Overview of DNA O-MAP workflow and label-free quantitative proteomics analysis of telomeres.

A) Schematic of DNA O-MAP. B) Fluorescent microscopy data showing the observed patterns of DNA (DAPI, left) and in situ biotinylation detected by staining with fluorescent streptavidin conjugates (middle, left) and overview of telomere targeted DNA O-MAP experiment. C) Significant gene sets identified by the Gene Set Enrichment Analysis of the proteins enriched by the telomere probe. D) DNA O-MAP telomeric proteins mapped onto the BioPlex interaction network32,33. The red box highlights shelterin complex proteins. Nodes are colored by the fold-enrichment compared to a no-primary-probe control shown in B, excluding unconnected nodes. E) Telomeric proteins observed in five previous datasets (PICh, C-BERST, CAPLOCUS, CAPTURE, BioID) superimposed onto Figure 1E, colored by the number of prior datasets where the protein was present and including unconnected nodes. Scale bars, 5 µm.

DNA O-MAP reveals distinct features of the sub-proteomes at peri-centromeric alpha satellites, telomeres, and the mitochondrial genome.

A) Workflow of DNA O-MAP integrated with sample multiplexing quantitative proteomics. B) Schematic of the three DNA loci examined in the TMT16plex experiment: peri-centromeric alpha satellites, telomeres, and mitochondrial genomes. C) Co-localization of DNA FISH and the streptavidin staining of the proteins biotinylated by DNA O-MAP targeting the peri-centromeric alpha satellites, telomeres, and mitochondrial genomes. Scale bar: 5 µm. D) Principal component analysis of scaled intensities of proteins enriched by the pan-alpha probe, telomere probe, mitochondrial genome oligo pool, and no-primary-probe control. E) Unsupervised hierarchical clustering of scaled intensities of proteins enriched by the pan-alpha probe, telomere probe, mitochondrial genome oligo pool, and no-primary-probe control. F) Log2 fold change of proteins compared to no-primary-probe control, grouped by HPA subcellular location. Significance calculated based on Welch’s t-test for pairwise comparisons (****: p-value <0.0001). G–J) Log2 fold change of proteins compared to mitochondrial probe enriched proteins for the RNA Polymerases (G), mtDNA nucleoid packaging proteins58 (H), Shelterin (I), and CENP-A nucleosomal complexes (J). Significance calculated based on Welch’s t-test for pairwise comparisons (p-value: *<0.05, **<0.01, ***<0.001, ****<0.0001).

DNA O-MAP efficiently labels single-copy chromatin loop anchors.

A) Workflow of DNA O-MAP integrated with biotin purification sequencing. B) Schematic of a pair of chromatin loop anchors on a hypothetical Hi-C map and 3-dimensional space. C) DNA FISH and the streptavidin staining of the proteins biotinylated by DNA O-MAP targeting anchors of chromatin loops on chromosome 3 and chromosome 19. D) Table listing the three anchors (Track 1-3) and no-primary-probe control (Track 4) biotinylated by DNA O-MAP and their expected anchors in contact in each track (top). Desthiobiotin purification sequencing signals across the 9-Mb region on chromosome 3 corresponding to the chr3 chromatin loop (middle). Desthiobiotin purification sequencing signals and pairwise contact map at 5-kb resolution across the 2.5-Mb region on chromosome 3 corresponding to the chr3 chromatin loop. Black circle on the contact map indicates the presence of a loop (bottom). E) Table listing the three chromatin loop anchors (Track 1-2) and no-primary-probe controls (Track 3-4) biotinylated by DNA O-MAP in duplicates and their expected anchors in contact in each track (top). Desthiobiotin purification sequencing signals across the 8-Mb region on chromosome 10 corresponding to the chr10 chromatin loop targeted (middle). Desthiobiotin purification sequencing signals and pairwise contact map at 5-kb resolution across the 1-Mb region on chromosome 10 corresponding to the chr10 chromatin loop. Black circle on the contact map indicates the presence of a loop (bottom). F) Desthiobiotin purification sequencing signals across the 7-Mb region on chromosome 19 corresponding to the chr19 chromatin loops targeted (top). Desthiobiotin purification sequencing signals and pairwise contact map at 5-kb resolution across the 1-Mb region on chromosome 19 corresponding to the chr19 chromatin loops. Black circles on the contact map indicate the presence of loops (bottom).

DNA O-MAP efficiently identifies the local proteome of the HOXA and HOXB gene clusters.

A) Schematic of DNA O-MAP being applied to the HOXA and HOXB gene clusters for identification of differentially enriched proteins. B) Representative images depicting overlap of FISH and Streptavidin labeling at HOXA and HOXB loci. C) Volcano plot of proteins identified at HOXA and HOXB loci. Each dot represents a single protein with proteins of interest called out in black. Green dots indicate proteins that passed significant enrichment thresholds with an absolute Log2 Fold Change greater than 1 (2-fold change) and corrected p-value < 0.05. D) ENCODE ChIP-seq data showing peak calls and p-values at HOXA and HOXB loci for selected enriched proteins, ZC3H13, SMARCB1, HDAC3, and TCF12. E) Schematic depicting the use of GSK126 with DNA O-MAP. F) Bar chart showing proteins with significantly altered abundance following treatment with GSK126 at HOXA. G) Bar chart showing proteins with significantly altered abundance at both HOXA and HOXB following treatment with GSK126 (Welch’s t-test, corrected p-value < 0.05).

DNA O-MAP elucidates the homolog-resolved chromosome X proteome.

A) Schematic of DNA O-MAP being applied to Xi and Xa for identification of differentially enriched proteins. B) Schematic showing the region of the X chromosome targeted by our primary hybridization probes. C) Representative images depicting overlap of Xist FISH and Xi Streptavidin labeling while spatially differentiated from Xa FISH. Scale bars are 10 uM. D) Volcano plot of proteins identified at Xa and Xi. Each dot represents a single protein with proteins of interest called out in black text. Green dots indicate proteins that passed significant enrichment thresholds with an absolute Log2 Fold Change greater than 1 (2-fold change) and corrected p-value < 0.1. E) Bar chart showing example proteins with significant enrichment at Xi in green and at Xa in blue. Corrected p-value < 0.1. F) ENCODE ChIP-seq data in mouse fibroblast cells at our targeted region of chromosome X for SMC3. G) Protein interaction networks of EIF and SWI/SNF complexes enriched at Xi. Node width is a function of corrected p-value and node color is a function of enrichment (Log2 Fold Change). H) Protein interaction network of the SWI/SNF complex from previously published RNA O-MAP of Xist. Node width is a function of -Log10(p-value) and node color is a function of enrichment (Log2 Fold Change).

Predicted genome-wide binding profile of the pan-alpha probe.

The intensity of red indicates the amount of predicted probe binding.

Replicate analysis of multi-target DNA O-MAP proteomics experiment.

A) Pearson correlation coefficient of the raw protein intensity values for each replicate of the analysis with hierarchical clustering on the rows and columns.

Quantification of DNA O-MAP labeling specificity and efficiency for pan-alpha, telomere, and mitochondria probes.

A) Fluorescent microscopy data showing co-localization of DNA FISH and streptavidin staining of the proteins biotinylated by DNA O-MAP using no primary probe or targeting the mitochondrial genome, telomeres, or alpha satellites. B) Representative images for both DNA FISH and streptavidin stain from each stage of quantifying specificity and labeling efficiency for probes targeting the mitochondrial genome. C) Table of results for the mitochondrial genome, telomeres, and alpha satellites showing iou, specificity, labelling efficiency, and cell count of images quantified for DNA FISH and streptavidin staining.

Relative quantitation for the multi-target DNA O-MAP proteomics experiment compared to no-probe control and mtDNA datasets.

Volcano plots from multiplexed proteomics experiments with proteins of interest highlighted. A-C) Fold-changes and significance calculated compared to no probe. D-F) Fold-changes and significance calculated compared to mtDNA probe.

Comparison of histone proteins between telomere and pan-alpha probes.

A) Log2 fold change of proteins compared to mitochondrial probe enriched histone complex proteins. Significance calculated based on Welch’s t-test for pairwise comparisons (p-value: *<0.05, **<0.01, ***<0.001, ****<0.0001). B) Volcano plot comparing the fold change of pan-alpha to the mtDNA probe with spindle proteins highlighted.

DNA O-MAP biotin purification sequencing of chr3 left, chr3 right, chr10 non-loop anchors, and no-primary-probe control.

A) Table listing the three anchors (Track 1-3) and no-primary-probe control (Track 4) biotinylated by DNA O-MAP and their expected contact anchors (left). Biotin purification sequencing signals across the 8-Mb region on chromosome 10 corresponding to the chr10 non-loop anchor targeted (right). B) Biotin purification sequencing signals across every chromosome in the genome for this experiment.

DNA O-MAP biotin purification sequencing of multiplexed targeting of chr3 left, chr10 right, chr19 right anchors, and no-primary-probe control in duplicates.

A) Table listing the three anchors (Track 1-2) and no-primary-probe control (Track 3-4) biotinylated by DNA O-MAP and their expected contact anchors (left). Biotin purification sequencing signals across the 9-Mb region on chromosome 3 corresponding chr3 left anchor targeted in Track 1-2 (right). B) Biotin purification sequencing signals across every chromosome in the genome for this experiment.

DNA O-MAP biotin purification sequencing enrichment score distribution of multiplexed targeting of chr3 left, chr10 right, chr19 right anchors.

The scatter plots illustrate the enrichment score distribution for experiments targeting specific regions of chromosome 3, 10, and 19. The x-axes show the log distance from the target in kilobase pairs (kp), and the y-axes show the enrichment scores. Data points highlighted in blue represent the primary target region and the data points highlighted in orange represent the contact regions.

DNA O-MAP elucidates differences between the HOXA and HOXB proximal proteomes and changes to their proteomes following inhibition of EZH2 with GSK126.

A) Heat map showing the Log2 fold change of all proteins that were scored as significantly enriched when comparing HOXA and HOXB proximal proteomes in K562 cells by DNA-OMAP. Adjusted p-value <0.05. B) Bar chart showing proteins with significantly altered abundance at HOXB, unique from HOXA, following treatment with GSK126 (Welch’s t-test, corrected p-value < 0.05).

DNA O-MAP elucidates the differential proteomes of the X chromosome at homolog resolution.

Heat map showing the Log2 fold change of all proteins that were scored as significantly enriched when comparing the active and inactive X chromosome homolog proximal proteomes in EY.T4 cells by DNA-OMAP. Adjusted p-value < 0.1.