A Novel Target of MicroRNA-29, Ring1 and YY1-binding Protein (Rybp), Negatively Regulates Skeletal Myogenesis

Abstract

Background: MicroRNA-29-YY1 regulatory circuitry functions during skeletal myogenesis. Results: A genome-wide search revealed Rybp as a novel target of miR-29, and it silences myogenic loci together with YY1. Conclusion: Rybp functions as a repressor of myogenesis. Significance: This study identifies a novel regulatory circuitry underlying muscle formation and highlights the intimate interplay among transcription factors, epigenetic regulators, and microRNAs.

Linc-YY1 promotes myogenic differentiation and muscle regeneration through an interaction with the transcription factor YY1

“…Furthermore, to extend these in vitro findings to in vivo muscle formation, we explored the function of Linc-YY1 in CTX-induced muscle regeneration. Treatment of regenerating muscles with siLinc-YY1 oligos following a scheme as described before 33 ( Fig. 4e) led to downregulation of Pax7, MyoD, Myogenin and embryonicMyHC (e-MyHC, a marker for regenerating fibres) at both messenger RNA and protein levels (Fig.…”

“…For western blotting analyses, total cell extracts were prepared in RIPA buffer 33,51 . The following dilutions were used for each antibody: myogenin (Santa Cruz Biotechnology; 1:2,000), MyoD (Santa Cruz Biotechnology; 1:2,000), a-Skeletal Actin (Sigma; 1:2,000), Troponin (Sigma; 1:2,000), MyHC (Sigma; 1:2,000), YY1 (Santa Cruz Biotechnology; 1:2,000), Ezh2 (Active Motif, 1:2,000), Suz12 (Abcam, 1:2,000), Eed (Millipore, 1:2,000), a-Tubulin (Sigma; 1:5,000), Pax 7 (Developmental Studies Hybridoma Bank; 1:2,000), eMyHC (Leica, 1:2,000) and glyceraldehydes 3-phosphate dehydrogenase (Santa Cruz Biotechnology; 1:5,000).…”

“…Immunofluorescence on cultured cells and single fibres was performed using the following antibodies: MyHC (Sigma; 1:350), Myogenin (Santa Cruz Biotechnology; 1:350), MyoD (Santa Cruz Biotechnology; 1:350) and Pax 7 (Developmental Studies Hybridoma Bank; 1:2,000). Frozen muscle sections were prepared by immersion in isopentene in liquid nitrogen 33,51 . Immunoflurescence staining on frozen muscle sections was performed using the following antibodies: MyoD (Santa Cruz, 1:100); Myogenin (Santa Cruz, 1:100) and Pax7 (DSHB, 1:100).…”

“…Immunoflurescence staining on frozen muscle sections was performed using the following antibodies: MyoD (Santa Cruz, 1:100); Myogenin (Santa Cruz, 1:100) and Pax7 (DSHB, 1:100). Haematoxylin and eosin staining was performed on frozen muscle sections (5 mm) 33,51 . Quantification of number of fibres with centrally located nuclei and IF positively stained cells was performed from a minimum of 20 randomly chosen fields, from 5-6 sections throughout the length of the muscle in 4-6 per group.…”

LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration

“…To test this notion, we depleted Dum in mouse limb muscles during injury-induced regeneration using intramuscular injection of siRNA oligos as described before [17][18][19]. As illustrated in Figure 3A, the injection of siDum or negative control oligos was performed three times on days 1/4, 2 and 4 post CTX injection and muscles were harvested at the designated times for analyses.…”