Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Summary

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

Abstract

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Introduction

In eukaryotic cells, DNA is highly condensed and organized into chromosomes.¹ The first level of DNA organization within a chromosome is the assembly of nucleosomes in which 147 bp of DNA is tightly wrapped around a histone octamer core.^2,3 Nucleosome particles assemble on a long DNA molecule forming a chromatin array which is then organized until a highly compact chromosome unit is formed.⁴ The disassembly of chromatin provides the access to free DNA required by critical cellular processes such as gene transcription and genome replication, suggesting that chroma....

Protocol

1. Continuous Dilution Assembly of Mono-nucleosomes

1. Generate and purify an approximately 400 bp DNA substrate that contains an off-centered Widom 601 nucleosome positioning sequence.⁵⁵
   NOTE: To limit the unwanted formation of di-nucleosomes, each ‘arm’ flanking the positioning sequence should not exceed ~150 bp.
   1. Use plasmid pGEM3Z-601 along with the designed primers and amplify the substrate DNA using PCR. For the 423 bp substrate with 122 and 154 bp arm lengths used here, use forward (5’-CAGTGAATTGTAATACGACTC-3’) and reverse (5’-ACAGCTATGACCATGATTAC-3’) primers.
   2. Add ....

Results

Mono-nucleosomes were first prepared for AFM imaging experiments using a continuous dilution assembly method (Figure 1). The prepared nucleosomes were then checked using discontinuous SDS-PAGE (Figure 2). A mica surface was next functionalized using APS, which captures nucleosomes at the surface while maintaining a smooth background for high-resolution imaging (Figure 3). Nucleosomes were deposited on APS-mica and were subsequently .......

Discussion

The protocol described above is rather straightforward and provide highly reproducible results, although a few important issues can be emphasized. Functionalized APS-mica is a key substrate for getting reliable and reproducible results. A high stability of APS-mica is one of the important features of this substrate that allows one to prepare the imaging substrate in advance for use that can be used at least two weeks after being prepared.^59,61 However, the surfac.......

Materials

Name	Company	Catalog Number	Comments
Plasmid pGEM3Z-601	Addgene, Cambridge, MA	26656
PCR Primers	IDT, Coralville, IA	Custom Order	(FP) 5'- CAGTGAATTGTAATACGACTC-3' (RP) 5'-ACAGCTATGACCATGATTAC-3'
DreamTaq polymerase	ThermoFischer Scientific, Waltham, MA	EP0701	Catalog number for 200 units
PCR purification kit	Qiagen, Hilden, Germany	28104	Catalog number for 50 units
Tris base	Sigma-Aldrich, St. Louis, MO	10708976001	Catalog number for 250 g
EDTA	ThermoFischer Scientific, Waltham, MA	15576028	Catalog number for 500 g
(CENP-A/H4)2, recombinant human	EpiCypher, Durham, NC	16-0010	Catalog number for 50 ug
H2A/H2B, recombinant human	EpiCypher, Durham, NC	15-0311	Catalog number for 50 ug
H3 Octamer, recombinant human	EpiCypher, Durham, NC	16-0001	Catalog number for 50 ug
Slide-A-Lyzer MINI Dialysis Device Kit, 10K MWCO, 0.1 mL	ThermoFischer Scientific, Waltham, MA	69574	Catalog number for 10 devices
Sodium Chloride	Sigma-Aldrich, St. Louis, MO	S9888-500G	Catalog number for 500 mg
Amicon Ultra-0.5 mL Centrifugal Filters	Millipore-sigma, Burlington, MO	UFC501008	Catalog number for 8 devices
HCl	Sigma-Aldrich, St. Louis, MO	258148-25ML	Catalog number for 25 mL
Tricine	Sigma-Aldrich, St. Louis, MO	T0377-25G	Catalog number for 25 g
SDS	Sigma-Aldrich, St. Louis, MO	11667289001	Catalog number for 1 kg
Ammonium Persulfate (AmmPS)	Bio-Rad, Hercules, CA	1610700	Catalog number for 10 g
30% Acrylamide/Bis Solution, 37.5:1	Bio-Rad, Hercules, CA	1610158	Catalog number for 500 mL
TEMED	Bio-Rad, Hercules, CA	1610800	Catalog number for 5 mL
4x Laemmli protein sample buffer for SDS-PAGE	Bio-Rad, Hercules, CA	1610747	Catalog number for 10 mL
2-ME	Sigma-Aldrich, St. Louis, MO	M6250-10ML	Catalog number for 10 mL
ageRuler Prestained Protein Ladder	ThermoFischer Scientific, Waltham, MA	26616	Catalog number for 500 uL
Bio-Safe™ Coomassie Stain	Bio-Rad, Hercules, CA	1610786	Catalog number for 1 L
Nonwoven cleanroom wipes: TX604 TechniCloth	TexWipe, Kernersvile, NC	TX604
Muscovite Block Mica	AshevilleMica, Newport News, VA	Grade-1
Aminopropyl silatrane (APS)	Synthesized as described in 22
HEPES	Sigma-Aldrich, St. Louis, MO	H4034-25G	Catalog number for 25 g
Scotch Tape	Scotch-3M, St. Paul, MN
TESPA-V2 afm probe (for static imaging)	Bruker AFM Probes, Camarillo, CA
MSNL-10 afm probe (for standard time-lapse imaing)	Bruker AFM Probes, Camarillo, CA
Aron Alpha Industrial Krazy Glue	Toagosei America, West Jefferson, OH	AA480	Catalog number for 2 g tube
MgCl2	Sigma-Aldrich, St. Louis, MO	M8266-100G	Catalog number for 100 g
Millex-GP Filter, 0.22 µm	Sigma-Aldrich, St. Louis, MO	SLGP05010	Catalog number for 10 devices
BL-AC10DS-A2 afm probe (for HS-AFM)	Olympus, Japan
Compound FG-3020C-20	FluoroTechnology Co., Ltd., Kagiya, Kasugai, Aichi, Japan
Compound FS-1010S135-0.5	FluoroTechnology Co., Ltd., Kagiya, Kasugai, Aichi, Japan
MultiMode Atomic Force Microscope	Bruker-Nano/Veeco, Santa Barbara, CA
High-Speed Time-Lapse Atomic Force Microsocopy	Toshio Ando, Nano-Life Science Institute, Kanazawa University, Kakuma-machi, Kanazawa, Japan