Decoding chromosome organization using proximity labeling and long-read sequencing

Kewei Xu; Yichen Zhang; James Baldwin-Brown; Thomas A. Sasani; Nitin Phadnis; Matthew P. Miller; Ofer Rog

doi:10.1016/j.jgg.2026.05.004

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Decoding chromosome organization using proximity labeling and long-read sequencing

doi: 10.1016/j.jgg.2026.05.004

a. School of Biological Sciences, University of Utah, Salt Lake City, UT 84112, USA;
b. Center for Cell and Genome Sciences, University of Utah, Salt Lake City, UT 84112, USA;
c. Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA;
d. Department of Biochemistry, University of Utah, Salt Lake City, UT 84112, USA

Funds:

Jian Huang for generating the ColabFold projection image for cohesin and tagged dam

scientific illustrator Maria Diaz de la Loza for graphical work

Peter Ames and Sandy Parkinson for E. coli strains

and Joe Carrier, Emily Parnell, Lexy von Diezmann, Jamie Gagnon, Michael Werner, Mike Shapiro, Julie Cooper, Aaron Quinlan, Richard Clark and Aaron Fleming for discussions and advice. This work is supported by NIGMS grants R35GM142749 (to MPM) and R35GM128804 (to OR).

All members of the Rog lab for discussions

Colin Dale and Li Szhen (Michelle) Teh for the use of PFGE machine

Luke Berkowitz for yeast strains

Received Date: 2026-02-13
Accepted Date: 2026-05-11
Rev Recd Date: 2026-05-11

Available Online: 2026-05-19

Abstract

Abstract

Genomic approaches have provided detailed insight into DNA-protein interactions and chromosome architecture. However, commonly deployed techniques do not preserve connectivity-based information, leaving large-scale genome organization poorly characterized. Here, we develop npDamID, an in vivo proximity-labeling technique that indelibly marks, and then decodes, protein-associated sites. npDamID tethers dam methyltransferase to a protein of interest, followed by Nanopore sequencing to identify methylated bases along reads > 100 kb. As proof-of-concept we analyze, in budding yeast, a conserved cohesin-based meiotic backbone that organizes chromatin into an array of loops. Our data recapitulates the pertinent features of known cohesin association patterns, and, importantly, exposes variability between cells. Analysis of single reads reveals distance-dependent short- and long-range correlation between adjacent methylated bases. Finally, an important advantage of our approach is the ability to define protein association patterns in repetitive regions. By anchoring ultra-long reads onto unique regions, we define in vivo cohesin association patterns within the ribosomal DNA locus. Our versatile technique promises to illuminate diverse chromosomal processes by providing a cumulative record of heterogeneous association patterns of chromosomal factors as well as the in vivo conformations of single chromosomes.
- Methylation,
- Nanopore,
- Cohesin,
- Meiosis,
- Proximity labeling,
- Budding yeast

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References(53)

References

Abdulhay, N.J., McNally, C.P., Hsieh, L.J., Kasinathan, S., Keith, A., Estes, L.S., Karimzadeh, M., Underwood, J.G., Goodarzi, H., Narlikar, G.J., et al., 2020. Massively multiplex single-molecule oligonucleosome footprinting. Elife 9, e59404.

Altemose, N., Maslan, A., Smith, O.K., Sundararajan, K., Brown, R.R., Mishra, R., Detweiler, A.M., Neff, N., Miga, K.H., Straight, A.F., et al., 2022. DiMeLo-seq: a long-read, single-molecule method for mapping protein-DNA interactions genome wide. Nat. Methods 19, 711-723.

Barton, R.E., Massari, L.F., Robertson, D., Marston, A.L., 2022. Eco1-dependent cohesin acetylation anchors chromatin loops and cohesion to define functional meiotic chromosome domains. Elife 11, e74447.

Blat, Y., Protacio, R.U., Hunter, N., Kleckner, N., 2002. Physical and functional interactions among basic chromosome organizational features govern early steps of meiotic chiasma formation. Cell 111, 791-802.

Brar, G.A., Yassour, M., Friedman, N., Regev, A., Ingolia, N.T., Weissman, J.S., 2012. High-resolution view of the yeast meiotic program revealed by ribosome profiling. Science 335, 552-557.

Carlile, T.M., Amon, A., 2008. Meiosis I is established through division-specific translational control of a cyclin. Cell 133, 280-291.

Challa, K., Lee, M.-S., Shinohara, M., Kim, K.P., Shinohara, A., 2016. Rad61/Wpl1 (Wapl), a cohesin regulator, controls chromosome compaction during meiosis. Nucleic Acids Res. 44, 3190-3203.

Chereji, R.V., Ramachandran, S., Bryson, T.D., Henikoff, S., 2018. Precise genome-wide mapping of single nucleosomes and linkers in vivo. Genome Biol. 19, 19.

Dekker, J., Rippe, K., Dekker, M., Kleckner, N., 2002. Capturing chromosome conformation. Science 295, 1306-1311.

Denis, E., Sanchez, S., Mairey, B., Beluche, O., Cruaud, C., Lemainque, A., Wincker, P., Barbe, V., 2018. Extracting high molecular weight genomic DNA from Saccharomyces cerevisiae. Protocol Exchange 2018.076.

Deshpande, A.S., Ulahannan, N., Pendleton, M., Dai, X., Ly, L., Behr, J.M., Schwenk, S., Liao, W., Augello, M.A., Tyer, C., et al., 2022. Identifying synergistic high-order 3D chromatin conformations from genome-scale nanopore concatemer sequencing. Nat. Biotechnol. 40, 1488-1499.

Fajish, G., 5th, Challa, K., Salim, S., Vp, A., Mwaniki, S., Zhang, R., Fujita, Y., Ito, M., Nishant, K.T., Shinohara, A., 2024. DNA double-strand breaks regulate the cleavage-independent release of Rec8-cohesin during yeast meiosis. Genes Cells 29, 86-98.

Fedeles, B.I., Singh, V., Delaney, J.C., Li, D., Essigmann, J.M., 2015. The AlkB Family of Fe(II)/α-ketoglutarate-dependent dioxygenases: repairing nucleic acid alkylation damage and beyond. J. Biol. Chem. 290, 20734-20742.

Furey, T.S., 2012. ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions. Nat. Rev. Genet. 13, 840-852.

Geier, G.E., Modrich, P., 1979. Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of Dpn I endonuclease. J. Biol. Chem. 254, 1408-1413.

Gilmour, D.S., Lis, J.T., 1984. Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes. Proc. Natl. Acad. Sci. U. S. A. 81, 4275-4279.

Hattman, S., Kenny, C., Berger, L., Pratt, K., 1978. Comparative study of DNA methylation in three unicellular eucaryotes. J. Bacteriol. 135, 1156-1157.

Heldrich, J., Milano, C.R., Markowitz, T.E., Ur, S.N., Vale-Silva, L.A., Corbett, K.D., Hochwagen, A., 2022. Two pathways drive meiotic chromosome axis assembly in Saccharomyces cerevisiae. Nucleic Acids Res. 50, 4545-4556.

Hong, S., Joo, J.H., Yun, H., Kleckner, N., Kim, K.P., 2019. Recruitment of Rec8, Pds5 and Rad61/Wapl to meiotic homolog pairing, recombination, axis formation and S-phase. Nucleic Acids Res. 47, 11691-11708.

Hook, P.W., Timp, W., 2023. Beyond assembly: the increasing flexibility of single-molecule sequencing technology. Nat. Rev. Genet. 24, 627-641.

Jiang, S., Cai, Z., Wang, Y., Zeng, C., Zhang, J., Yu, W., Su, C., Zhao, S., Chen, Y., Shen, Y., et al., 2024. High plasticity of ribosomal DNA organization in budding yeast. Cell Rep. 43, 113742.

Kind, J., Pagie, L., de Vries, S.S., Nahidiazar, L., Dey, S.S., Bienko, M., Zhan, Y., Lajoie, B., de Graaf, C.A., et al., 2015. Genome-wide maps of nuclear lamina interactions in single human cells. Cell 163, 134-147.

Klein, F., Mahr, P., Galova, M., Buonomo, S.B., Michaelis, C., Nairz, K., Nasmyth, K., 1999. A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis. Cell 98, 91-103.

Lengronne, A., Katou, Y., Mori, S., Yokobayashi, S., Kelly, G.P., Itoh, T., Watanabe, Y., Shirahige, K., Uhlmann, F., 2004. Cohesin relocation from sites of chromosomal loading to places of convergent transcription. Nature 430, 573-578.

Li, H., 2018. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094-3100.

Li, H., Durbin, R., 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754-1760.

Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., 1000 Genome Project Data Processing Subgroup, 2009. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078-2079.

Marcus, J., Dixon-Luinenburg, O., Gamarra, N., Schwartz, J.P., Rozenwald, M., Maslan, A., Urnov, F., Straight, A.F., Altemose, N., Ioannidis, N.M., et al., 2026. Integrated analysis of multimodal long-read epigenetic assays. bioRxiv 2025.11.09.687458.

McIntyre, A.B.R., Alexander, N., Grigorev, K., Bezdan, D., Sichtig, H., Chiu, C.Y., Mason, C.E., 2019. Single-molecule sequencing detection of N6-methyladenine in microbial reference materials. Nat. Commun. 10, 579.

Nebel, B.R., Coulon, E.M., 1962. The fine structure of chromosomes in pigeon spermatocytes. Chromosoma 13, 272-291.

Panizza, S., Mendoza, M.A., Berlinger, M., Huang, L., Nicolas, A., Shirahige, K., Klein, F., 2011. Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination. Cell 146, 372-383.

Rattner, J.B., Goldsmith, M.R., Hamkalo, B.A., 1981. Chromosome organization during male meiosis in Bombyx mori. Chromosoma 82, 341-351.

Sakuno, T., Hiraoka, Y., 2022. Rec8 cohesin: a structural platform for shaping the meiotic chromosomes. Genes 13, 200.

Salim, D., Gerton, J.L., 2019. Ribosomal DNA instability and genome adaptability. Chromosome Res. 27, 73-87.

Sati, S., Cavalli, G., 2017. Chromosome conformation capture technologies and their impact in understanding genome function. Chromosoma 126, 33-44.

Schalbetter, S.A., Fudenberg, G., Baxter, J., Pollard, K.S., Neale, M.J., 2019. Principles of meiotic chromosome assembly revealed in S. cerevisiae. Nat. Commun. 10, 4795.

Schwartzman, O., Tanay, A., 2015. Single-cell epigenomics: techniques and emerging applications. Nat. Rev. Genet. 16, 716-726.

Shipony, Z., Marinov, G.K., Swaffer, M.P., Sinnott-Armstrong, N.A., Skotheim, J.M., Kundaje, A., Greenleaf, W.J., 2020. Long-range single-molecule mapping of chromatin accessibility in eukaryotes. Nat. Methods 17, 319-327.

Simpson, J.T., Workman, R.E., Zuzarte, P.C., David, M., Dursi, L.J., Timp, W., 2017. Detecting DNA cytosine methylation using nanopore sequencing. Nat. Methods 14, 407-410.

Sun, X., Huang, L., Markowitz, T.E., Blitzblau, H.G., Chen, D., Klein, F., Hochwagen, A., 2015. Transcription dynamically patterns the meiotic chromosome-axis interface. Elife 4, e07424.

Vader, G., Blitzblau, H.G., Tame, M.A., Falk, J.E., Curtin, L., Hochwagen, A., 2011. Protection of repetitive DNA borders from self-induced meiotic instability. Nature 477, 115-119.

van Steensel, B., Delrow, J., Henikoff, S., 2001. Chromatin profiling using targeted DNA adenine methyltransferase. Nat. Genet. 27, 304-308.

van Steensel, B., Henikoff, S., 2000. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat. Biotechnol. 18, 424-428.

Virtanen, P., Gommers, R., Oliphant, T.E., Haberland, M., Reddy, T., Cournapeau, D., Burovski, E., Peterson, P., Weckesser, W., Bright, J., et a., 2020. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat. Methods 17, 261-272.

Watanabe, Y., Nurse, P., 1999. Cohesin Rec8 is required for reductional chromosome segregation at meiosis. Nature 400, 461-464.

Weng, Z., Ruan, F., Chen, Weitian, Chen, Z., Xie, Y., Luo, M., Xie, Z., Zhang, C., Wang, J., Sun, Y., et al., 2023. BIND&MODIFY: a long-range method for single-molecule mapping of chromatin modifications in eukaryotes. Genome Biol. 24, 61.

Xu, L., Ajimura, M., Padmore, R., Klein, C., Kleckner, N., 1995. NDT80, a meiosis-specific gene required for exit from pachytene in Saccharomyces cerevisiae. Mol. Cell. Biol. 15, 6572-6581.

Yatskevich, S., Rhodes, J., Nasmyth, K., 2019. Organization of Chromosomal DNA by SMC Complexes. Annu. Rev. Genet. 53, 445-482.

Yue, J.-X., Li, J., Aigrain, L., Hallin, J., Persson, K., Oliver, K., Bergstrom, A., Coupland, P., Warringer, J., Lagomarsino, M.C., et al., 2017. Contrasting evolutionary genome dynamics between domesticated and wild yeasts. Nat. Genet. 49, 913-924.

Yue, X., Xie, Z., Li, M., Wang, K., Li, X., Zhang, X., Yan, J., Yin, Y., 2022. Simultaneous profiling of histone modifications and DNA methylation via nanopore sequencing. Nat. Commun. 13, 1-14.

Zhang, L., Liang, Z., Hutchinson, J., Kleckner, N., 2014. Crossover patterning by the beam-film model: analysis and implications. PLoS Genet. 10, e1004042.

Zhou, T., Zhang, R., Ma, J., 2021. The 3D Genome Structure of Single Cells. Annu. Rev. Biomed. Data Sci. 4, 21-41.

Zickler, D., Kleckner, N., 2023. Meiosis: Dances Between Homologs. Annu. Rev. Genet. 57, 1-63.

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