Nano-Surveillance: Tracking Individual Molecules in a Sea of Chromatin

Abstract

Chromatin is the epigenomic platform for diverse nuclear processes such as DNA repair, replication, transcription, telomere, and centromere function. In cancer cells, mutations in key processes result in DNA amplification, chromosome translocations, and chromothripsis, severely distorting the natural chromatin state. In normal and diseased states, dozens of chromatin effectors alter the physical integrity and dynamics of chromatin at the level of both single nucleosomes and arrays of nucleosomes folded into 3-dimensional shapes. Integrating these length scales, from the 10 nm sized nucleosome to mitotic chromosomes, whilst jostling within the crowded environment of the cell, cannot yet be achieved by a single technology. In this review, we discuss tools that have proven powerful in the investigation of nucleosome and chromatin fiber dynamics. We also provide a deeper focus into atomic force microscopy (AFM) applications that can bridge diverse length and time scales. Using time course AFM, we observe that chromatin condensation by H1.5 is dynamic, whereas using nano-indentation force spectroscopy we observe that both histone variants and nucleosome binding partners alter material properties of individual nucleosomes. Finally, we demonstrate how high-speed AFM can visualize plasmid DNA dynamics, intermittent nucleosome-nucleosome contacts, and changes in nucleosome phasing along a contiguous chromatin fiber. Altogether, the development of innovative technologies holds the promise of revealing the secret lives of nucleosomes, potentially bridging the gaps in our understanding of how chromatin works within living cells and tissues.

Single Molecule Analysis of CENP-A Chromatin by High-Speed Atomic Force Microscopy

“…This novel single-molecule technique is powerful, and similar to live cell imaging (Ashwin et al, 2019; Specht et al, 2017) except that nucleosomes can be directly visualized without the need of a fluor-tag. Recently, we showed by HS-AFM that, at the qualitative level, H3 nucleosomes are mobile and make intermittent contact with other H3 nucleosomes (Melters and Dalal, 2021). We therefore set out to quantify the positional dynamics of individual nucleosomes in the context of chromatin by determining the diffusion constant (D) from plots of the mean square displacement (MSD) as a function of time (Figure 1A).…”

“…A prime candidate is the linker histone H1, which is known to efficiently compact H3 chromatin and restrict transcription (Clausell et al, 2009; Prendergast and Reinberg, 2021; Saha and Dalal, 2021). Previously we published HS-AFM movies of H3 chromatin and H3 chromatin with linker histone variant H1.5 (Melters and Dalal, 2021). Similar as described above, we tracked individual H3 nucleosomes in real-time without (Figure 2A, Supplemental movie 3) and with H1.5 (Figure 2B; Supplemental movie 4) and calculated the MSDs and diffusion constants as described above.…”

Binding Dynamics of Disordered Linker Histone H1 with a Nucleosomal Particle

“…A comprehensive review of powerful experimental tools for probing linker histones in chromatin environment can be found in Melters et al . [71], where the authors successfully visualized the dynamics of nucleosome arrays with H1.5 in real time by high-speed AFM.…”

“…We note with excitement that the advent of high-speed AFM [63][64][65][66][67] provides precisely the kinetic handle needed to complement H1 fast dynamics observed in FRAP studies [5,68] and H1 off/on dyad classic steady-state biochemistry experiments [69,70] glean insights into linker histone biology as it relates to chromatin spacing and folding. A comprehensive review of powerful experimental tools for probing linker histones in chromatin environment can be found in Melters et al [71], where the authors successfully visualized the dynamics of nucleosome arrays with H1.5 in real time by high-speed AFM.…”

A glitch in the snitch: the role of linker histone H1 in shaping the epigenome in normal and diseased cells

“…However, the authors also demonstrate that both fibre conformations can exist in a dynamic equilibrium which can be modulated by minor changes in the ionic environment [ 50 ]. It is noteworthy that H1 exists as variants in most organisms which may result in differing preferences of nucleosomal localization and elicit varying degrees and dynamism of chromatin compaction [ 62 ].…”