miR-29 in Myoblast Conversion to Myofibroblast were then used to measure the enrichment fold of MyoD on four putative binding sites, M1/M2, M3 and M4. (C, D and E) ChIP-PCR assays were performed as above to examine the binding of YY1, Ezh2 and H3K27me3 to putative YY1 binding sites, Y1, Y2, Y3 or Y6. Enrichment folds are shown with respect to IgG control where normalized PCR values were set to 1. Data are plotted as mean 6 S.D. (F) Upper: 10T1/2 cells were transfected with 0.25 mg of miR-29-promoter-luc reporter plasmid along with 0.5 mg YY1 plasmid and Smad3 plasmid (0, 0.20, 0.50, 1.00, or 2.00 mg). 24 hr post- transfection, cells were treated with TGF-b for 48 hrs at which time luciferase activities were determined. (G) The transfections were performed as above with 0.5 mg of MyoD plasmid and Smad3 expression plasmid (0, 0.20, 0.50, 1.00, or 2.00 mg). (H) The transfections were performed as above with indicated plasmids (0.5 mg of MyoD, YY1 or Smad3 plasmids were used). (I) C2C12 cells were transfected with siRNA oligos knocking down Smad3, MyoD or YY1. The expression of Lims1 was examined by Western blotting using Tubulin as a loading control. *p,0.05. ** p,0.01. doi:10.1371/journal.pone.0033766.g005 29. The subsequent experimental data confirmed that Lims1 is a direct target of miR-29. Considering that myofibroblast differen- tiation is dependent on cell adhesion [26,27], down-regulation of Lims1 probably mediates the suppressive role of miR-29 during myoblast conversion to myofibroblast. These data thus add a novel target to the growing list of miR-29 targets, and implicate miR-29 as a potent regulator in many cellular processes involving cell adhesion factors such as cell migration, cell invasion and cell survival. Collectively, our transcriptome analysis demonstrated that the two main functions of miR-29 in muscle development are to increase myogenic differentiation and to suppress fibrogenic differentiation. As the major inducer of fibrotic cascade, TGF-b signaling has been shown to induce the conversion of C2C12 into myofibro- blasts while inhibiting the myogenic differentiation. The down- stream molecular mechanisms are not fully understood. Our studies identify a novel pathway through which miR-29 regulates TGF-b signaling induced transdifferentiation. In line with a recent study demonstrating that TGF-b controls miR-29 to inhibit myogenic differentiation [13], we also found that TGF-b can attenuate the pro-myogenic actions of miR-29. Our results, however, for the first time demonstrated that miR-29 also regulates TGF-b induced transdifferentiation, thus establishing the dual roles of TGF-b-miR-29 axis in both myogenic and fibrogenic differentiation of muscle cells. Our findings provide novel insights in understanding the pathologic fibrosis of skeletal muscle. Muscle fibrosis is a major pathological hallmark of chronic myopathies most often muscular dystrophies, which are inherited disorders characterized by muscle degeneration and associated progressive wasting and weakness. In the most severe cases, such as Duchenne muscular dystrophy (DMD), the absence of dystrophin protein leads to sarcolemmar permeability, influx of calcium, and activation of proteases to cause myofiber necrosis and degeneration. This is followed to some extent by regeneration but the complete regeneration is prevented by excessive synthesis and deposition of ECM proteins, which eventually leads to fibrosis [28,29,30]. Thus, fibrosis is a prominent pathological hallmark of skeletal muscle in patients with DMD and contributes to progressive muscle dysfunction and the lethal phenotype of DMD [31,32]. Unfortunately, the molecular mechanisms under- lying muscle fibrogenesis is not fully understood. TGF-b signaling is elevated in dystrophic muscles and is speculated to be the major inducer of muscle fibrogenesis but the underlying mechanisms are still unclear. Interestingly, miR-29 was found to be down-regulated in dystrophic muscles in concomitant with the increased TGF-b signaling (our unpublished data). Our findings thus fuels the interesting hypothesis that loss of miR-29 through TGF-b signaling promotes transdifferentiation of myoblasts into myofi- broblasts, which represents a novel contributing route to muscle fibrogenesis in dystrophic muscles. Very recently, Ardite E. et al discovered that miR-21 is also involved in fibrosis of DMD [33], highlighting the critical roles that miRNAs in general play in muscle fibrogenesis. It is believed that Smad proteins mediate gene activation or repression as a result of promoter-specific interactions with transcriptional activators or co-repressors which compensate for its weak intrinsic binding affinity for their target elements (SBE). In contrast to the well-documented cooperation of Smads with sequence-specific factors to activate transcription, the mechanisms underlying Smad-mediated transcriptional repression are only beginning to emerge [12,34,35]. Here we uncover a novel mechanism by which Smad3 exerts its function through synergetic interfering with MyoD association and harnessing YY1/Ezh2 repressive complex. Previously, Liu et al demonstrated that Smad3 acts downstream of TGF-b to repress MyoD-dependent activation through physically interacting with MyoD thus interfering with its formation of an active MyoD/E protein complex and its subsequent binding to multimerized E-box sequence [12,35]. In agreement with the above findings, our results also revealed disengagement of MyoD from multiple E-boxes with TGF-b activation of Smad3 binding. Furthermore, in our case, inhibition of MyoD binding on miR-29 promoter seems to be dependent on Smad3 association with proximal SBE as all the identified E-boxes are in the vicinity of SBEs (Fig. 5A). In addition to the above mechanism, we present evidence for a new layer of repression through recruitment of YY1/Ezh2 repressive complex on multiple sites of miR-29 promoter. Given that three of the four YY1 binding sites, Y2, Y3, and Y6 (Fig. 5A), are not adjacent to SBEs, it is very likely that the recruitment is independent on Smad3 binding. Nevertheless, one of the identified YY1 sites, Y1, was very close to a SBE, S1, suggesting that additional mechanism dependent on Smad3 binding may exist. Further studies are needed to test the above hypotheses. Therefore, the above two modes of actions exert reinforcing levels of control on miR-29 transcription, ensuring its down-regulation during the fibrogenic differentiation of myoblasts. The application of this new mechanism may extend beyond miR-29 promoter and represent a general mode of TGF-b/Smad3 repression in skeletal muscle differentiation considering many myofibrillar genes were also regulated by MyoD and YY1/Ezh2 complex [5,25]. Together with others’ findings, our data suggest that diverse mechanisms lead to transcriptional repression in response to TGF-b. Taken together, our results identified miR-29 as a pleiotropic molecule in muscle cells. As modeled in Figure 6, during normal muscle regeneration, miR-29 level is elevated through replacing a repressive YY1/Ezh2/HDAC1 complex by a MyoD/SRF activating complex on its promoter, leading to successful myogenic differentiation [5]; However, during the transdifferentiation, activated TGF-b signaling induces Smad3 translocation into nucleus where it binds to miR-29 promoter, resulting in MyoD dissociation as well as YY1/Ezh2 stabilization. This causes a loss of miR-29 expression and increased expression of Collagens and Lims1, leading to the transdifferentiation of myoblasts into myofibroblasts. Materials and Methods Cell Mouse C2C12 myoblasts were obtained from ATCC and cultured in DMEM supplemented with 10% FBS, 2 mM L- PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e33766