DNA O-MAP uncovers the molecular neighborhoods associated with specific genomic loci

Yuzhen Liu
Christopher D McGann
Mary Krebs
Thomas A Perkins Jr.
Rose Fields
Conor K Camplisson
David Z Nwizugbo
Chris Hsu
Shayan C Avanessian
Ashley F Tsue
Evan E Kania
David M Shechner
Brian J Beliveau author has email address
Devin K Schweppe author has email address

Department of Genome Sciences, University of Washington, Seattle, WA, USA
Brotman Baty Institute for Precision Medicine, Seattle, WA, USA
Molecular and Cellular Biology Program, University of Washington, Seattle, WA, USA
Department of Pharmacology, University of Washington, Seattle, WA, USA
Institute of Stem Cell and Regenerative Medicine, University of Washington, USA

https://doi.org/10.7554/eLife.102489.1

Open access
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Figures and data

Overview of DNA O-MAP workflow and label-free quantitative proteomics analysis of telomeres.
A) Schematic of DNA O-MAP. B) Overview of telomere targeted DNA O-MAP experiment. C) Fluorescent microscopy data showing the observed patterns of DNA (DAPI, left) and in situ biotinylation detected by staining with fluorescent streptavidin conjugates (middle, left)l. D) Significant gene sets identified by the Gene Set Enrichment Analysis of the proteins enriched by the telomere probe. E) DNA O-MAP telomeric proteins mapped onto the BioPlex interaction network^28,29. The red box highlights shelterin complex proteins. Nodes are colored by the fold-enrichment compared to a no-primary-probe control shown in C, excluding unconnected nodes. F) 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 proteins⁴² (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).

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