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ChromEMT: visualizing and reconstructing chromatin ultrastructure and 3D organization in situ | Nature Protocols
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ChromEMT: visualizing and reconstructing chromatin ultrastructure and 3D organization in situ

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Abstract

Structure determines function. The discovery of the DNA double-helix structure revealed how genetic information is stored and copied. In the mammalian cell nucleus, up to two meters of DNA is compacted by histones to form nucleosome/DNA particle chains that form euchromatin and heterochromatin domains, chromosome territories and mitotic chromosomes upon cell division. A critical question is what are the structures, interactions and 3D organization of DNA as chromatin in the nucleus and how do they determine DNA replication timing, gene expression and ultimately cell fate. To visualize genomic DNA across these different length scales in the nucleus, we developed ChromEMT, a method that selectively enhances the electron density and contrast of DNA and interacting nucleosome particles, which enables nucleosome chains, chromatin domains, chromatin ultrastructure and 3D organization to be imaged and reconstructed by using multi-tilt electron microscopy tomography (EMT). ChromEMT exploits a membrane-permeable, fluorescent DNA-binding dye, DRAQ5, which upon excitation drives the photo-oxidation and precipitation of diaminobenzidine polymers on the surface of DNA/nucleosome particles that are visible in the electron microscope when stained with osmium. Here, we describe a detailed protocol for ChromEMT, including DRAQ5 staining, photo-oxidation, sample preparation and multi-tilt EMT that can be applied broadly to reconstruct genomic DNA structure and 3D interactions in cells and tissues and different kingdoms of life. The entire procedure takes ~9 days and requires expertise in electron microscopy sample sectioning and acquisition of multi-tilt EMT data sets.

Key points

  • ChromEMT is a method that selectively enhances the electron density and contrast of DNA and interacting nucleosome particles, enabling chromatin ultrastructure and 3D organization to be imaged and reconstructed by using multi-tilt electron microscopy tomography.

  • This approach enables chromatin to be stained specifically and its ultrastructure to be reconstructed and visualized across a different set of length scales in the nucleus.

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Fig. 1: The sequence of DNA, assembly with histones into nucleosome particles, interactions between nucleosome polymers in 3D topological domains, with the nuclear architecture and chromosome territories span multiple length scales, and together determine genome accessibility and function.
Fig. 2: Overview of the ChromEMT method to visualize and reconstruct chromatin ultrastructure in interphase and mitotic cells.
Fig. 3: Photo-oxidation station setup examples.
Fig. 4: Time series and transmitted light images of DRAQ5 photo-oxidation and DAB precipitation.
Fig. 5: Correlated light and TEM images of ChromEM-stained chromosomes in mitosis.
Fig. 6: Cellular and nuclear ultrastructure is well preserved in ChromEM sample preparation.
Fig. 7: Multi-tilt EMT data-acquisition scheme.
Fig. 8: ChromEMT enables the ultrastructure of DNA/nucleosome particles and chromatin chains to be visualized and resolved in the nucleus at nucleosome resolutions and megabase scales.
Fig. 9: ChromEMT enables chromatin chains, interactions, 3D organization and domains to be visualized, resolved and reconstructed in the context of the nuclear architecture.

Data availability

The EM tomograms are deposited in the Cell Image Library at http://www.cellimagelibrary.org/groups/49801.

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Acknowledgements

Funding was supported by grants from the W. M. Keck Foundation and NIH 5U01EB021247 to C.C.O. and M.H.E. C.C.O. was supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute. Salk core services were supported by P30CA014195 from NCI. All EM studies were carried out at the NCMIR, an NIGMS-supported Research Resource supported by NIH under GM103412 and an NIDA Award U01DA047731 to M.H.E.

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Authors and Affiliations

  1. Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA

    Horng D. Ou, Akiko Inagaki & Clodagh C. O’Shea

  2. National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, CA, USA

    Sebastien Phan, Thomas J. Deerinck & Mark H. Ellisman

  3. Department of Neurosciences, University of California San Diego, School of Medicine, La Jolla, CA, USA

    Sebastien Phan, Thomas J. Deerinck & Mark H. Ellisman

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Contributions

The detailed methods are based and extend upon the contributions of the authors listed in the initial publication of the ChromEMT method and its application in the visualization and 3D reconstruction of chromatin in interphase nuclei and mitotic chromosomes47. C.C.O. and M.H.E. jointly supervised this work. H.D.O. performed sample preparation and photo-oxidation. T.J.D. performed sample sectioning. S.P. performed EM tomogram acquisition and reconstruction. The initial draft of this manuscript was written by H.D.O., T.J.D. and S.P. with editing from C.C.O. and M.H.E. A.I. contributed to the writing and editing of the text and figures, and C.C.O. contributed to finalizing the manuscript for publication.

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Correspondence to Mark H. Ellisman or Clodagh C. O’Shea.

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Key references using this protocol

Ou, H. D. et al. Science 357, eaag0025 (2017): https://doi.org/10.1126/science.aag0025

Ou, H. D. et al. Methods 90, 39–48 (2015): https://doi.org/10.1016/j.ymeth.2015.06.002

Phan, S. et al. Adv. Struct. Chem. Imaging 2, 8 (2016): https://doi.org/10.1186/s40679-016-0021-2

Ngo, J. T. et al. Nat. Chem. Biol. 12, 459–465 (2016): https://doi.org/10.1038/nchembio.2076

Ou, H. D. et al. Cell 151, 304–319 (2012): https://doi.org/10.1016/j.cell.2012.08.035

Supplementary information

Supplementary Information

Supplementary Information 1

Supplementary Video 1

Quiescent SAECs were fixed and labeled with ChromEM, and 250-nm sections were cut and imaged by eight-tilt EMT. The reconstructed EMT volume is 1,968 nm (X) by 1,877 nm (Y) by 169 nm (Z), with a voxel value of 1 nm by 1 nm by 1 nm. There are one hundred sixty-nine 1-nm-thick serial computational TSs numbered #0–#168. To visualize chromatin as a continuum from the top to the bottom of the nuclear volume, sequential TSs can be reconstructed and visualized as a continuum (TS 10–110). 3D reconstruction and surface rendering of chromatin (magenta) and the nuclear membrane (green) are shown in the second half of the movie.

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Ou, H.D., Phan, S., Deerinck, T.J. et al. ChromEMT: visualizing and reconstructing chromatin ultrastructure and 3D organization in situ. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01071-2

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