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Measuring Cytological Proximity of Chromosomal Loci to Defined Nuclear Compartments with TSA-seq

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Spatial Genome Organization

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2532))

Abstract

Distinct nuclear structures and bodies are involved in genome intranuclear positioning. Measuring proximity and relative distances of genomic loci to these nuclear compartments, and correlating this chromosome intranuclear positioning with epigenetic marks and functional readouts genome-wide, will be required to appreciate the true extent to which this nuclear compartmentalization contributes to regulation of genome functions. Here we present detailed protocols for TSA-seq, the first sequencing-based method for estimation of cytological proximity of chromosomal loci to spatially discrete nuclear structures, such as nuclear bodies or the nuclear lamina. TSA-seq uses Tyramide Signal Amplification (TSA) of immunostained cells to create a concentration gradient of tyramide–biotin free radicals which decays exponentially as a function of distance from a point-source target. Reaction of these free radicals with DNA deposits tyramide–biotin onto DNA as a function of distance from the point source. The relative enrichment of this tyramide-labeled DNA versus input DNA, revealed by DNA sequencing, can then be used as a “cytological ruler” to infer relative, or even absolute, mean chromosomal distances from immunostained nuclear compartments. TSA-seq mapping is highly reproducible and largely independent of the target protein or antibody choice for labeling a particular nuclear compartment. Our protocols include variations in TSA labeling conditions to provide varying spatial resolution as well as enhanced sensitivity. Our most streamlined protocol produces TSA-seq spatial mapping over a distance range of ~1 micron from major nuclear compartments using ~10–20 million cells.

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References

  1. Takizawa T, Meaburn KJ, Misteli T (2008) The meaning of gene positioning. Cell 135(1):9–13. https://doi.org/10.1016/j.cell.2008.09.026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Bickmore WA (2013) The spatial organization of the human genome. Annu Rev Genomics Hum Genet 14:67–84. https://doi.org/10.1146/annurev-genom-091212-153515

    Article  CAS  PubMed  Google Scholar 

  3. Chen Y, Zhang Y, Wang Y et al (2018) Mapping 3D genome organization relative to nuclear compartments using TSA-Seq as a cytological ruler. J Cell Biol 217:4025–4048. https://doi.org/10.1083/jcb.201807108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zhang L, Zhang Y, Chen Y et al (2020) TSA-seq reveals a largely conserved genome organization relative to nuclear speckles with small position changes tightly correlated with gene expression changes. Genome Res 31:251–264. https://doi.org/10.1101/gr.266239.120

    Article  Google Scholar 

  5. Guelen L, Pagie L, Brasset E et al (2008) Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature 453(7197):948–951. https://doi.org/10.1038/nature06947

    Article  CAS  PubMed  Google Scholar 

  6. Kind J, Pagie L, de Vries SS et al (2015) Genome-wide maps of nuclear lamina interactions in single human cells. Cell 163:134–147. https://doi.org/10.1016/j.cell.2015.08.040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. van Steensel B, Belmont AS (2017) Lamina-associated domains: links with chromosome architecture, heterochromatin, and gene repression. Cell 169(5):780–791. https://doi.org/10.1016/j.cell.2017.04.022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hall LL, Smith KP, Byron M et al (2006) Molecular anatomy of a speckle. Anat Rec A Discov Mol Cell Evol Biol 288(7):664–675. https://doi.org/10.1002/ar.a.20336

    Article  PubMed  PubMed Central  Google Scholar 

  9. Chen Y, Belmont AS (2019) Genome organization around nuclear speckles. Curr Opin Genet Dev 55:91–99. https://doi.org/10.1016/j.gde.2019.06.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Spector DL (2001) Nuclear domains. J Cell Sci 114(16):2891

    Article  CAS  Google Scholar 

  11. Spector DL (2006) SnapShot: cellular bodies. Cell 127(5):1071. https://doi.org/10.1016/j.cell.2006.11.026

    Article  PubMed  Google Scholar 

  12. Ferrai C, de Castro IJ, Lavitas L et al (2010) Gene positioning. Cold Spring Harb Perspect Biol 2(6):a000588. https://doi.org/10.1101/cshperspect.a000588

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Geyer PK, Vitalini MW, Wallrath LL (2011) Nuclear organization: taking a position on gene expression. Curr Opin Cell Biol 23(3):354–359. https://doi.org/10.1016/j.ceb.2011.03.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Feuerborn A, Cook PR (2015) Why the activity of a gene depends on its neighbors. Trends Genet 31(9):483–490. https://doi.org/10.1016/j.tig.2015.07.001

    Article  CAS  PubMed  Google Scholar 

  15. Feric M, Vaidya N, Harmon Tyler S et al (2016) Coexisting liquid phases underlie nucleolar subcompartments. Cell 165:1686–1697. https://doi.org/10.1016/j.cell.2016.04.047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Yamazaki T, Souquere S, Chujo T et al (2018) Functional domains of NEAT1 architectural lncRNA induce Paraspeckle assembly through phase separation. Mol Cell 70(6):1038–1053e1037. https://doi.org/10.1016/j.molcel.2018.05.019

    Article  CAS  PubMed  Google Scholar 

  17. Rai AK, Chen JX, Selbach M et al (2018) Kinase-controlled phase transition of membraneless organelles in mitosis. Nature 559(7713):211–216. https://doi.org/10.1038/s41586-018-0279-8

    Article  CAS  PubMed  Google Scholar 

  18. Strom AR, Brangwynne CP (2019) The liquid nucleome - phase transitions in the nucleus at a glance. J Cell Sci 132(22):jcs235093. https://doi.org/10.1242/jcs.235093

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hondele M, Sachdev R, Heinrich S et al (2019) DEAD-box ATPases are global regulators of phase-separated organelles. Nature 573(7772):144–148. https://doi.org/10.1038/s41586-019-1502-y

    Article  CAS  PubMed  Google Scholar 

  20. Sabari BR, Dall'Agnese A, Boija A et al (2018) Coactivator condensation at super-enhancers links phase separation and gene control. Science 361(6400):eaar3958. https://doi.org/10.1126/science.aar3958

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Cho W-K, Spille J-H, Hecht M et al (2018) Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science 361(6400):412. https://doi.org/10.1126/science.aar4199

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Boija A, Klein IA, Sabari BR et al (2018) Transcription factors activate genes through the phase-separation capacity of their activation domains. Cell 175(7):1842–1855e1816. https://doi.org/10.1016/j.cell.2018.10.042

    Article  CAS  PubMed  Google Scholar 

  23. Boehning M, Dugast-Darzacq C, Rankovic M et al (2018) RNA polymerase II clustering through carboxy-terminal domain phase separation. Nat Struct Mol Biol 25(9):833–840. https://doi.org/10.1038/s41594-018-0112-y

    Article  CAS  PubMed  Google Scholar 

  24. Lu H, Yu D, Hansen AS et al (2018) Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II. Nature 558(7709):318–323. https://doi.org/10.1038/s41586-018-0174-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Guo YE, Manteiga JC, Henninger JE et al (2019) Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature 572(7770):543–548. https://doi.org/10.1038/s41586-019-1464-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Saitoh N, Spahr CS, Patterson SD et al (2004) Proteomic analysis of interchromatin granule clusters. Mol Biol Cell 15(8):3876–3890. https://doi.org/10.1091/mbc.E04-03-0253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Galganski L, Urbanek MO, Krzyzosiak WJ (2017) Nuclear speckles: molecular organization, biological function and role in disease. Nucleic Acids Res 45(18):10350–10368. https://doi.org/10.1093/nar/gkx759

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Dopie J, Sweredoski MJ, Moradian A et al (2020) Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins. J Cell Biol 219(9):e201910207. https://doi.org/10.1083/jcb.201910207

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Su J-H, Zheng P, Kinrot SS et al (2020) Genome-scale imaging of the 3D organization and transcriptional activity of chromatin. Cell 182(6):1641–1659.e1626. https://doi.org/10.1016/j.cell.2020.07.032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Takei Y, Yun J, Ollikainen N et al (2020) Global architecture of the nucleus in single cells by DNA seqFISH+ and multiplexed immunofluorescence. bioRxiv:2020.2011.2029.403055. https://doi.org/10.1101/2020.11.29.403055

  31. Robinett CC, Straight A, Li G et al (1996) In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition. J Cell Biol 135(6):1685–1700. https://doi.org/10.1083/jcb.135.6.1685

    Article  CAS  PubMed  Google Scholar 

  32. Hepperger C, Otten S, von Hase J et al (2007) Preservation of large-scale chromatin structure in FISH experiments. Chromosoma 116(2):117–133. https://doi.org/10.1007/s00412-006-0084-2

    Article  CAS  PubMed  Google Scholar 

  33. Landt SG, Marinov GK, Kundaje A et al (2012) ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res 22(9):1813–1831. https://doi.org/10.1101/gr.136184.111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Vogel MJ, Peric-Hupkes D, van Steensel B (2007) Detection of in vivo protein-DNA interactions using DamID in mammalian cells. Nat Protoc 2(6):1467–1478. https://doi.org/10.1038/nprot.2007.148

    Article  CAS  PubMed  Google Scholar 

  35. Skene PJ, Henikoff S (2017) An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife 6:e21856. https://doi.org/10.7554/eLife.21856

    Article  PubMed  PubMed Central  Google Scholar 

  36. Skene PJ, Henikoff JG, Henikoff S (2018) Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat Protoc 13(5):1006–1019. https://doi.org/10.1038/nprot.2018.015

    Article  CAS  PubMed  Google Scholar 

  37. Kaya-Okur HS, Wu SJ, Codomo CA et al (2019) CUT&tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10(1):1930. https://doi.org/10.1038/s41467-019-09982-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. van Schaik T, Vos M, Peric-Hupkes D et al (2020) Cell cycle dynamics of lamina-associated DNA. EMBO Rep 21(11):e50636. https://doi.org/10.15252/embr.202050636

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Pickersgill H, Kalverda B, de Wit E et al (2006) Characterization of the Drosophila melanogaster genome at the nuclear lamina. Nat Genet 38(9):1005–1014. https://doi.org/10.1038/ng1852

    Article  CAS  PubMed  Google Scholar 

  40. Briand N, Collas P (2020) Lamina-associated domains: peripheral matters and internal affairs. Genome Biol 21(1):85. https://doi.org/10.1186/s13059-020-02003-5

    Article  PubMed  PubMed Central  Google Scholar 

  41. Ilik İA, Malszycki M, Lübke AK et al (2020) SON and SRRM2 are essential for nuclear speckle formation. Elife 9:e60579. https://doi.org/10.7554/eLife.60579

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Fei J, Jadaliha M, Harmon TS et al (2017) Quantitative analysis of multilayer organization of proteins and RNA in nuclear speckles at super resolution. J Cell Sci 130(24):4180. https://doi.org/10.1242/jcs.206854

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Quinodoz SA, Ollikainen N, Tabak B et al (2018) Higher-order inter-chromosomal hubs shape 3D genome organization in the nucleus. Cell 174(3):744–757e724. https://doi.org/10.1016/j.cell.2018.05.024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Quinodoz SA, Bhat P, Ollikainen N et al (2020) RNA promotes the formation of spatial compartments in the nucleus. bioRxiv:2020.2008.2025.267435. https://doi.org/10.1101/2020.08.25.267435

  45. Chen W, Yan Z, Li S et al (2018) RNAs as proximity-labeling media for identifying nuclear speckle positions relative to the genome. iScience 4:204–215. https://doi.org/10.1016/j.isci.2018.06.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Bobrow MN, Harris TD, Shaughnessy KJ et al (1989) Catalyzed reporter deposition, a novel method of signal amplification: application to immunoassays. J Immunol Methods 125(1–2):279–285

    Article  CAS  Google Scholar 

  47. Raap AK, van de Corput MPC, Vervenne RAM et al (1995) Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides. Hum Mol Genet 4(4):529–534. https://doi.org/10.1093/hmg/4.4.529

    Article  CAS  PubMed  Google Scholar 

  48. Gao XD, Tu LC, Mir A et al (2018) C-BERST: defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2. Nat Methods 15(6):433–436. https://doi.org/10.1038/s41592-018-0006-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Myers SA, Wright J, Peckner R et al (2018) Discovery of proteins associated with a predefined genomic locus via dCas9–APEX-mediated proximity labeling. Nat Methods 15(6):437–439. https://doi.org/10.1038/s41592-018-0007-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Fazal FM, Han S, Parker KR et al (2019) Atlas of subcellular RNA localization revealed by APEX-Seq. Cell 178(2):473–490e426. https://doi.org/10.1016/j.cell.2019.05.027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Kurihara M, Kato K, Sanbo C et al (2020) Genomic profiling by ALaP-Seq reveals transcriptional regulation by PML bodies through DNMT3A exclusion. Mol Cell 78(3):493–505.e498. https://doi.org/10.1016/j.molcel.2020.04.004

    Article  CAS  PubMed  Google Scholar 

  52. Tran JR, Paulson DI, Moresco JJ et al (2021) An APEX2 proximity ligation method for mapping interactions with the nuclear lamina. J Cell Biol 220(1):e202002129. https://doi.org/10.1083/jcb.202002129

    Article  CAS  PubMed  Google Scholar 

  53. Hopman AHN, Ramaekers FCS, Speel EJM (1998) Rapid synthesis of biotin-, Digoxigenin-, Trinitrophenyl-, and Fluorochrome-labeled Tyramides and their application for in situ hybridization using CARD amplification. J Histochem Cytochem 46(6):771–777. https://doi.org/10.1177/002215549804600611

    Article  CAS  PubMed  Google Scholar 

  54. http://wiki.xenbase.org/xenwiki/index.php/Flourescin_Tyramide_Synthesis

  55. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with bowtie 2. Nat Methods 9(4):357–359. https://doi.org/10.1038/nmeth.1923

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We thank Drs. William Brieher, Brian Freeman, K.V. Prasanth, and Lisa Stubbs (UIUC, Urbana, IL) for helpful suggestions in developing these protocols. We also thank Belmont laboratory members for reagents and suggestions. We thank the UIUC Biotechnology center for guidance with DNA sonication and sequencing library preparation. We thank Drs. Jian Ma, Bas van Steensel, David Gilbert, and Huimin Zhao from the Belmont 4DN NOFIC U54 Center and other members of the 4D-Nucleome Consortium for helpful suggestions and feedback. This work was supported by National Institutes of Health grants R01GM58460 (ASB) and U54 DK107965 (ASB).

Author contributions: ASB conceptualized the TSA-seq idea and supervised the development of TSA-seq 1.0 and 2.0. YC developed TSA-seq 1.0 with protocols for tyramide–biotin labeling, TSA cell labeling, genomic DNA purification and fragmentation, dot blot and biotinylated DNA bead pulldown, contributed by LZ for Condition 3 (DTT). LZ developed TSA-seq 2.0, added protocols for TSA adherent cell labeling and Drosophila DNA spike in controls, and optimized protocols/methods for TSA labeling, genomic DNA purification and fragmentation, dot blot, and sequencing library construction. LZ, ASB, and YC prepared the manuscript.

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Author notes

  1. Yu Chen

    Present address: Department of Molecular and Cell Biology, Li Ka Shing Center for Biomedical and Health Sciences, Howard Hughes Medical Institute, University of California, Berkeley, CA, USA

  2. Liguo Zhang and Yu Chen contributed equally as co-first authors.

Authors and Affiliations

  1. Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Liguo Zhang, Yu Chen & Andrew S. Belmont

  2. Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Andrew S. Belmont

  3. Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Andrew S. Belmont

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  1. Liguo Zhang
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  2. Yu Chen
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  3. Andrew S. Belmont
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Correspondence to Andrew S. Belmont .

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  1. Institut de génétique et de biologie moléculaire et cellulaire (IGBMC), Illkirch, France

    Tom Sexton

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Zhang, L., Chen, Y., Belmont, A.S. (2022). Measuring Cytological Proximity of Chromosomal Loci to Defined Nuclear Compartments with TSA-seq. In: Sexton, T. (eds) Spatial Genome Organization. Methods in Molecular Biology, vol 2532. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2497-5_8

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  • DOI: https://doi.org/10.1007/978-1-0716-2497-5_8

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