Myogenic miRs. (A) Time course of miR-1 and miR-206 induction during C₂C₁₂ myogenesis. Total RNA from C₂C₁₂ cells in GM or DM for zero (d0), one (d1), two (d2), three (d3), or four (d4) days was subject to Northern blot analysis with a ³²P end-labeled miR-1, miR-206, or U6 probe. ³³P-labeled 10-bp RNA ladder (Ambion) is shown on the left. The miR-1 blot (Top) was stripped and reprobed sequentially for miR-206 (Middle) and U6 (Bottom). Mature and precursor microRNAs are labeled with an arrow and an arrowhead, respectively. (B) Same as in A except that a new set of samples was probed with a ³²P end-labeled miR-133 probe, stripped, and reprobed with a ³²P end-labeled U6 probe. (C) RNA was isolated from growing, undifferentiated primary human myoblasts in SkGM2 proliferation media (U) or from primary human myoblasts differentiated for 2 weeks in 2% horse serum-containing media (D). The position of the microRNA is indicated on the left. The membrane first was probed with miR-1, stripped, and subsequently probed for miR-133, miR-206 and U6 as indicated below the blots. ³³P-labeled 10-bp RNA ladder (Ambion) is shown on the left for the miR-1 blot. (D) Myogenin induction (by using quantitative RT-PCR) in the differentiated sample indicates efficient differentiation of the primary human myoblasts along the skeletal myogenic lineage. The same RNA samples was used for C and D.

Determination of the loci contributing to mature microRNAs. DNase I-treated RNA from either undifferentiated (day 0/GM) or differentiated (day 4/DM) C₂C₁₂ cells was used for RT-PCR. Genomic DNA (gen) was used as a positive control; lack of a signal from reactions without reverse transcriptase (− lanes) shows that there was no genomic DNA contamination. The RNA species being detected is indicated. (A) Intronic miRs. miR-1-2 and miR-133a-1 are located on chromosome 18 and are intronic (i); they are robustly induced during myogenesis (ii Lower and iii Lower). Mindbomb is not induced during myogenesis (ii Upper and iii Upper). (B and C) Intergenic microRNAs. (B) miR-1-1 and miR-133a-2 are located on chromosome 2 and are intergenic (i); they are also robustly induced during myogenesis (ii and iii). (C) miR-133b is located on chromosome 1 and is intergenic (i); it is not up-regulated as dramatically as the other miR-133 isoforms (ii). (D) Amplification of Mindbomb mRNA (Upper) confirms the lack of inducibility during myogenesis; GAPDH amplification (Lower) reveals equal loading of input RNA. Similar results were obtained in a duplicate experiment; results from one set are shown for consistency.

miR-1, miR-133, and miR-206 induction is specific to myogenesis. (A) C₂C₁₂ cells were grown in GM or 2% horse serum (DM) in the presence or absence of 300 ng/ml BMP2. RNA from treated cells were harvested at the indicated times and probed for miR-1, miR-133, miR-206, and U6 small RNAs. Mature and precursor microRNAs are labeled with an arrow and arrowhead, respectively. (B) The same RNA samples used in A was DNase I treated and subject to RT-PCR for the detection of a myogenic marker (myogenin) or a osteoblastic marker (osteocalcin) and a housekeeping gene (GAPDH). Separate RT-PCR reactions were performed but were all loaded in the same well for simultaneous detection by using ethidium bromide staining. RT reactions performed without reverse transcriptase yielded no signal after PCR, and these samples are loaded in the “−” lanes.

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.