Characterization of the relationship between Myc and miR-294. (a) cMyc (blue) and nMyc (yellow) bind the miR-290 cluster promoter. ChIP-seq data reads6 were aligned to the mm9 assembly of the genome and peaks were generated with Findpeaks16. Vertical hash marks denote the positions of the miR-290 cluster miRNAs. (b) Quantitative RT-PCR for total mature miR-294 expression in control (V6.5) ES cells, MEFs, and MEFs infected with viruses expressing Sox2 (S), Oct4 (O), Klf4 (K) or cMyc (M). RNA was collected on days 2 and 6. N=3. Horizontal black bars indicate Ct > 40. Sno202 was used as input control. Data was normalized to ES cells. (c) H3K4me3 (green) and H3K27me3 (red) surrounding the miR-290 cluster in MEFs. Chip-seq data7 were analyzed as described in a. (d) Quantitative RT-PCR for total mature miR-294 expression in control (V6.5) ES cells (E), MEFs (M), MEFs infected with either Oct4, Sox2, Klf4 (OSK); Oct4, Sox2, Klf4, and cMyc (OSKM); or OSK+miR-294, and established iPS lines resulting from these conditions (iPS). RNA was collected on days 2, 6, and 10 of reprogramming. Three independent experiments are shown. Horizontal black bars indicate Ct > 40. Sno202 was used as input control. Data was normalized to ES cells. (e) Total cell number during reprogramming. Cells were counted on day 7 after infection with OSKM or OSK +/− miRNA mimic. Concentrations of miR-294 mimic: 1.6, 16 and 160nM. Concentration of miR-1 mimic: 160nM. (f) GFP negative colonies in presence of cMyc. Oct4-GFP+, ES-like colonies (black arrow) and GFP-negative, nonES-like colonies (white arrow). (g) Quantification of number of day 10 GFP-negative colonies after infection with OSKM or OSK +/− miR-294 mimic. All error bars indicate standard deviation of N=3.

The ESCC miRNAs promote three-factor, but not four-factor induced pluripotency. (a) Fold increase of day 10 Oct-GFP+ colonies with retroviruses expressing Oct4, Sox2, and Klf4 (OSK) together with 16nM miRNA mimic relative to transfection reagent only (Mock). N=3. Raw data in Supplementary Table 1. (b) Sequence of miR-290 cluster, miR-302d, and miR-294 seed sequence mutant. Bold indicates seed sequence. Capitals indicate point mutations. Grey box highlights ESCC seed-sequence. (c) Fold increase in day 10 Oct4-GFP+ colonies with addition of mimic to OSK in the presence (light grey) or absence (dark grey) of cMyc retrovirus. Bars represent the number of GFP+ colonies after mimic transfection divided by the number of GFP+ colonies after mock transfection. N= 6, 26, 2, 5, & 3 left to right. Asterisk indicates p-value ≤ 0.0001. Raw data for bars 1&2 in Supplementary Table 2. (d) Percent day 10 Oct4-GFP colonies for OSK plus 1.6, 16 and 160nM transfected miR-294 mimic or 160nM miR-1 relative to OSKM. (e) Quantitative RT-PCR for endogenous pluripotency markers in control (V6.5) ES cells, MEFs, and miR-294-iPS lines. N=3, 3, & 5. RPL7 was used as input control. Data was normalized to ES expression. (f) Quantitative RT-PCR for exogenous Oct4, Sox2, and Klf4 in MEFs 6 days after viral infection, control (V6.5) ES cells, and MEFs (each N=3) and 5 individual miR-294-iPS lines. Horizontal black bars indicate Ct > 40. RPL7 was used as input control. Data was normalized to MEF expression 6 days after viral infection. (g) X-gal staining demonstrates miR-294-iPS chimeric contribution to ectoderm (neural tissue, N), endoderm (lung, L), and mesoderm (cartilage, C). (h) GFP expression in genital ridges of E12.5 chimera demonstrates Oct4-GFP miR-294-iPS contribution to germline. All error bars indicate standard deviation.