. We were thus intrigued to explore the underlying mechanisms through which Smad3 represses miR-29 transcriptional activity. To test whether Smad3 can directly bind to miR-29 promoter, we searched for its binding site on miR-29b/c promoter [5]. Indeed, three SBEs were discovered in the proximal promoter region (24849, 22741, and 2692 bp upstream of the transcriptional start site). Next, using ChIP-PCR assays, we detected an induction of Smad3 binding by TGF-b treatment at all three predicted SBEs (Fig. 4G), indicating a TGF-b-induced Smad3 nuclear translocation and subsequent association to miR-29 promoter. Smad3 regulates miR-29 promoter through inhibiting MyoD binding and enhancing YY1/Polycomb recruitment Previously, Liu et al. demonstrated that Smad3 inhibits MyoD transcriptional activity through disruption of its binding to E-box sites of muscle genes [12]. We thus asked whether Smad3 repression on miR-29 promoter could be executed in a similar fashion as MyoD has been implicated as an activator of miR-29 at the onset of myogenic differentiation [5]. Four putative MyoD binding E-boxes were identified (Fig. 5A, M1, M2, M3 and M4). As shown in Figure 5B, an association of MyoD with these sites was detected in differentiated myotubes without TGF-b treatment. However, the binding was largely suppressed by TGF-b. In addition to MyoD regulation, we have previously demonstrated that miR-29 promoter is epigenetically silenced in undifferentiated myoblasts by an YY1/Polycomb repressive complex through recruitment to an YY1 binding CCAT box, and removal of this complex is necessary for the myogenic program to occur [25]. This promoted us to ask whether TGF-b silencing miR-29 can be mediated by YY1/Polycomb complex. A search for putative YY1 binding sites uncovered a total of six sites (Fig. 5A, Y1–Y6). According to our previous findings [5], Y6 was competent for YY1 binding in undifferentiated myoblasts whereas Y3, Y4, Y5 were not. Y1 and Y2 represent two new sites previously untested. Subsequent ChIP-PCR assays revealed no enrichment of YY1 on any site in differentiated cells without TGF-b treatment (Fig. 5C), which is in agreement with the activation status of miR-29. However, an increase of enrichment was found at Y1, Y2, Y3 and Y6 after TGF-b treatment, indicating that TGF-b indeed enhanced YY1 binding on multiple locations. Yet, no binding on Y4 and Y5 was detected in both untreated and treated cells (data not shown). Additional ChIP-PCR assays showed marked increase of Ezh2 binding at all four YY1 sites (Fig. 5D); consequently, increased levels of H3K27me3 were detected (Fig. 5E), suggesting that TGF-b treatment stabilizes YY1 binding and recruitment of Ezh2 and subsequent histone modification on multiple regions, which leads to silencing of miR-29 promoter. To substantiate the above findings from ChIP assays, reporter assays using miR-29-promoter-Luc plasmid were performed. As shown in Figure 5F, ectopic expression of YY1 repressed miR-29 reporter activities and the repression is enhanced with co-transfection of Smad3 at a dose-dependent manner, suggesting a repressive synergy between YY1 and Smad3. Ectopic expression of MyoD, on the other hand, strongly trans-activated the reporter, and this activation was repressed by Smad3 co-expression at a dose- dependent manner (Fig. 5G), suggesting Smad3 inhibits MyoD activation. Moreover, addition of YY1 further abrogated MyoD activation (Fig. 5H), indicating that the two mechanisms probably co-act. Collectively, the above results suggest the inhibitory action of TGF-b/Smad3 on miR-29 transcription is exerted through dual mechanisms by blocking MyoD binding and enhancing YY1/ Ezh2 association. In keeping with the earlier findings, we found that knockdown of either Smad3 or YY1 down-regulated Lims1 expression whereas knockdown of MyoD up-regulated its expression, suggesting Lims1 is under regulation of TGF-b/ Smad3/YY1/MyoD axis (Fig. 5I). Discussion In the current study, we present evidences for the pleiotropic roles of miR-29 in skeletal muscle cells. To our knowledge, this is the first report to describe the global effects of miR-29 on cellular transcriptome. In line with a previous study analyzing transcrip- tome and targetome of miR-155 expressing cells, our results PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e33766