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Hi-CO: 3D genome structure analysis with nucleosome resolution

Abstract

The nucleosome is the basic organizational unit of the genome. The folding structure of nucleosomes is closely related to genome functions, and has been reported to be in dynamic interplay with binding of various nuclear proteins to genomic loci. Here, we describe our high-throughput chromosome conformation capture with nucleosome orientation (Hi-CO) technology to derive 3D nucleosome positions with their orientations at every genomic locus in the nucleus. This technology consists of an experimental procedure for nucleosome proximity analysis and a computational procedure for 3D modeling. The experimental procedure is based on an improved method of high-throughput chromosome conformation capture (Hi-C) analysis. Whereas conventional Hi-C allows spatial proximity analysis among genomic loci with 1–10 kbp resolution, our Hi-CO allows proximity analysis among DNA entry or exit points at every nucleosome locus. This analysis is realized by carrying out ligations among the entry/exit points in every nucleosome in a micrococcal-nuclease-fragmented genome, and by quantifying frequencies of ligation products with next-generation sequencing. Our protocol has enabled this analysis by cleanly excluding unwanted non-ligation products that are abundant owing to the frequent genome fragmentation by micrococcal nuclease. The computational procedure is based on simulated annealing-molecular dynamics, which allows determination of optimized 3D positions and orientations of every nucleosome that satisfies the proximity ligation data sufficiently well. Typically, examination of the Saccharomyces cerevisiae genome with 130 million sequencing reads facilitates analysis of a total of 66,360 nucleosome loci with 6.8 nm resolution. The technique requires 2–3 weeks for sequencing library preparation and 2 weeks for simulation.

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Fig. 1: An overview of the Hi-CO procedure.
Fig. 2: A workflow of the sequenced read analysis to derive the Hi-CO matrix.
Fig. 3: Optimization of the MNase digestion step.
Fig. 4: Schematic representation of the DNA adaptors.
Fig. 5: Excision of a gel piece containing the ligation products.
Fig. 6: Size distribution of the DNA library.
Fig. 7: The nucleosome model for the SA-MD simulation.
Fig. 8: Trends in probabilities of read pairs as a function of genomic distance.
Fig. 9: Typical Hi-CO contact matrix.
Fig. 10: 3D nucleosome folding structure.

Data availability

All data presented in this study are generated using the raw data shown in our previous publication12 that are available at BioProject accession number PRJNA427106. Source data are provided with this paper.

Code availability

Software for SA-MD simulation and a LabVIEW code for analyzing paired-end sequencing reads to output the Hi-CO matrix are available at https://doi.org/10.6084/m9.figshare.13176101.v1. Sample Hi-CO data measuring nucleosome contacts for chromosome 1 of the S. cerevisiae genome are available at the same link.

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Acknowledgements

This work was supported by PRESTO; Japan Science and Technology Agency (JPMJPR15F7); grants-in-aid for Scientific Research (A) (20H00460), Challenging Exploratory Research (26650055), Challenging Pioneering Research (19H05545 and 20K20458) and Scientific Research on Innovative Areas (23115005); Japan Society for the Promotion of Science; and grants from the RIKEN Epigenome Manipulation Project, RIKEN Incentive Research Projects, Astellas Foundation for Research on Metabolic Disorders, Suntory Rising Stars Encouragement Program in Life Sciences (SunRiSE), the Takeda Science Foundation, and the Mochida Memorial Foundation for Medical and Pharmaceutical Research. D.P. acknowledges additional support from a RIKEN Foreign Postdoctoral Researcher fellowship.

Author information

Affiliations

Authors

Contributions

M.O. developed the experimental protocol. M.O. and Y.T. developed the sequence analysis method. M.O., T.A. and Y.T. developed the simulation method. M.O., T.A., D.G.P. and Y.T. wrote the manuscript.

Corresponding author

Correspondence to Yuichi Taniguchi.

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The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

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Related links

Key reference using this protocol

Ohno, M. et al. Cell 176, 520–534 (2019): https://doi.org/10.1016/j.cell.2018.12.014

Extended data

Extended Data Fig. 1 DNA fragments from input and ChIP samples.

ag, Acrylamide gel images of MNase-digested genomes before (input) and after (ChIP) immunoprecipitation, in a replicate experiment for Fig. 3b using histone H3 antibody (a), or experiments using antibodies against other core histones (H2B and H4) (b), using histone H4 antibody at different MNase digestion conditions (c), using a GFP antibody for analyzing a strain containing GFP-fused histone H4 at different cell cycle phases (df) and using a his-tag antibody for analyzing a strain expressing his-tag-fused histone H4 (g). In c, the arrows indicate the conditions described in the protocol.

Extended Data Fig. 2 Hi-CO matrix with colors swapped.

a,b, In addition to a Hi-CO matrix with the original color scheme (a), a matrix where the cyan and yellow color are swapped (b) is shown. Data at YDL127W gene locus are used to generate the matrix.

Supplementary information

Source data

Source Data Fig. 8

Raw data of probability graph in a.

Source Data Fig. 9

Raw data of contact matrix.

Source Data Fig. 10

Raw data of XYZ coordinates of nucleosome models.

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Ohno, M., Ando, T., Priest, D.G. et al. Hi-CO: 3D genome structure analysis with nucleosome resolution. Nat Protoc 16, 3439–3469 (2021). https://doi.org/10.1038/s41596-021-00543-z

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