Interplay between charge distribution and DNA in shaping HP1 paralog phase separation and localization

Reviewer #1 (Public Review):

Summary:

HP1 plays a pivotal role in orchestrating chromatin packaging through the creation of biomolecular condensates. The existence of distinct homologs offers an intriguing avenue for investigating the interplay between genetic sequence and condensate formation. In this study, the authors conducted extensive coarse-grained simulations to delve into the phase separation behavior of HP1 paralogs. Additionally, the researchers delved into the captivating possibility of various HP1 paralogs co-localizing within assemblies composed of multiple components. Importantly, the study also delved into the critical role of DNA in finely tuning this complex process.

Strengths:

I applaud the authors for their methodical approach in conducting simulations aimed at dissecting the contributions of hinges, CTE, NTE, and folded regions. The comprehensive insights unveiled in Figure 3 compellingly substantiate the significance of these protein components in facilitating the process of phase separation.

This systematic exploration has yielded several innovative revelations. Notably, the authors uncovered a nuanced interplay between the folded and disordered domains. Although disordered regions have traditionally been linked to driving phase separation through their capacity for forming multivalent interactions, the authors have demonstrated that the contribution of the CD cannot be overlooked, as it significantly impacts the saturation concentration.

The outcomes of this study serve to elucidate the intricate mechanisms and regulatory aspects governing HP1 LLPS.

Weaknesses:

The authors do not provide an assessment of the quantitative precision of their model. To illustrate, HP1a is anticipated to undergo phase separation primarily under low salt concentrations. Does the model effectively capture this sensitivity to salt conditions? Regrettably, the specific salt conditions employed in the simulations are not explicitly stated. While I anticipate that numerous findings in the manuscript remain valid, it could be beneficial to acknowledge potential limitations tied to the simulations. For instance, might the absence of quantitative precision impact certain predictions, such as the CD's influence on phase separation?

Reviewer #2 (Public Review):

In this paper, Phan et al. investigate the properties of human HP1 paralogs, their interactions and abilities to undergo liquid-liquid phase separation. For this, they use a coarse-grained computational approach (validated with additional all-atom simulations) which allows to explore complex mixtures. Matching (wet-lab) experimental results, HP1 beta (HP1b) exhibits different properties from HP1 alpha and gamma (HP1a,g), in that it does not phase separate. Using domain switch experiments, the authors determine that the more negatively charged hinge in HP1b, compared to HP1a and HP1g, is mainly responsible for this effect. Exploring heterotypic complexes, mixtures between HP1 subtypes and DNA, the authors further show that HP1a can serve as a scaffold for HP1b to enter into condensed phases and that DNA can further stabilize phase separated compartments. Most interestingly, they show that a multicomponent mixture containing DNA, and HP1a and HP1b generates spatial separation between the HP1 paralogs: due to increased negative charge of DNA within the condensates, HP1b is pushed out and accumulates at the phase boundary. This represents an example how complex assemblies could form in the cell.
Overall, this is purely computational work, which however builds on extensive experimental results (including from the authors). The methods showcase how coarse-grained models can be employed to generate and test hypotheses how proteins can condense. Applied to HP1 proteins, the results from this tour-de-force study are consistent and convincing, within the experimental constraints. Moreover, they generate further models to test experimentally, in particular in light of multicomponent mixtures.

There are, of course, some limitations to these models.

First, the CG models employed probably will not be able to pick up more complex structure-driven interactions (i.e. specific binding of a peptide in a protein cleft, including defined H-bonds, or induced structural elements). Some of those interactions (i.e. beyond charge-charge or hydrophobics) may also play a role in HP1, and might be ignored here. There is also the question of specificity, i.e. how can diverse phases coexist in cells, when the only parameters are charge and hydrophobicity? Does the arrangement of charges in the NTD, hinges and CTDs matter or are only the average properties important?

Second, the authors fix CSD-CSD dimers, whereas these interactions are expected to be quite dynamic. In the particular example of HP1 proteins, having dimerization equilibria may change the behavior of complex mixtures significantly, e.g. in view of the proposed accumulation of HP1b at a phase boundary. This point would warrant more discussion in the paper. Moreover, the biological plausibility of such a behavior would be interesting. Is there any experimental data supporting such assemblies?