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. 2020 Jan 25;48(7):3789–3805. doi: 10.1093/nar/gkaa035

Figure 7.

Figure 7.

circSamd4 binds PUR proteins, derepressing Mhc gene transcription. (A) Activity of the Mhc promoter, as determined by monitoring luciferase activity after transfecting 500 ng of reporter pMH2-LUC-Renilla (Figure 6C) into C2C12 cells that had been transfected 24 h earlier with parent vector pcDNA3 or with vectors expressing circSamd4 or circSamd4-Δ3 (left graph). Eight h after transfection of the reporter construct, the cells were switched to DM media for 24 h and promoter activity was measured; as above (Figure 6C), GLuc was normalized to RLuc and these ratios were plotted relative to the ratios measured in the empty vector group (right graph). (B) The levels of MHC in the different transfection groups shown in panel (A) were assessed by western blot analysis and HSP90 levels were shown as the loading control. (C–F) ChIP-qPCR analysis of the association of PURA and PURB with the Mhc proximal promoter region (−270/−450). Following crosslinking (Materials and Methods), antibodies recognizing PURA (C, E) or PURB (D, F) or control nonspecific rabbit IgG antibodies were used to immunoprecipitate sheared chromatin from C2C12 myoblasts in which circSamd4 was silenced (C, D) or overexpressed (E, F). Specific oligonucleotide primers were used to amplify the region of interaction with PURA and PURB. ChIP-qPCR data were calculated using percentage of input; the TTS region of the neuron-specific gene Syn2 (region +200 TTS), which encodes the protein SYN2 (Synapsin), was assayed as a non-target negative control. (G) Summary model. In myoblasts (GM), virtually undetectable levels of circSamd4 permit the repression of the Mhc promoter by PURA and PURB. As myogenesis progresses (DM), the rise in circSamd4 leads to binding of a fraction of PURA and PURB away from the Mhc promoter, thereby helping to derepress Mhc transcription and enabling the accumulation of Mhc mRNA and the production of MHC, a key protein in mature myotubes. Data in (A, C–F) are the means ±SEM from three or more independent experiments. *P < 0.05, **P < 0.005.