Cell Stem Cell, Volume 21

Supplemental Information

Transient and Permanent Reconfiguration

of Chromatin and Transcription Factor

Occupancy Drive Reprogramming

Anja S. Knaupp, Sam Buckberry, Jahnvi Pflueger, Sue Mei Lim, Ethan Ford, Michael R.Larcombe, Fernando J. Rossello, Alex de Mendoza, Sara Alaei, Jaber Firas, Melissa L.Holmes, Shalima S. Nair, Susan J. Clark, Christian M. Nefzger, Ryan Lister, and Jose M.Polo

Figure S1. Experimental design and validation experiments. Related to Figure 1. (A) Representative bright field images (scale bar: 200 µm) and FACS blots of reprogramming cultures on day 0, 3, 6, 9, 12 and at the iPSCs state. The populations collected for profiling are indicated in red insets in the FACS blots. For day 3 and 6, refractory cells (Thy1+/SSEA1-) and reprogramming cells (SSEA1+/Thy1-) were FACS isolated. For day 9 and 12, reprogramming intermediates were further purified based on c-Kit to obtain SSEA1+/c-Kit+/Thy1- cells. IPSCs were sorted for Oct4-GFP. Percentages given for subpopulations indicate the percent of all viable cells. Post sort analysis suggests at least 90% purity for each population collected for profiling. (B) Representative histology sections from teratomas derived from dox-independent iPSCs showing derivatives of all three germ layers. Scale bar: 200 μm. (C) Heatmap of ATAC-seq read counts in ATAC-seq peaks with columns ordered by hierarchical clustering indicates high concordance between biological replicates. Replicates are denoted by r1 or r2 for each reprogramming timepoint. (D) Smoothed scatterplots show that the correlation between biological replicates for identical timepoints (rows 1 and 2) is higher than the correlation for the same samples between different timepoints (row 3). (E) Global increase in chromatin accessibility during reprogramming. Points on plots show the counts for each class for biological replicates, with the line indicating the average. Bases plot shows the cumulative ATAC-seq peak width. Peaks plot shows the number of ATAC-seq peaks called for each sample. Promoters plot indicates the number of ATAC-seq peaks intersecting promoters at each timepoint. Silent promotes plot indicates the number of ATAC-seq peaks intersecting promoter regions of genes not showing detectable levels of expression during reprogramming. TES plot show the number of ATAC-seq peaks intersecting transcription end sites. (F) Close-up of Pou5f1 RNA-seq normalized read counts (CPM) shown in Fig. 1F with the 5’ UTR shown in blue as an indication of endogenous transcript signal.

Figure S2. Profiling of chromatin accessibility during reprogramming shows early, transient and late changes in accessibility. Related to Figure 2. (A) Heatmaps of ATAC-seq signal, DNA methylation level and Oct4/Sox2 occupancy in clusters defined in Figure 2A. Heatmaps are ordered by descending ATAC-seq signal intensity. (B) ATAC-seq signal examples for the different cluster types and time point mean gene expression of the nearest gene. (C) Stacked bar plots show fraction of total CpGs displaying high (≥0.8), intermediate (>0.2 and <0.8), low (>0 and ≤0.2) and no (0) DNA methylation during reprogramming, line plot shows levels of global DNA methylation for each timepoint. (D) Average expression levels for genes that encode enzymes involved in DNA methylation (Dnmt1, Dnmt3a and Dnmt3b) and demethylation (Tet1, Tet2 and Tet3). (E) Heatmaps of ATAC-seq and Oct4/Sox2/Klf4 ChIP-seq data from Chronis et al. 2017 in clusters defined in Figure 2A which are ordered by descending ATAC-seq signal intensity the same as Fig. S2A. (F) CTCF ChIP-seq signal in ATAC-seq clusters where the motif was predicted to be enriched. Data are from mouse ESCs (Minajigi et al. 2015). (G) Reprogramming efficiency of MEFs upon shRNA mediated knockdown of Cebpb, Fosb and Fos (blue) or overexpression of Sox9 (green). Data are presented as mean +/- SEM of at least three replicates. (H) Averaged Sox9 expression.

Figure S3. Analysis of Oct4-Sox2 binding. Related to Figure 3. (A) Oct4 and Sox2 ChIP-seq peak fold enrichment for peaks with the Oct4-Sox2 motif present and absent. (B) Aggregate signal of DNA methylation levels at Oct4/Sox2 bound loci for clusters described in Figures 3D-H. Clusters for motif present (P1-P4) and motif absent (A1-A4) are denoted in lower left corner of panels. (C) Mean expression of nearest gene at Oct4/Sox2 bound loci for clusters described in Figure 3D-H. Shaded ribbon represents the standard deviation and numbers in the upper left of plots indicate the number of genes in the group. (D) Klf4 ChIP-seq binding density in motif absent clusters. Data from Chronis et al. (2017) for 48h after OKSM induction, pre-iPSCs and ESCs. (E) Heatmaps of Sox2 ChIP-seq signal and ATAC-seq signal. Heatmaps are grouped by K-means clustering (k=2) of ATAC-seq signal in MEFs and day 3 SSEA1+ reprogramming intermediates. (F) Distribution of Oct4 and Sox2 ChIP-seq peaks at day 3 of reprogramming for chromatin regions open and closed in MEFs. (G) Fraction of day 3 Sox2 ChIP-seq peaks featuring full Oct4-Sox2 motif or partial Sox2 motif. (H) Distribution of NucleoATAC nucleosome occupancy scores for Sox2 bound motifs at day 3 in regions with closed chromatin in MEFs. (I) Nucleosome density, Tn5 insertion enrichment and ChIP-seq signal at Sox2 bound motifs on day 3 of reprogramming in SSEA1+ cells for four nucleosome occupancy classes. Tn5 insertion signal shows a distinct footprint at bound motifs (x=0). (J) Western blot analysis of Oct4 and histone H3 as housekeeper control in iPSCs and iPSCs treated with dox for 3 and 6 days. Oct4 protein levels are graphed relative to housekeeper and no dox controls. (K) Heatmap of Oct4 and Sox2 ChIP-seq binding density for iPSCs after dox treatment in the corresponding clusters in Figures 3D-H.

Figure S4. Differences in reprogramming and refractory cells. Related to Figure 4. (A) Hierarchical clustering of gene expression for genes detected as differentially expressed at day 6 of reprogramming between SSEA+ (d6) and Thy1+ cells (d6T). Normalized expression values have been z-score scaled for each gene. (B) Binding density heatmap of Oct4 and Sox2 in relation to TSS and TES of differentially expressed genes. (C) Principal component plot and (D) Heatmap of normalized ATAC-seq read counts in ATAC-seq peaks during reprogramming and compared with data from Chronis et al. (2017). References Minajigi, A., Froberg, J., Wei, C., Sunwoo, H., Kesner, B., Colognori, D., Lessing, D., Payer, B., Boukhali, M., Haas, W., et al. (2015). Chromosomes. A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation. Science 349.