Skeletal muscle differentiation induces wide-ranging nucleosome repositioning in muscle gene promoters.

In a previous report, we demonstrated that Cbx1, PurB and Sp3 inhibited cardiac muscle differentiation by increasing nucleosome density around cardiac muscle gene promoters. Since cardiac and skeletal muscle express many of the same proteins, we asked if Cbx1, PurB and Sp3 similarly regulated skeletal muscle differentiation. In a C2C12 model of skeletal muscle differentiation, Cbx1 and PurB knockdown increased myotube formation. In contrast, Sp3 knockdown inhibited myotube formation, suggesting that Sp3 played opposing roles in cardiac muscle and skeletal muscle differentiation. Consistent with this finding, Sp3 knockdown also inhibited various muscle-specific genes. The Cbx1, PurB and Sp3 proteins are believed to influence gene-expression in part by altering nucleosome position. Importantly, we developed a statistical approach to determine if changes in nucleosome positioning were significant and applied it to understanding the architecture of muscle-specific genes. Through this novel statistical approach, we found that during myogenic differentiation, skeletal muscle-specific genes undergo a set of unique nucleosome changes which differ significantly from those shown in commonly expressed muscle genes. While Sp3 binding was associated with nucleosome loss, there appeared no correlation with the aforementioned nucleosome changes. In summary, we have identified a novel role for Sp3 in skeletal muscle differentiation and through the application of quantifiable MNase-seq have discovered unique fingerprints of nucleosome changes for various classes of muscle genes during myogenic differentiation.

Modifying miRs for effective reprogramming of fibroblasts to cardiomyocytes.

Reprogramming scar fibroblasts into cardiomyocytes has been proposed to reverse the damage associated with myocardial infarction. However, the limited improvement in cardiac function calls for enhanced strategies. We reported enhanced efficacy of our miR reprogramming cocktail miR combo (miR-1, miR-133a, miR-208a, and miR-499) via RNA-sensing receptor stimulation. We hypothesized that we could combine RNA-sensing receptor activation with fibroblast reprogramming by chemically modifying miR combo. To test the hypothesis, miR combo was modified to enhance interaction with the RNA-sensing receptor Rig1 via the addition of a 5'-triphosphate (5'ppp) group. Importantly, when compared with unmodified miR combo, 5'ppp-modified miR combo markedly improved reprogramming efficacy in vitro. Enhanced reprogramming efficacy correlated with a type-I interferon immune response with strong and selective secretion of interferon ß (IFNß). Antibody blocking studies and media replacement experiments indicated that 5'ppp-miR combo utilized IFNß to enhance fibroblast reprogramming efficacy. In conclusion, miRs can acquire powerful additional roles through chemical modification that potentially increases their clinical applications.

Rig1 receptor plays a critical role in cardiac reprogramming via YY1 signaling.

We discovered that innate immunity plays an important role in the reprogramming of fibroblasts into cardiomyocytes. In this report, we define the role of a novel retinoic acid-inducible gene 1 Yin Yang 1 (Rig1:YY1) pathway. We found that fibroblast to cardiomyocyte reprogramming efficacy was enhanced by specific Rig1 activators. To understand the mechanism of action, we performed various transcriptomic, nucleosome occupancy, and epigenomic approaches. Analysis of the datasets indicated that Rig1 agonists had no effect on reprogramming-induced changes in nucleosome occupancy or loss of inhibitory epigenetic motifs. Instead, Rig1 agonists were found to modulate cardiac reprogramming by promoting the binding of YY1 specifically to cardiac genes. To conclude, these results show that the Rig1:YY1 pathway plays a critical role in fibroblast to cardiomyocyte reprogramming.