(A) An rVISTA schematic showing the degree of sequence conservation between human and mouse chromosome (Chr) 1 in a region upstream of the miR-29b/c cluster. Predicted YY1, MyoD, myogenin, SRF, and Mef2 sites are displayed. (B) EMSA performed from C2C12 MB or MT with probes corresponding to YY1 sites A-D. With MB extracts, a supershift EMSA was performed using YY1 antisera. Arrows denote YY1/DNA bound complexes. (C) ChIP assays for YY1 was performed with chromatin from C2C12 MB or MT. Precipitated DNA was amplified with oligonucleotides spanning regions A-D. Total inputs are indicated. (D) ChIPs as in (C) were repeated for Ezh2, H3K27, HDAC-1, SRF or Mef2. (E) MB were transfected with either an YY1 expression plasmid (pCMV-YY1) or YY1 siRNA oligos and then induced to differentiate for 24h, at which time miR-29b and miR-29c were measured by qRT-PCR and normalized to U6. Fold changes are shown with respect to control siRNA transfected cells where miR-29 levels were set to a value of 1. (F) MB were transfected with a miR-29b/c-enhancer-Luc reporter and maintained as MB or differentiated in MT for 48h, at which time luciferase activities were determined. (G) MB were transfected with a miR-29b/c-enhancer-Luc reporter, along with plasmids for YY1, SRF or Mef2. Cells were then switched differentiation conditions and luciferase activity was subsequently measured. (H) MB were transfected with the miR-29b/cenhancer- Luc reporter (YY1 wt) or with an enhancer reporter containing a deletion mutation in the YY1 “D” site (YY1 mut), and subsequently differentiated for 48h (MT) at which time luciferase activity was determined (left graph). Separate transfections were performed with YY1 wt and YY1 mut reporters along with an YY1 expression plasmid (pCMV-YY1) or YY1 siRNA. Cells were subsequently differentiated for 48h at which time luciferase was determined. Vector siRNA oligo transfected cells were used as a control (right graph). All luciferase data were normalized to β-galactosidase protein and represent the average of three independent experiments ± S.D.