JoVE Journal > Biochemistry
Summary
Abstract
Introduction
Protocol
Risultati
Discussion
Divulgazioni
Acknowledgements
Materials
Riferimenti
Traduzione automatica
Biochimica

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Published: March 09, 2022
doi:
10.3791/63501
, , ,
1Department of Biological Sciences,Ulsan National Institute of Science and Technology, 2Center for Genomic Integrity,Institute for Basic Science (IBS)

Summary

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

Abstract

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

Introduction

Eukaryotic DNA is packaged into a higher-order structure known as chromatin1,2. Nucleosome is the fundamental unit of chromatin, which consists of approximately 147 bp DNA wrapped around the octameric core histones3,4. Chromatin plays a critical role in eukaryotic cells; for example, the compact structure protects DNA from endogenous factors and exogenous threats5. Chromatin structure changes dynamically according to the cell cycle phase and environmental stimuli, and these changes control protein access during DNA transactions such as replication, transcription, and repair6. Chromatin dynamics are also important for genomic stability and epigenetic information.

Chromatin is dynamically regulated by various factors, including histone tail modifications and chromatin organizers such as chromatin remodelers, polycomb group proteins, and histone chaperones7. Histone chaperones coordinate the assembly and disassembly of nucleosomes via deposition or detachment of core histones8,9. Defects in histone chaperones induce genome instability and cause developmental disorders and cancer9,10. Various histone chaperones do not need chemical energy consumption like ATP hydrolysis to assemble or disassemble nucleosomes9,11,12,13. Recently, researchers reported that bromodomain-containing AAA+ (ATPase associated with diverse cellular activities) ATPases play a role in chromatin dynamics as histone chaperones14,15,16,17. Human ATAD2 (ATPase family AAA domain-containing protein 2) promotes chromatin accessibility to enhance gene expression18. As a transcriptional co-regulator, ATAD2 regulates the chromatin of oncogenic transcriptional factors14, and the overexpression of ATAD2 is related to poor prognosis in many types of cancer19. Yta7, the Saccharomyces cerevisiae (S. cerevisiae) homolog of ATAD2, decreases nucleosome density in chromatin15. In contrast, Abo1, the Schizosaccharomyces pombe (S. pombe) homolog of ATAD2, increases nucleosome density16. Using a unique single-molecule imaging technique, DNA curtain, whether Abo1 contributes to nucleosome assembly or disassembly is addressed17,20.

Traditionally, the biochemical properties of biomolecules have been examined by bulk experiments such as the electrophoretic mobility shift assay (EMSA) or co-immunoprecipitation (co-IP), in which a large number of molecules are probed, and their average properties are characterized21,22. In bulk experiments, molecular sub-states are veiled by the ensemble-average effect, and probing biomolecular interactions is restricted. In contrast, single-molecule techniques circumvent the limitations of bulk experiments and enable the detailed characterization of biomolecular interactions. In particular, single-molecule imaging techniques have been widely used to study DNA-protein and protein-protein interactions23. One such technique is DNA curtain, a unique single-molecule imaging technique based on microfluidics and total internal reflection fluorescence microscopy (TIRFM)24,25. In a DNA curtain, hundreds of individual DNA molecules are anchored to the lipid bilayer, which permits the two-dimensional motion of DNA molecules due to lipid fluidity. When hydrodynamic flow is applied, DNA molecules move along the flow on the bilayer and get stuck at a diffusion barrier, where they are aligned and stretched. While DNA is stained with intercalating agents, fluorescently labeled proteins are injected, and TIRFM is used to visualize protein-DNA interactions in real-time at a single-molecule level23. The DNA curtain platform facilitates the observation of protein movements such as diffusion, translocation, and collision26,27,28. Moreover, DNA curtain can be used for protein mapping on DNA with defined positions, orientations, and topologies or applied to the study of phase separation of protein and nucleic acids29,30,31.

In this work, the DNA curtain technique is used to provide evidence for the function of chaperones through direct visualization of specific proteins. Moreover, because DNA curtain is a high-throughput platform, it facilitates an extent of data collection sufficient for statistical reliability. Here, it is described how to conduct the DNA curtain assay in detail to investigate the molecular role of S. pombe bromodomain-containing AAA+ ATPase Abo1.

Protocol

1. Preparation of the flow cell Prepare a cleaned fused silica slide containing nano-trench patterns following previously published report25. Drill two holes with 1 mm diameter in a cleaned fused silica slide (Figure 1A) using a diamond-coated drill bit (see Table of Materials). Deposit 250 nm of thick aluminum (Al) on the slide using DC sputter32 (see Table of Materia…

Representative Results

This work describes the procedure for flow cell preparation for the DNA curtain assay (Figure 1A). The DNA curtain assay facilitated the study of histone H3-H4 dimer assembly on DNA by Abo1. First, DNA curtain formation was checked by staining DNA molecules with YOYO-1, an intercalating dye. Green lines were shown in parallel arrays, indicating that YOYO-1 intercalated into DNA molecules, which were well-aligned and stretched at a diffusion barrier under hydrodynamic flow (<strong class="xfi…

Discussion

As a single-molecule imaging technique, DNA curtain has been used extensively to probe DNA metabolic reactions43. DNA curtain is a hybrid system that concatenates lipid fluidity, microfluidics, and TIRFM. Unlike other single-molecule techniques, DNA curtain enables high-throughput real-time visualization of protein-DNA interactions. Therefore, the DNA curtain technique is suitable for probing the mechanism behind molecular interactions, including sequence-specific association, protein movement alo…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

The authors appreciate the kind support for Abo1 and Cy5-H3-H4 by Professor Ji-Joon Song, Carol Cho, Ph.D., and Juwon Jang, Ph.D., in KAIST, South Korea. This work is supported by the National Research Foundation Grant (NRF-2020R1A2B5B01001792), intramural research fund (1.210115.01) of Ulsan National Institute of Science and Technology, and the Institute for Basic Science (IBS-R022-D1).

Materials

1 mL luer-lock syringe BecktonDickinson 301321
1' x 3' fused-silca slide glass G. Finkenbeiner 1 inch x 3 inch rectangular and 1 mm thickness
10 mL luer-lock syringe BecktonDickinson 302149
18:1 (Δ9-Cis) PC (DOPC) Avanti 850375 This is a component of biotinylated lipid stock
18:1 Biotinyl cap PE Avanti 870273 This is a component of biotinylated lipid stock
18:1 PEG2000 PE Avanti 880130 This is a component of biotinylated lipid stock
3 mL luer-lock syringe BecktonDickinson 302832
6-way sample injection valve IDEX MX series II
950K PMMA All-resist 671.04
Acetone SAMCHUN A1759
Adenosine 5'-triphosphate disodium salt hydrate (ATP) Sigma A2383
Aluminum (Al) TASCO, South Korea LT50AI414 Diameter 4 inch, thickness 1/4 inch
Amicon Ultra centrifugal filter, MWCO 10 kDa Millipore Z648027
Ampicillin Mbcell MB-A4128 Antibiotics
AZ 300 MIF developer Merck 10454110521 Used for removing aluminum
Blade DORCO DN52 12 mm x 6 m
Boron trichloride (BCl3) UNIONGAS Purity: >99.99%
Bovine serum albumin (BSA) Sigma A7030
Catalase Sigma C40-1g This is a component of 100x gloxy stock
Chlorine (Cl2) UNIONGAS Purity: >99.99%
Clear double-sided tape 3M 313770
D-(+)-glucose Sigma G7528
DC sputter Sorona SRM-120 Used for deposition aluminum on a slide
Diamond-coated drill bit Eurotool DIB-211.00 Used for making holes in a fusced silica slide
DL-Dithiothreitol (DTT) Sigma D0632
Dove-prism Korea Electro-Optics Co. Ltd. 1906-106 Custom-made fused-silica dove prism with anti-reflection coating
Drill Dremel Dremel 3000 Used for making holes in a fusced silica slide
Electron Bean Lithography Nanobeam Ltd. NB3
Ethylene-diamine-tetraacetic acid (EDTA) Sigma EDS-1KG
Fingertight fittings IDEX F-300 It is connected with "PFA Tubing Natural" to form luer-lock tubing
Flangeless male nut IDEX P-235 It is connected with "PFA Tubing Natural" to form luer-lock tubing
Freeze Dryer, HyperCOOL Labogene HC3110 Used for lyophilizing liquid proteins
Glucose oxidase Sigma G2133-50KU This is a component of 100x gloxy stock
Guanidinium hydrochloride Acros Organics 364790025
Hamilton syringe Hamilton Company 80065 This syringe is used for sample injection
Hellmanex III Sigma Z805939
HiLoad 26/600 SuperdexTM 200 pg Cytiva 28-9893-36 Used for FPLC (size exclusion)
Hot plate stirrer Corning PC-420D
Hydrochloric acid Sigma H1759 Used for Tris-HCl
Index matching oil ZEISS 444970-9000-000
Inductively coupled plasma-reactive ion etching Top Technology Ltd. FabStar
Isopropyl β-D-1-thiogalactopyranoside (IPTG) Glentham Life Sciences GC6586-100g Used for induction of β-galactosidase activity
Lambda phage DNA NEB N0311
LB broth BD difco 244610 Media for E.coli cell growth
Luer adapter 10-32 IDEX P-659 This connects luer-lock syringe and tubing
Magnesium chloride hexahydrate fisher bioreagents BP214
Methyl isobutyl ketone (MIBK) KAYAKU ADVANCED MATERIALS Used for developing solution
Microscope (Eclipse Ti2) Nikon Eclipse Ti2 Inverted fluorescence microscope
Microscope glass coverslip MARIENFELD 101142 22 x 50 mm (No. 1)
Microscope slide DURAN GROUP DU.2355013 Slide glass ground edge 45°, plain 26 x 76 mm
Nanoport IDEX N-333-01
Objective lens Nikon CFI Plan Apochromat VC 60XC WI Immersion type: water, magnification: 60x, correction: 18, working distance: 0.29 (0.31-0.28)
One Shot BL21 (DE3)pLysS Chemically Competent E. coli Thermo Fisher Scientific C6060-03 Competent cell for overexpressing proteins
Oxygen (O2) NOBLEGAS, South Korea Purity: >99.99%
PFA tubing natural IDEX 1512L It is connected with "Fingertight Fittings" to form luer-lock tubing
Phenylmethylsulfonyl fluoride (PMSF) Roche 11359061001 Protease inhibitor
Sephacryl S-200 High Resolution Cytiva 17-0584-01 Used for FPLC (size exclusion)
Shut-off valve IDEX P-732
Sodium acetate Sigma 791741
Sodium chloride (NaCl) Sigma S3014
Sodium hydroxide (NaOH) Sigma s5881
Spectra/Por molecularporous membrane tubing, MWCO 6-8 kDa Spectrum laboratories 132660
Streptavidin Thermo Fisher Scientific S888
Sulfur tetralfluoride (SF4) NOBLEGAS, South Korea Purity: >99.99%
Syringe pump KD Scientific 78-8210
Tetrafluoromethane (CF4) NOBLEGAS, South Korea Purity: >99.99%
TritonX-100 Sigma T9284
Trizma base Sigma T1503 Used for Tris-HCl
TSKgel SP-5PW TOSOH 14715 Used for FPLC (ion exchange)
Union assembly IDEX P-760 This connects tubings
Urea Sigma U5378
Vacuum oven Jeio Tech OV-11
YOYO-1 Thermo Fisher Scientific Y3601 This intercalation dye is diluted in DMSO
β-mercaptoethanol (BME) Sigma M6250
DOWNLOAD MATERIALS LIST

Riferimenti

  1. Woodcock, C. L., Ghosh, R. P. Chromatin higher-order structure and dynamics. Cold Spring Harbor Perspectives in Biology. 2 (5), 000596 (2010).
  2. Kim, K., Eom, J., Jung, I. Characterization of structural variations in the context of 3D chromatin structure. Molecules and Cells. 42 (7), 512-522 (2019).
  3. Kornberg, R. D. Chromatin structure: a repeating unit of histones and DNA. Science. 184 (4139), 868-871 (1974).
  4. McGhee, J. D., Felsenfeld, G. Nucleosome structure. Annual Review of Biochemistry. 49, 1115-1156 (1980).
  5. Takata, H., et al. Chromatin compaction protects genomic DNA from radiation damage. PLOS One. 8 (10), 75622 (2013).
  6. Ehrenhofer-Murray, A. E. Chromatin dynamics at DNA replication, transcription and repair. European Journal of Biochemistry. 271 (12), 2335-2349 (2004).
  7. Peterson, C. L., Almouzni, G. Nucleosome dynamics as modular systems that integrate DNA damage and repair. Cold Spring Harbor Perspectives in Biology. 5 (9), 012658 (2013).
  8. Torigoe, S. E., Urwin, D. L., Ishii, H., Smith, D. E., Kadonaga, J. T. Identification of a rapidly formed nonnucleosomal histone-DNA intermediate that is converted into chromatin by ACF. Molecules and Cells. 43 (4), 638-648 (2011).
  9. Gurard-Levin, Z. A., Quivy, J. P., Almouzni, G. Histone chaperones: assisting histone traffic and nucleosome dynamics. Annual Review of Biochemistry. 83, 487-517 (2014).
  10. Burgess, R. J., Zhang, Z. Histone chaperones in nucleosome assembly and human disease. Nature Structural & Molecular Biology. 20 (1), 14-22 (2013).
  11. Das, C., Tyler, J. K., Churchill, M. E. The histone shuffle: histone chaperones in an energetic dance. Trends in Biochemical Sciences. 35 (9), 476-489 (2010).
  12. Hammond, C. M., Stromme, C. B., Huang, H., Patel, D. J., Groth, A. Histone chaperone networks shaping chromatin function. Nature Reviews Molecular Cell Biology. 18 (3), 141-158 (2017).
  13. De Koning, L., Corpet, A., Haber, J. E., Almouzni, G. Histone chaperones: an escort network regulating histone traffic. Nature Structural & Molecular Biology. 14 (11), 997-1007 (2007).
  14. Zou, J. X., Revenko, A. S., Li, L. B., Gemo, A. T., Chen, H. W. ANCCA, an estrogen-regulated AAA+ ATPase coactivator for ERalpha, is required for co-regulator occupancy and chromatin modification. Proceedings of the National Academy of Sciences of the United States of America. 104 (46), 18067-18072 (2007).
  15. Lombardi, L. M., Davis, M. D., Rine, J. Maintenance of nucleosomal balance in cis by conserved AAA-ATPase Yta7. Genetica. 199 (1), 105-116 (2015).
  16. Gal, C., et al. Abo1, a conserved bromodomain AAA-ATPase, maintains global nucleosome occupancy and organisation. EMBO Reports. 17 (1), 79-93 (2016).
  17. Cho, C., et al. Structural basis of nucleosome assembly by the Abo1 AAA+ ATPase histone chaperone. Nature Communications. 10 (1), 5764 (2019).
  18. Morozumi, Y., et al. Atad2 is a generalist facilitator of chromatin dynamics in embryonic stem cells. Journal of Molecular Cell Biology. 8 (4), 349-362 (2016).
  19. Zhang, M., Zhang, C., Du, W., Yang, X., Chen, Z. ATAD2 is overexpressed in gastric cancer and serves as an independent poor prognostic biomarker. Clinical and Translational Oncology. 18 (8), 776-781 (2016).
  20. Kang, Y., Cho, C., Lee, K. S., Song, J. J., Lee, J. Y. Single-molecule imaging reveals the mechanism underlying histone loading of schizosaccharomyces pombe AAA+ ATPase Abo1. Molecules and Cells. 44 (2), 79-87 (2021).
  21. Fried, M. G. Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. Electrophoresis. 10 (5-6), 366-376 (1989).
  22. Kessler, S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. Journal of Immunology. 115 (6), 1617-1624 (1975).
  23. Lu, H. P. Single-molecule study of protein-protein and protein-DNA interaction dynamics. Methods in Molecular Biology. 305, 385-414 (2005).
  24. Fazio, T., Visnapuu, M. L., Wind, S., Greene, E. C. DNA curtains and nanoscale curtain rods: high-throughput tools for single molecule imaging. Langmuir. 24 (18), 10524-10531 (2008).
  25. Kang, Y., et al. High-throughput single-molecule imaging system using nanofabricated trenches and fluorescent DNA-binding proteins. Biotechnology and Bioengineering. 117 (6), 1640-1648 (2020).
  26. Cheon, N. Y., Kim, H. S., Yeo, J. E., Scharer, O. D., Lee, J. Y. Single-molecule visualization reveals the damage search mechanism for the human NER protein XPC-RAD23B. Nucleic Acids Research. 47 (16), 8337-8347 (2019).
  27. Lee, J. Y., Finkelstein, I. J., Arciszewska, L. K., Sherratt, D. J., Greene, E. C. Single-molecule imaging of FtsK translocation reveals mechanistic features of protein-protein collisions on DNA. Molecules and Cells. 54 (5), 832-843 (2014).
  28. Kang, H. J., et al. TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution. Nucleic Acids Research. 49 (1), 269-284 (2021).
  29. Zhou, H., et al. Mechanism of DNA-induced phase separation for transcriptional repressor VRN1. Angewandte Chemie International Edition. 58 (15), 4858-4862 (2019).
  30. Visnapuu, M. L., Greene, E. C. Single-molecule imaging of DNA curtains reveals intrinsic energy landscapes for nucleosome deposition. Nature Structural & Molecular Biology. 16 (10), 1056-1062 (2009).
  31. Stigler, J., Camdere, G. O., Koshland, D. E., Greene, E. C. Single-molecule imaging reveals a collapsed conformational state for DNA-bound cohesin. Cell Reports. 15 (5), 988-998 (2016).
  32. Thornton, J. A. Sputter Coating- Its Principles and Potential. SAE Transactions. 82, 1787-1805 (1973).
  33. Grigorescu, A. E., Hagen, C. W. Resists for sub-20-nm electron beam lithography with a focus on HSQ: state of the art. Nanotechnology. 20 (29), 292001 (2009).
  34. Meir, A., Kong, M., Xue, C., Greene, E. C. DNA curtains shed light on complex molecular systems during homologous recombination. Journal of Visualized Experiments. (160), e61320 (2020).
  35. Cold Spring Harbor Protocols. Gloxy. Vol. 2. Cold Spring Harbor Protocols. , (2007).
  36. Gracey, L. E., et al. An in vitro-identified high-affinity nucleosome-positioning signal is capable of transiently positioning a nucleosome in vivo. Epigenetics Chromatin. 3 (1), 13 (2010).
  37. Subtil-Rodriguez, A., Reyes, J. C. BRG1 helps RNA polymerase II to overcome a nucleosomal barrier during elongation, in vivo. EMBO Reports. 11 (10), 751-757 (2010).
  38. Lancrey, A., et al. Nucleosome positioning on large tandem DNA repeats of the ‘601’ sequence engineered in Saccharomyces cerevisiae. bioRxiv. , (2021).
  39. Perales, R., Zhang, L., Bentley, D. Histone occupancy in vivo at the 601 nucleosome binding element is determined by transcriptional history. Molecular and Cellular Biology. 31 (16), 3485-3496 (2011).
  40. Lowary, P. T., Widom, J. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. Journal of Molecular Biology. 276 (1), 19-42 (1998).
  41. Rossmann, K. Point spread-function, line spread-function, and modulation transfer function. Tools for the study of imaging systems. Radiology. 93 (2), 257-272 (1969).
  42. Blainey, P. C., et al. Nonspecifically bound proteins spin while diffusing along DNA. Nature Structural & Molecular Biology. 16 (12), 1224-1229 (2009).
  43. Collins, B. E., Ye, L. F., Duzdevich, D., Greene, E. C. DNA curtains: novel tools for imaging protein-nucleic acid interactions at the single-molecule level. Methods in Cell Biology. 123, 217-234 (2014).
  44. Shi, X., Lim, J., Ha, T. Acidification of the oxygen scavenging system in single-molecule fluorescence studies: in situ sensing with a ratiometric dual-emission probe. Analytical Chemistry. 82 (14), 6132-6138 (2010).
  45. Rasnik, I., McKinney, S. A., Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. Nature Methods. 3 (11), 891-893 (2006).
  46. Aitken, C. E., Marshall, R. A., Puglisi, J. D. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophysical Journal. 94 (5), 1826-1835 (2008).
  47. Teif, V. B., Rippe, K. Nucleosome mediated crosstalk between transcription factors at eukaryotic enhancers. Physical Biology. 8 (4), 044001 (2011).

Tags

Keywords: DNA CurtainSingle-molecule ImagingChromatin DynamicsHistone ChaperonesTIRF MicroscopyBiomolecule InteractionsLambda DNAStreptavidinBiotinylated LipidYOYO-1 DyeMicrofluidic System
check_url/it/63501?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citazione di questo articolo
Kang, Y., Bae, S., An, S., Lee, J. Y. Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique. J. Vis. Exp. (181), e63501, doi:10.3791/63501 (2022).
Scarica citazione
Ristampe e autorizzazioni

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image