Electrostatic interactions in nucleosome and higher-order structures are regulated by protonation state of histone ionizable residue

Reviewer #1 (Public review):

Summary:

This is a valuable study probing the impact of pH and cancer mutations on nucleosome interactions and higher-order chromatin structures.

Strengths:

The study is comprehensive, covering all the titratable residues of nucleosomes and all known cancer mutations. The analysis was rigorously carried out within the feasibility of current computational capabilities. The methods used in this study are also solid. The results of this study can enhance our understanding of higher-order chromatin organizations and their modulation by various genetic and epigenetic changes.

Weaknesses:

The interpretation and illustration of the data need improvement, such as the change of protonation states of titratable residues on the nucleosome-protein interactions and higher-order chromatin structures.

Reviewer #2 (Public review):

Summary:

The paper by Zhang et al. has two parts.

The first one presents a comprehensive study of the nucleosome pKs, including their shifts from reference values in solution. They also explore changes in the protonation states of the histone residue in response to the formation of various nucleosome complexes, including higher-order nucleosome structures. The overall conclusion is that pH-induced changes in histone residue protonation states modulate nucleosome surface electrostatic potentials, and influence nucleosome-partner protein interactions. Proton uptake or release often accompanied by nucleosome-partner protein interactions affects their binding processes.

In the second part, the authors study the effect of 1266 recurrent histone cancer mutations on the nucleosome surface electrostatics: they show a significant subset of these has a major effect on the nucleosome-partner interactions, with the potential to regulate nucleosome self-association, thereby affecting higher-order chromatin structures.

Strengths:

The main strengths of this work are its technical rigor, comprehensive nature, and novelty of several of its aspects. For example, I am not aware of another work that analyzed pK shifts in the nucleosome in such level of detail, and on for so many different structures. The same for pK shifts upon nucleosome-partner binding. The analysis of pK shifts in nucleosome-nucleosome binding is likely completely new. The authors use an established methodology, check it against experiment at least in some instances, and, very importantly, base their conclusions on many different structures. The specific pK-related numbers they report are believable.

Regarding the second part of the work: the specific connection made between a subset of cancer-associated mutations and the major electrostatic changes in the nucleosome is novel and should be of interest to a broad community. The authors conclude that cancer mutations can also regulate nucleosome self-association, modulating the organization and dynamics of higher-order chromatin structures.

The detailed and comprehensive analysis of the cancer-associated mutations, including their partitioning into multiple relevant categories, is of value in its own right.

Weaknesses:

The main weakness of the first (pK-related) part of this work is the lack of relevance to specific conditions in most living cells of higher eukaryotes. The problem is that the nucleosome resides in the nucleus, where the pH is very tightly controlled, and for good reasons. See e.g. Casey, J., Grinstein, S., and Orlowski, J. ``Sensors and regulators of intracellular pH." Nature Rev. Mol. Cell. Biology. (2009). Parker, M. D., and Boron, W. F. ``The divergence, actions, roles, and relatives of sodium-coupled bicarbonate transporters.", Physiol. Rev. (2013). While intracellular pH does deviate from about 7.2, the naturally occurring deviations are only of the order of 0.3 pH units. In that respect, what the authors call "physiological" range of 6.5 to 7.5 is still too broad, let alone the "slightly basic (pH 5 to 6.5) or ``slightly acidic" (pH 7.5 to 9) conditions, as defined by the authors. It is hard to imagine a situation where intra-nuclear pH changes from e.g. "slightly acidic" to neutral in a live cell nucleus.

This said, there is nothing wrong with studying the response of the nucleosome structures to these large variations of pH, which can be reproduced in-vitro. It is the relevance of the findings to in-vivo conditions that are highly questionable.

The second part of the work - the effect of cancer mutations - is free from this major defect. In the opinion of this reviewer, it can (and should) stand on its feet, as a separate work.

However, the lack of specific, testable (preferably quantitative) biologically relevant predictions is a weakness of both parts. For example, in "Discussion" the authors state that "Histone ionizable residues are highly sensitive to cellular pH fluctuations, leading to changes in their protonation states and consequent alterations in nucleosome surface electrostatic potentials and interactions." This statement is certainly true, based on what is already known about the effect of pH on protein-DNA (or protein-protein) association, from previous works. But what are the specific predictions here?