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Superresolution imaging of chromatin fibers to visualize epigenetic information on replicative DNA

Nature Protocols volume 15, pages 1188–1208 (2020)Cite this article

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Abstract

During DNA replication, the genetic information of a cell is copied. Subsequently, identical genetic information is segregated reliably to the two daughter cells through cell division. Meanwhile, DNA replication is intrinsically linked to the process of chromatin duplication, which is required for regulating gene expression and establishing cell identities. Understanding how chromatin is established, maintained or changed during DNA replication represents a fundamental question in biology. Recently, we developed a method to directly visualize chromatin components at individual replication forks undergoing DNA replication. This method builds upon the existing chromatin fiber technique and combines it with cell type–specific chromatin labeling and superresolution microscopy. In this method, a short pulse of nucleoside analog labels replicative regions in the cells of interest. Chromatin fibers are subsequently isolated and attached to a glass slide, after which a laminar flow of lysis buffer extends the lysed chromatin fibers parallel with the direction of the flow. Fibers are then immunostained for different chromatin-associated proteins and mounted for visualization using superresolution microscopy. Replication foci, or ‘bubbles,’ are identified by the presence of the incorporated nucleoside analog. For researchers experienced in molecular biology and superresolution microscopy, this protocol typically takes 2–3 d from sample preparation to data acquisition, with an additional day for data processing and quantification.

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Fig. 1
Fig. 2: A flow chart of the chromatin fiber image acquisition and data analysis procedure.
Fig. 3: Superresolution imaging of chromatin fibers resolves sister chromatids.
Fig. 4: Distributions of old histone-enriched H3K27me3 and new histone-enriched H3K14Ac on Kc cell–derived chromatin fibers.
Fig. 5: Comparison of histone distribution patterns in fly and yeast using distinct methods.

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Data availability

All cell lines and fly lines described in this protocol can be obtained upon request. Data for graphs shown in Fig. 4c, 4f is available in Supplementary Table 1. Other data generated during this study are available from the corresponding author upon request.

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Acknowledgements

We thank Chuanhe Yu, Xu Hua and Zhiguo Zhang as well as Nataliya Petryk and Anja Groth for helpful discussions of this manuscript. We thank Shelby Blythe and Eric Wieschaus for the PCNA-EGFP fly line. We thank Barbara Mellone and Sharon Pavanacherry for suggestions on the chromatin fiber technique. We thank Johns Hopkins Integrated Imaging Center for confocal and Airyscan imaging and Carnegie Institute Imaging Center for STED microscopy work. This work was supported by NIH grants 5T32GM007231 and F31GM115149-01A1 (M.W.), NIH grant T32GM007231 (J.S.), NIH grant R01GM33397 (J.G.G.), and NIH grants R35GM127075 and R01GM112008, the Howard Hughes Medical Institute, the David and Lucile Packard Foundation and Johns Hopkins University startup funds (X.C.)

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Author notes

  1. These authors contributed equally: Yingying Li, Jonathan Snedeker.

Authors and Affiliations

  1. Department of Biology, The Johns Hopkins University, Baltimore, MD, USA

    Matthew Wooten, Yingying Li, Jonathan Snedeker & Xin Chen

  2. Department of Embryology, Carnegie Institution for Science, Baltimore, MD, USA

    Zehra F. Nizami & Joseph G. Gall

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  1. Matthew Wooten
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Contributions

Conceptualization, M.W., J.S., Z.F.N., J.G.G. and X.C.; methodology, M.W., J.S., Y.L., Z.F.N., J.G.G. and X.C.; investigation, M.W. and Y.L.; writing original draft, M.W. and X.C.; funding acquisition, J.G.G. and X.C.; supervision, J.G.G. and X.C.

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Correspondence to Xin Chen.

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Peer review information Nature Protocols thanks Robert Duronio, Bing Zhu and Corella Casas-Delucchi for their contribution to the peer review of this work.

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Key reference using this protocol

Wooten, M. et al. Nat. Struct. Mol. Biol. 26, 732–743 (2019): https://doi.org/10.1038/s41594-019-0269-z

Integrated supplementary information

Supplementary Fig. 1 Modified 50-ml conical tube.

A modified 50-ml conical tube with a small hole made at the bottom with a 25-gauge hypodermic needle.

Supplementary Fig. 2 Modified 50-ml conical tube with lysis buffer.

Modified 50-ml conical tube containing 25 ml of lysis buffer with the cap screwed on and angled at 35° in preparation for lysis and fiber-generation steps.

Supplementary Fig. 3 50-ml conical tube zoomed-in view.

A zoom-in picture of Supplementary Fig. 2.

Supplementary Fig. 4 Humid chamber closed.

A humid chamber made with a P1000 tip box. The lid is wrapped with tin foil to minimize light exposure to slides placed inside. The lid should be placed on when slides are inside. The humid chamber can contain four slides comfortably.

Supplementary Fig. 5 Humid chamber open.

A humid chamber with the lid off. Note that a wet paper towel is placed at the bottom of the tip box shortly before adding the slides, which are placed on top of the tip rack.

Supplementary information

Supplementary Information

Supplementary Figs. 1–5, Supplementary Tables 1 and 2 and Supplementary Methods.

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Wooten, M., Li, Y., Snedeker, J. et al. Superresolution imaging of chromatin fibers to visualize epigenetic information on replicative DNA. Nat Protoc 15, 1188–1208 (2020). https://doi.org/10.1038/s41596-019-0283-y

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