In Diverse Conditions Intrinsic Chromatin Condensates Have Liquid-like Material Properties

Abstract

SUMMARYEukaryotic nuclear DNA is wrapped around histone proteins to form nucleosomes, which further assemble to package and regulate the genome. Understanding of the physical mechanisms that contribute to higher order chromatin organization is limited. Previously, we reported the intrinsic capacity of chromatin to undergo phase separation and form dynamic liquid-like condensates, which can be regulated by cellular factors. Recent work from Hansen, Hendzel, and colleagues suggested these intrinsic chromatin condensates are solid in all but a specific set of conditions. Here we show that intrinsic chromatin condensates are fluid in diverse solutions, without need for specific buffering components. Exploring experimental differences in sample preparation and imaging between these two studies, we suggest what may have led Hansen, Hendzel, and colleagues to mischaracterize the innate properties of chromatin condensates. We also describe how liquid-like in vitro behaviors can translate to the locally dynamic but globally constrained movement of chromatin in cells.

Advanced Surface Passivation for High-Sensitivity Studies of Biomolecular Condensates

“…Preparation of mPEG/BSA passivated surface mPEG passivation was performed as previously described (9). Briefly, #1.5H glass slides (Thorlabs) or #1.5H 384-well microscopy plates (Cellvis) were washed with 5% Hellmanex at 37˚C for 4 hours and then rinsed copiously with H2O.…”

“…Undesirable interactions between condensates and glass surfaces represent a significant methodological challenge in these experiments. Such interactions can alter condensate material properties and generate appreciable background (4,8,9). Yet complete inhibition of surface adhesion is also undesirable, since many experiments are impaired by condensate movement.Effective surface passivation, balancing these effects, is crucial to robust, quantitative biochemical studies of condensates.The most common passivation technique used in condensate biochemistry involves coating glass surfaces with methoxy polyethylene glycol (mPEG) and Bovine Serum Albumin (BSA) (4, 8).However, this method has substantial limitations.…”

Chromatin accessibility: methods, mechanisms, and biological insights

“…Multi-scale simulations have identified nucleosome ‘breathing’ (partial unwrapping of DNA) at physiological salt as a factor that drives multi-valent interactions between nucleosomes and causes chromatin to form heterogeneous condensates rather than ordered 30-nm fibers [ 230 ]. The extent of chromatin liquidity inside the droplets under certain buffer conditions is under some debate, but there are a variety of physiological-like conditions at which liquid-like behavior is robustly observed without the need for any special bundlers or crowding agents [ 215 , 228 , 231 , 232 ].…”

Orientation-Independent-DIC imaging reveals that a transient rise in depletion force contributes to mitotic chromosome condensation

“…To our knowledge, we are the first to demonstrate that the depletion force/macromolecular crowding converts fibrous chromatin condensates formed by cations into liquid droplets in vitro . Previous reports on chromatin liquid droplet formation have thus far only used synthetic nucleosomes such as 12-mer arrays, which have uniform properties in the length, spacing, size, and modifications (54, 56, 61). The liquid droplets we observed using native chromatin reminded us of the chromatin liquid droplets in living mitotic cells when Schneider et al injected cells with the restriction enzyme AluI to fragment chromatin (15).…”