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

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 chromatin is a highly dynamic system.^5,6,7 Understanding the dynamic properties of DNA at various chromatin levels is critically important for elucidating genetic processes at the molecular level where mistakes can lead to cell death or the development of diseases such as cancer.⁸ A chromatin property of great importance is the dynamics of nucleosomes.^9,10,11,12 The high stability of these particles has allowed for the structural characterization by crystallographic techniques.² What these studies lack are the dynamic details of nucleosomes such as the mechanism of DNA unwrapping from the histone core; the dynamic pathway of which is required for transcription and replication processes.^{7,9,13,14,15,16} Furthermore, special proteins termed remodeling factors have been shown to facilitate the disassembly of nucleosomal particles¹⁷; however, the intrinsic dynamics of nucleosomes is the critical factor in this process that contributes to the entire disassembly process.^14,16,18,19

Single-molecule techniques such as single-molecule fluorescence^19,20,21, optical trapping (tweezers)^13,18,22,23 and AFM^{10,14,15,16,24,25,26} have been instrumental in understanding the dynamics of nucleosomes. Among these methods, AFM benefits from several unique and attractive features. AFM allows one to visualize and characterize individual nucleosomes as well as the longer arrays²⁷. From AFM images, important characteristics of nucleosome structure such as the length of DNA wrapped around the histone core can be measured ^10,14,26,28; a parameter that is central to the characterization of nucleosome unwrapping dynamics. Past AFM studies have revealed nucleosomes to be highly dynamic systems and that DNA can spontaneously unwrap from the histone core¹⁴. The spontaneous unwrapping of DNA from nucleosomes was directly visualized by AFM operating in the time-lapse mode when the imaging is done in aqueous solutions ^14,26,29.

The advent of the high-speed time-lapse AFM (HS-AFM) instrumentation made it possible to visualize the nucleosome unwrapping process at the millisecond time-scale ^14,15,24. Recent HS-AFM ^16,30 studies of centromere specific nucleosomes revealed several novel features of the nucleosomes compared with the canonical type. Centromere nucleosomes constitute of a centromere, a small part of the chromosome critically important for chromosome segregation³¹. Unlike canonical nucleosomes in bulk chromatin, the histone core of centromere nucleosomes contains CENP-A histone instead of histone H3^32,33. As a result of this histone substitution, DNA wrapping in centromere nucleosomes is ~120 bp instead of the ~147 bp for canonical nucleosomes; a difference that can lead to distinct morphologies of the centromere and canonical nucleosomes arrays³⁴, suggesting that centromere chromatin undergoes higher dynamics compared with the bulk one. The novel dynamics displayed by centromere nucleosomes in HS-AFM^16,30 studies exemplify the unique opportunity provided by this single-molecule technique to directly visualize the structural and dynamic properties of nucleosomes. Examples of these features will be briefly discussed and illustrated at the end of the paper. This progress was made due to the development of novel protocols for AFM imaging of nucleosomes as well as the modifications of existing methods. The goal of the protocol described here is to make these exciting advances in single-molecule AFM nucleosome studies accessible to anyone who would like to utilize these techniques in their chromatin investigations. Many of the techniques described are applicable to problems beyond the study of nucleosomes and can be used for investigations of other protein and DNA systems of interest. A few examples of such applications can be found in publications^{35,36,37,38,39,40,41,42,43,44,45,46,47,48,49} and prospects of AFM studies of various biomolecular systems are given in reviews^{29,50,51,53,54}.