Visualizing Epigenetic Modifications and Their Spatial Proximities in Single Cells Using Three DNA-encoded Amplifying FISH Imaging Strategies: BEA-FISH, PPDA-FISH and Cell-TALKING

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2024 Sep 4

PMID 39232201

Authors

Feng Chen

Xinyin Li

Min Bai

Yongxi Zhao

Affiliations

Soon will be listed here.

Abstract

Epigenetic modifications and spatial proximities of nucleic acids and proteins play important roles in regulating physiological processes and disease progression. Currently available cell imaging methods, such as fluorescence in situ hybridization (FISH) and immunofluorescence, struggle to detect low-abundance modifications and their spatial proximities. Here we describe a step-by-step protocol for three DNA-encoded amplifying FISH-based imaging strategies to overcome these challenges for varying applications: base-encoded amplifying FISH (BEA-FISH), pairwise proximity-differentiated amplifying FISH (PPDA-FISH) and cellular macromolecules-tethered DNA walking indexing (Cell-TALKING). They all use the similar core principle of DNA-encoded amplification, which transforms different nonsequence molecular features into unique DNA barcodes for in situ rolling circle amplification and FISH analysis. This involves three key reactions in fixed cell samples: target labeling, DNA encoding and rolling circle amplification imaging. Using this protocol, these three imaging strategies achieve in situ counting of low-abundance modifications alone, the pairwise proximity-differentiated visualization of two modifications and the exploration of multiple modifications around one protein (one-to-many proximity), respectively. Low-abundance modifications, including 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil and 5-formyluracil, are clearly visualized in single cells. Various combinatorial patterns of nucleic acid modifications and/or histone modifications are found. The whole protocol takes ~2-4 d to complete, depending on different imaging applications.

References

Zhao L, Song J, Liu Y, Song C, Yi C . Mapping the epigenetic modifications of DNA and RNA. Protein Cell. 2020; 11(11):792-808. PMC: 7647981. DOI: 10.1007/s13238-020-00733-7. View

Conibear A . Deciphering protein post-translational modifications using chemical biology tools. Nat Rev Chem. 2023; 4(12):674-695. DOI: 10.1038/s41570-020-00223-8. View

Weinberg D, Papillon-Cavanagh S, Chen H, Yue Y, Chen X, Rajagopalan K . The histone mark H3K36me2 recruits DNMT3A and shapes the intergenic DNA methylation landscape. Nature. 2019; 573(7773):281-286. PMC: 6742567. DOI: 10.1038/s41586-019-1534-3. View

Xia B, Han D, Lu X, Sun Z, Zhou A, Yin Q . Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale. Nat Methods. 2015; 12(11):1047-50. PMC: 4626315. DOI: 10.1038/nmeth.3569. View

Bernstein B, Mikkelsen T, Xie X, Kamal M, Huebert D, Cuff J . A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell. 2006; 125(2):315-26. DOI: 10.1016/j.cell.2006.02.041. View

Voigt P, LeRoy G, Drury 3rd W, Zee B, Son J, Beck D . Asymmetrically modified nucleosomes. Cell. 2012; 151(1):181-93. PMC: 3498816. DOI: 10.1016/j.cell.2012.09.002. View

Blanco E, Gonzalez-Ramirez M, Alcaine-Colet A, Aranda S, Di Croce L . The Bivalent Genome: Characterization, Structure, and Regulation. Trends Genet. 2019; 36(2):118-131. DOI: 10.1016/j.tig.2019.11.004. View

Gu T, Hao D, Woo J, Huang T, Guo L, Lin X . The disordered N-terminal domain of DNMT3A recognizes H2AK119ub and is required for postnatal development. Nat Genet. 2022; 54(5):625-636. PMC: 9295050. DOI: 10.1038/s41588-022-01063-6. View

Simpson J, Workman R, Zuzarte P, David M, Dursi L, Timp W . Detecting DNA cytosine methylation using nanopore sequencing. Nat Methods. 2017; 14(4):407-410. DOI: 10.1038/nmeth.4184. View

10.

Bartee D, Thalalla Gamage S, Link C, Meier J . Arrow pushing in RNA modification sequencing. Chem Soc Rev. 2021; 50(17):9482-9502. DOI: 10.1039/d1cs00214g. View

11.

Wang Y, Zhang X, Liu H, Zhou X . Chemical methods and advanced sequencing technologies for deciphering mRNA modifications. Chem Soc Rev. 2021; 50(24):13481-13497. DOI: 10.1039/d1cs00920f. View

12.

Ku W, Nakamura K, Gao W, Cui K, Hu G, Tang Q . Single-cell chromatin immunocleavage sequencing (scChIC-seq) to profile histone modification. Nat Methods. 2019; 16(4):323-325. PMC: 7187538. DOI: 10.1038/s41592-019-0361-7. View

13.

Weiner A, Lara-Astiaso D, Krupalnik V, Gafni O, David E, Winter D . Co-ChIP enables genome-wide mapping of histone mark co-occurrence at single-molecule resolution. Nat Biotechnol. 2016; 34(9):953-61. DOI: 10.1038/nbt.3652. View

14.

Zhong S, Li Z, Jiang T, Li X, Wang H . Immunofluorescence Imaging Strategy for Evaluation of the Accessibility of DNA 5-Hydroxymethylcytosine in Chromatins. Anal Chem. 2017; 89(11):5702-5706. DOI: 10.1021/acs.analchem.7b01428. View

15.

Pfaffeneder T, Spada F, Wagner M, Brandmayr C, Laube S, Eisen D . Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA. Nat Chem Biol. 2014; 10(7):574-81. DOI: 10.1038/nchembio.1532. View

16.

Larsson C, Koch J, Nygren A, Janssen G, Raap A, Landegren U . In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nat Methods. 2005; 1(3):227-32. DOI: 10.1038/nmeth723. View

17.

Choi H, Chang J, Trinh L, Padilla J, Fraser S, Pierce N . Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat Biotechnol. 2010; 28(11):1208-12. PMC: 3058322. DOI: 10.1038/nbt.1692. View

18.

Trcek T, Chao J, Larson D, Park H, Zenklusen D, Shenoy S . Single-mRNA counting using fluorescent in situ hybridization in budding yeast. Nat Protoc. 2012; 7(2):408-19. PMC: 4112553. DOI: 10.1038/nprot.2011.451. View

19.

Cao X, Chen F, Xue J, Zhao Y, Bai M, Zhao Y . Hierarchical DNA branch assembly-encoded fluorescent nanoladders for single-cell transcripts imaging. Nucleic Acids Res. 2022; 51(3):e13. PMC: 9943671. DOI: 10.1093/nar/gkac1138. View

20.

Cao X, Yu H, Xue J, Bai M, Zhao Y, Li Y . RNA-Primed Amplification for Noise-Suppressed Visualization of Single-Cell Splice Variants. Anal Chem. 2020; 92(13):9356-9361. DOI: 10.1021/acs.analchem.0c01734. View