Figure 5

MSC interferes with the ability of MyoD to positively regulate the microRNA miR-206 by blockading a necessary MyoD-binding site. (A) Site-specific ChIP for MSC in the miR-206 promoter and at a control locus. (B) MSC ChIP as in (A) in RD cells infected either with an empty retrovirus (Control), or RD cells differentiated through expression of RUNX1 (RUNX1). (C) Screenshot from the human UCSC Genome Browser of the region that corresponds to the miR-206 promoter. Mapped reads from ChIP-seq for MyoD in RD and HFF cells are indicated, with the number on the left-hand y-axis indicating the number of reads mapped at the peak of occupancy. The location of E-boxes are indicated at the bottom of the panel by the black rectangles. Vertical lines are drawn through the apparent highest points of occupancy for MyoD (red) and MSC (green) in RD cells. (D) Electrophoretic mobility shift assays using in vitro transcribed and translated proteins as indicated and probes that correspond to either the E-box located at the peak of MyoD binding as indicated by the red mark in 5C, or the E-box located at the peak of MSC binding, indicated by the green mark. The position of MyoD:E and MSC:E heterodimers are indicated by the arrows. The lane marked 2x E12 indicates protein mixtures that included double the amount of E12 compared to other lanes, and the triangles indicate decreasing amounts of either MSC or MyoD in the mixtures as other proteins were maintained at constant levels and total protein amounts were balanced with translation of empty CS2. (E) Luciferase assays in RD cells with constructs as indicated below the figure using either the miR-206 promoter luciferase reporter (206) or one in which the E-box that the peak of MSC occupancy is located over has been mutated (206 MSC-binding Ebox mutant). Control indicates transfection with an empty plasmid. All luciferase assays were normalized to the results from a co-transfected renilla plasmid. (F) qPCR for CKM from RD cells transduced with either an empty virus (control), or one expressing either the MD ~ E or MD ~ E2/5 forced dimer. Cells were differentiated for 24 h before collection of RNA for use in qPCR. (G) qPCR for RUNX1 from the RD cells assayed in E. (H) qPCR for pri-miR-206 from the RD cells assayed in E and F. For all ChIPs, relative enrichment is calculated as the ratio of the % of input amplified with antibody to the % of input amplified with no antibody. The control locus is located at hemoglobin beta, a silent gene in myogenic cells. Corrected relative enrichment (5B) is the ratio of enrichment at miR-206 to enrichment at the control locus. All graphs represent the mean ± SEM of at least three independent experiments. All qPCR of gene expression was corrected to TIMM17b and control cells set to 1. * : P <0.05; ** : P < 0.01; *** : P < 0.001; † : P = 0.058.