Background

Nucleosomes are the basic units of compaction of eukaryotic DNA into chromatin and function as regulators of gene readout and activity (Bowman and Poirier, 2015; Sadakierska-Chudy and Filip, 2015; Baldi et al., 2020). Canonical nucleosome core particles consist of two copies each of the four histones H2A, H2B, H3, and H4 assembled into a histone octamer that is tightly wrapped by 147 bp of DNA (Luger et al., 1997; Richmond and Davey, 2003). Accessibility to the genetic code for readout and processing is facilitated by (partial) unwrapping of nucleosomal DNA and can be achieved either by active processes involving, e.g., RNA polymerase or nucleosome chaperones that exert forces and torques on the nucleosomes (Sirinakis et al., 2011; Mueller-Planitz et al., 2013; Narlikar et al., 2013; Ordu et al., 2016), or spontaneously by thermal fluctuations (Li and Widom, 2004). Using single-molecule micromanipulation techniques such as optical tweezers, the energetics of force-induced nucleosome unwrapping has been probed at high resolution (Mihardja et al., 2006; Hall et al., 2009; Schlingman et al., 2014; Ngo et al., 2015; Kaczmarczyk et al., 2020). However, the unwrapping landscape in the absence of force has been more difficult to access.

Atomic force microscopy (AFM) is a powerful tool to probe nucleosome structure and interactions due to its capability to image molecular complexes at the single-molecule level, label-free, and with sub-nanometer resolution, well suited to visualize the DNA and protein components of nucleosomes (Shlyakhtenko et al., 2009; Bintu et al., 2011; Miyagi et al., 2011; Katan et al., 2015; Ordu et al., 2016). Recent improvements in hardware make fast imaging of thousands of molecules possible, and combination with automated image analysis enables highly quantitative and reproducible studies of DNA and nucleoprotein complexes (Dufrêne et al., 2017; Ando, 2018; Brouns et al., 2018; Würtz et al., 2019; Bangalore et al., 2020).

By combining large field of view AFM imaging and automated image analysis of DNA and nucleosomes, we have recently elucidated the nucleosomal wrapping landscape for passive invasion of nucleosomes with linker DNA, in contrast to the previous force-induced unwrapping assays (Konrad et al., 2021). While we have demonstrated the strength of our methodology by quantitatively capturing the conformational ensemble of wild-type and CENP-A nucleosomes – centromeric nucleosomes where histone H3 is replaced with the CENP-A variant – the methodology can be easily adapted to study a wide range of open questions such as the effect of post-translational modifications on nucleosome wrapping or the impact of DNA sequence on nucleosome positioning on the single-molecule level.

The current protocol describes all steps necessary to study DNA and nucleosomes by AFM imaging, starting with the surface deposition of the molecules and ending with the quantitative image analysis after AFM imaging. The protocol describes AFM imaging of dry samples in air. However, it can be readily adapted for AFM measurements in liquid. In liquid, the deposition protocol and imaging parameters have to be adjusted. In particular, instead of drying the surface after depositing the sample, the sample buffer solution remains on the surface for imaging. Examples of how to perform liquid AFM measurements have been published previously (Bussiek et al., 2003; Brouns et al., 2018). Subsequent analysis of the AFM images might also require adjustment of the image analysis parameters (see AFM image analysis).