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Supplementary Figure 1 Reproducibility of G4 ChIP-seq and open chromatin profiling. (ah) Area-proportional Venn diagrams of BG4 enriched regions as identified by MACS2.0 and (ir), scatter plots showing the correlation in tag density of G4 ChIP-, ATAC- and FAIRE-seq peaks between biological replicates in HaCaT (a-c; i-m), HaCaT treated with Entinostat (d-e; n-o) and NHEK (f-h; p-r). Each dot in the scatter plot represents the tag density of one or more G4 ChIP or ATAC or FAIRE peak with the same value. Sequence fragment density or tag density (t.d.) was calculated by counting sequenced fragments in 1kb windows. Tag density is presented in log10 scale.

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Nature Genetics: doi:10.1038/ng.3662

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Supplementary Figure 2 Enrichment analysis of different G4 structural classes, Multiple EM for Motif Elicitation (MEME) and Find Individual Motif Occurrences (FIMO) density analysis of G4 ChIP-seq and nucleosome-depleted regions. (a) The total number of G4 ChIP-

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Nature Genetics: doi:10.1038/ng.3662

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seq regions in each of the indicated structural class of G4s are shown. Loop size 1–3, 4–5 and 6– 7, indicates that at least one loop of this length is present in the G4; long loop indicates a G4 with any loop of length >7 (up to 12 for any loop and 21 for the middle loop); simple bulge indicates a G4 with a bulge of 1–7 bases in one G-run or multiple 1-base bulges; 2-tetrads / complex bulge indicates G4s with two G-bases per G-run or several bulges of 1–5 bases; and other indicates other G4-types that do not fall into the former categories. (b) Fold enrichment for each structural class in (a) compared to random (average of 10 randomizations). Higher enrichment values mean higher likelihood to be present among the G4 ChIP-seq peak sequences. Error bars are s.e.m. (c) Fold enrichment of G4 motifs compared to highly similar motifs with less G4 forming potential. G4 forming motifs are compared to similar motifs either lacking one G-run (first 3 sets of bars) or having one G less in the G-run compared to their G4 forming counterpart. H = either the base A, C or T. (d) G4 ChIP peaks enriched by the BG4 antibody revealed three distinct motifs, a G-rich (E-value = 2.6e-050), the AP-1 (E-value = 5.3e-045) binding motif and an unrelated motif (E-value = 9.9e-024). (e) Motif density, motif occurrence per residue, in the G4 ChIP-seq and nucleosomedepleted peaks were analyzed via FIMO. Notably, only the G-rich motif showed an increased density in the G4 ChIP-seq peak fraction relative to the other motifs and a reduced density in nucleosome-depleted regions. (f) The most intense/ significant (q-value) 10,560 FAIRE sites were intersected with the 10,560 high-confident G4 ChIP-seq HaCaT sites to reveal the FAIRE accessibility distribution with and without the presence of G4 ChIP-seq peaks. Average number of FAIRE peaks and OQs density (OQs/peak) are shown for the different fractions across replicates above the box-plots.

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Nature Genetics: doi:10.1038/ng.3662

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Supplementary Figure 3 G4 ChIP-qPCR of selected G4 ChIP-seq peaks validates the specificity of the G4 ChIP protocol and the presence of G4s in nucleosome-depleted regions. ChIP-qPCR shows BG4, BG4 pre-blocked with either 20 eq. of the MYC G4 promoter sequence d(TGA GGG TGG GTA GGG TGG GTA A) or the ssDNA sequence d(GGC ATA GTG CGT GGG CG), DNase and RNase A treated BG4 as well as control antibody enrichments for a selected set of 11 different genomic regions. All amplified regions (Supplementary Table 3) overlap with at least one OQ, except the negative G4 control region ESR1. Only RPA3/RPA3OS,

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Nature Genetics: doi:10.1038/ng.3662

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MAZ/KIF22, SPRED2, IRF2, KIF14 and GAPDH and chr1_365 (non-coding regulatory region) display an open chromatin signature as determined by ATAC-/FAIRE-seq. The control antibody used was a phage-display antibody recognizing TACE (ADAM17) that was generated from the same library as BG465. The ESR1 regulatory region was used as internal reference to normalize promoter enrichments (error bars display standard deviation; N = 3).

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Nature Genetics: doi:10.1038/ng.3662

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Nature Genetics: doi:10.1038/ng.3662

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Supplementary Figure 4 BG4/1H6 G4-antibodies show significant colocalization with euchromatin but not with heterochromatin. HaCaT cells were paraformaldehyde fixed, RNase A treated and stained with BG4 (a) or 1H6 (b) and co-stained with antibodies against H3K9me3, H3K4me3 or RNA polymerase 2 (RNA Pol2). Confocal Z stack images, (15 x z steps, 0.280 ?m apart) were captured using a Leica TCS SP8 microscope. The central slice of the Z-stack is displayed in the representative images. Merge is between BG4/1H6 and histone mark/RNA polymerase II channels only and Z shows a cross section through the Z-stack in the YZ plane. Scale bar shown is 5 ?m. (c) Colocalization between the BG4/1H6 and H3K9me3, H3K4me3 or RNA Pol2 signal was determined by calculating the first Manders’ coefficient, M1 (percentage of total BG4/1H6 signal coinciding with histone/RNA Pol2 signal). Graphs show the mean corrected M1 coefficient (M1diff), where the expected value (assuming random signal distribution within the nucleus) has been subtracted, for all fields of view, or within biological replicates. A two-way t-test was performed for each condition across the mean values of M1diff for the three biological replicates. (P<0.05 = *; P<0.01 = **). Each biological replicate (N = 3) equates to 60–150 cells. Values above 0 are suggestive of positive colocalization and values below 0 of negative colocalization relative to what is expected by chance.

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Nature Genetics: doi:10.1038/ng.3662

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Supplementary Figure 5 Structural analysis of identified nucleosome-depleted sequences that overlap with OQs and G4 ChIP-seq peaks. (a) The total number of different G4 structures is shown for OQs regions overlapping G4 ChIP-seq peaks, at nucleosome-depleted regions. Loop size 1–3, 4–5 and 6–7, indicates that at least one loop of this length is present in the G4; long loop size indicates a G4 with any loop of length >7; bulges indicates a G4 with a bulge of 1–7 bases in one G-run or multiple 1-base bulges; 2-tetrads / complex bulge indicates G4s with two G-bases per G-run or several bulges of 1–5 bases; and other indicates other G4-types that do not fall into the former categories. (b) The total number of different G4 structures is shown for OQs regions not overlapping ChIP-seq peaks, at nucleosome-depleted regions. (c) Bar plot showing the ratio of OQs with and without a G4 ChIP-seq peak for each class shown in (a) and (b), calculated as ratio of the fraction of each class. Higher ratio values mean higher representation among the OQs regions with G4 ChIP-seq peaks when compared to the OQs regions without G4 ChIP-seq peaks.

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Nature Genetics: doi:10.1038/ng.3662

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(d) Area-proportional Venn diagram showing overlap in high-confidence G4 ChIP-seq peaks with OQs present in nucleosome-depleted chromatin (i.e. the union of high-confidence ATAC/FAIRE-seq peaks) and all ATAC-/FAIRE-seq peaks signature). (i.e. those with or without a OQs

Supplementary Figure 6 G4 ChIP-seq association with cancer-related genes. (a) Example IGV screenshot for the DDIT3 gene is shown. Tracks are shown for G4 ChIP-seq (top, red) and control input below (red); nucleosome-depleted regions by ATAC and FAIRE-seq (tracks 3 and 4, black) and OQs (Pyridostatin derived) on the reverse (-ss) and forward strand (+ss), respectively (tracks 5 and 6, purple)16. (b) Comparison of G4 ChIP-seq peak density (peaks per Mb) for cancer-related genes, both oncogenes (red) and tumor suppressors (purple) compared to normal genes (grey). P-values shown for each pair are from the Wilcoxon rank sum test. (c) Top 187 genes with the highest G4 ChIP-seq peak density having peak density of at least 50 and gene length of at least 1 kb, sorted by G4 peak density. The insets show from left to right the top 15

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Nature Genetics: doi:10.1038/ng.3662

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genes for all combined genes, for oncogenes only and tumor suppressors only, respectively (see Supplementary Table 1).

Supplementary Figure 7 G4 ChIP-seq association with SCNAs. (a) example IGV screenshot for the BCL2L1 region is shown. Tracks are shown for G4 ChIP-seq (top, red) and control input below (red); nucleosome-depleted regions by ATAC and FAIRE-seq (tracks 3 and 4, black); OQs (Pyridostatin derived) on the reverse (-ss) and forward strand (+ss), respectively (tracks 5 and 6, purple)16. (b) Comparison of G4 ChIP-seq peak density (peaks per Mb) for amplifications (n=54, left plot) and deletions (n=54, right plot) compared to random peak regions (average of 5 randomizations). The dashed black lines indicate twice the average genome-wide peak density (avg_density = 3.91). P-values shown for each pair are from the 2-sample test for equality of proportions (Chi-squared), calculated considering the fraction of data points above the dashed

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Nature Genetics: doi:10.1038/ng.3662

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lines. (c) Top 50 SCNA regions with the highest G4 ChIP-seq peak density (see Supplementary Table 2). The dashed red line indicates the average genome-wide peak density.

Supplementary Figure 8 HaCaT cells exhibit differential gene expression after Entinostat treatment. (a) Differential Binding Analysis (DBA) showing significant (FDR < 0.05) differences in gene expression (log2 fold change vs. log2 counts per million), between Entinostattreated versus untreated HaCaT cells. Red dots represent genes that are significantly increased/decreased in their expression level. Grey continues line indicates trend. (b) Distribution of mRNA levels are shown for promoter associated genes that feature an ATAC-seq peak with a OQs signature (2,884 genes), in comparison to genes that exhibit a promoter G4 ChIP peak, ATAC-seq and OQs feature (4,940 genes) in Entinostat treated HaCaTs. **** indicates significance (P<0.0001; unpaired two-tailed T-test).

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Nature Genetics: doi:10.1038/ng.3662

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Supplementary Figure 9 NHEK form significantly more nucleosome-depleted regions, but less G4s than HaCaT cells. (a) Differential binding analysis showing significant (FDR <0.05) differences in gene expression (left), G4 ChIP-seq (middle) and ATAC-seq (right) peak intensities between NHEK versus HaCaT cells (log2 fold change vs. log2 counts per million). Red dots represent genes that are significantly increased/decreased in their expression/fragment counts. (b) Area-proportional Venn diagram of the union of high-confidence ATAC-/FAIRE-seq peaks between NHEK and HaCaT. (c) Representative example of immunofluorescence microscopy visualization of G4 foci in the nuclei of HaCaT and NHEK cells (red dots) using the BG4 antibody.

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Kwok, ?H. ?F. ?et ?al. ?Development ?of ?a ?'mouse ?and ?human ?cross-?‐reactive' ?affinity-?‐ matured ?exosite ?inhibitory ?human ?antibody ?specific ?to ?TACE ?(ADAM17) ?for ?cancer ? immunotherapy. ?Protein ?Eng ?Des ?Sel ?27, ?179-?‐190 ?(2014). ?

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Nature Genetics: doi:10.1038/ng.3662

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