Supplementary Figure 8: c-MYC cooperates with NCoR/SMRT to derail reprogramming and this can be overcome by conjugating the VP16 co-activator domain to OCT4 or SOX2.

(a) Heatmap showing the overlap of NCoR/SMRT bound sites (day 9 OSKM reprogramming) versus c-MYC (48 h OSKM reprogramming and pre-iPSCs). The heatmap was generated by creating a non-redundant list of overlapping peak summits within 400 bp. Each row of the heatmap is a genomic locus either bound (red) or not bound (white) by the indicated factor. Only sites bound by NCoR/SMRT (day 9 OSKM reprogramming) are shown. The number of sites bound by the different factors and time points is indicated. Data are taken from GSE90895, the same for panels b-e. (b) GO analysis of the ChIP-seq peaks co-bound by NCoR/SMRT and c-MYC in 48 h OSKM reprogramming or pre-iPSCs (panel a). Gene sets for GO analysis were collected by annotating all genes within 2 kb and the closest gene within 20 kb of a binding site, duplicate genes were removed. (c) NCoR/SMRT and c-MYC are bound to the Klf2 locus during reprogramming and in ESCs. Genomic view of NCoR/SMRT and c-MYC binding during reprogramming (day 9 OSKM reprogramming, 48 h OSKM reprogramming and pre-iPSCs) and in ESCs. (d) Pileup tag density plots for NCoR/SMRT bound sites in day 9 OSKM reprogramming versus the binding sites for OCT4, SOX2, KLF4, and c-MYC in ESCs (left panel). Plots are centered on NCoR/SMRT binding sites, the tag density of the indicated OSKM factors is shown. Data are normalized to library size. Pair-wise Venn overlap for NCoR/SMRT bound sites in day 9 OSKM reprogramming versus OSKM bound sites in ESCs (right panel). Peaks were considered overlapping if their summits were within 400 bp. (e) As in panel d, but comparing NCoR/SMRT bound sites in ESCs to OSKM bound sites in ESCs. (f) Flow cytometry on day 6 after co-transducing the Oct4 distal enhancer-driven DsRed reporter in MEFs with empty vector, OSK or OSKM in serum+Vc. Percent indicates the proportion of cells positive for DsRed. (g) Number of Oct4-GFP⁺ colonies on day 16 in MEFs transduced with OSKM, treated with TSA across the indicated reprogramming windows in serum+Vc. Data are the mean of 3 technical replicates from 1 of 2 representative experiments. (h) Number of Oct4-GFP⁺ colonies on day 16 in MEFs transduced with OSKM and inducible HDAC3-YF treated with doxycycline across the indicated reprogramming windows in serum+Vc. Data are the mean ± s.e.m. (n=3 biological replicates with 3 technical replicates each), P value was calculated using one-tailed unpaired Student’s t-test. (i) Phase contrast and fluorescence images for detecting Oct4 distal enhancer-driven DsRed reporter in MEFs co-transduced with OSKM and the indicated engineered reprogramming factors OCT4-VP16 and SOX2-VP16 on day 6 in serum+Vc. Scale bar, 100 μm. (j) Number of GFP⁺ colonies on day 18 (serum) or day 16 (serum+Vc) in MEFs transduced with either full length versions of OSKM, or O_VP16SKM, OS_VP16KM, O_VP16S_VP16KM, where the VP16 domain is fused to the indicated reprogramming factor. Data are the mean ± s.e.m. (n=3 biological replicates with 3 technical replicates each), P value was calculated using two-tailed unpaired Student’s t-test, ** indicates P value <0.01. (k) Schematic representing how c-MYC directs the NCoR/SMRT-HDAC3 co-repressor complex to conflicting purposes during reprogramming. At somatic loci, recruitment of NCoR/SMRT is beneficial in the earlier stages of reprogramming, as it seemingly works to suppress the somatic gene program, which may be independent of HDAC3. However, at pluripotency loci and potentially other loci, recruitment of NCoR/SMRT-HDAC3 is deleterious, working to restrain reprogramming.