Fig. 4.

Myogenin and MyoD binding to upstream regions of microRNAs. ChIP-on-chip analyses detects specific binding of MyoD (black lines) and myogenin (gray lines) in the upstream regions of myogenic microRNAs: miR133a-2 (A), miR-206 (B), miR-133a-1 and miR-1-2 (C), and lack thereof in the upstream region of a hepatic microRNA, miR-122a (D). Arrows below the microRNA indicate the direction of transcription. (E) The results of a PCR-based assay to confirm enrichment of sequences bound by myogenin and MyoD in the upstream regions of all of the microRNAs listed in A–D plus two conserved E boxes upstream of miR-1-1 (bottom set). Myogenin (Myog) and pancreatic amylase (Amy2) promoters were used as positive and negative controls, respectively, for myogenic factor binding. In each of the sets of four lanes, the first three lanes correspond to genomic DNA inputs of 90, 30, and 10 ng of DNA from whole-cell lysates. The last lane corresponds to an input of 10 ng of immunoenriched DNA from MyoD or myogenin immunoprecipitations. Shaded boxes under peak regions indicate likely binding sites for the myogenic factors and are listed in Data Set 1. Because the average size of the labeled fragments hybridized to the DNA arrays is ≈300 bp, we cannot resolve sites below this distance.