Histone variants shape the chromatin states in Arabidopsis

Reviewer #1 (Public Review):

Jamge et al. sought to identify the relationships between histone variants and histone modifications in Arabidopsis by systematic genomic profiling of 13 histone variants and 12 histone modifications to define a set of "chromatin states". They find that H2A variants are key factors defining the major chromatin types (euchromatin, facultative heterochromatin, and constitutive heterochromatin) and that loss of the DDM1 chromatin remodeler leads to loss of typical constitutive heterochromatin and replacement of this state with features common to genes in euchromatin and facultative heterochromatin. This study deepens our understanding of how histone variants shape the Arabidopsis epigenome and provides a wealth of data for other researchers to explore.

Strengths:
1. The manuscript provides convincing evidence supporting the claims that: A) Arabidopsis nucleosomes are homotypic for H2A variants and heterotypic for H3 variants, B) that H3 variants are not associated with specific H2A variants, and C) H2A variants are strongly associated with specific histone post-translational modifications (PTMs) while H3 variants show no such strong associations with specific PTMs. These are important findings that contrast with previous observations in animal systems and suggest differences in plant and animal chromatin dynamics.

2. The authors also performed comprehensive epigenomic profiling of all H2A, H2B, and H3 variants and 12 histone PTMs to produce a Hidden Markov Model-based chromatin state map. These studies revealed that histone H2A variants are as important as histone PTMs in defining the various chromatin states, which is unexpected and of high significance.

3. The authors show that in ddm1 mutants, normally heterochromatic transposable element (TE) genes lose H2A.W and gain H2A.Z, along with the facultative heterochromatin and euchromatin signatures associated with H2A.Z at silent and expressed genes, respectively.

Weaknesses:
1. Following up on the finding that H2A.Z replaces H2A.W at TE genes in ddm1 mutants, the authors provide in vitro evidence that DDM1 binds to H2A.Z-H2B dimers. These results are taken together to conclude that DDM1 normally removes H2A.Z-H2B dimers from nucleosomes at TE genes and replaces them with H2A.W-H2B dimers. However, the evidence for this model is circumstantial and such a model raises a variety of other questions that are not addressed by the authors. For example: if DDM1 does remove H2A.Z from TE genes, how does H2A.Z normally come to occupy these sites, given that they are highly DNA methylated and that H2A.Z is known to anticorrelate with DNA methylation in plants and animals? Given that H2A.Z does not accumulate in TEs in h2a.w mutants, how would H2A.X and H2A instead become enriched at these sites if DDM1 cannot bind these forms of H2A? Given that there are no apparent regions with common sequence between H2A.Z and H2A.W variants that are not also shared with other H2A classes, how would DDM1 selectively bind to H2A.W-H2B and H2A.Z-H2B dimers to the exclusion of H2A(.X)-H2B dimers?

Reviewer #2 (Public Review):

Jamge et al. set out to delineate the relationship between histone variants, histone modifications and chromatin states in Arabidopsis seedlings and leaves. A strength of the study is its use of multiple types of data: the authors present mass-spec, immunoblotting and ChIP-seq from histone variants and histone modifications. They confirm the association between certain marks and variants, in particular for H2A, and nicely describe the loss of constitutive heterochromatin in the ddm1 mutant.

The support for some of the conclusions is weak. The title of the discussion, "histone variants drive the overall organization of chromatin states" implies a causation which wasn't investigated, and overstates the finding that some broad chromatin states can be further subdivided when one considers histone variants (adding variables to the model).

Adding variables to a ChromHMM model naturally increases the complexity of the models that can be built, however it is difficult to objectively define which level of complexity is optimal. The differences between states may be subtle to the point that they may be considered redundant. The authors claim that the sub-states they define are biologically important, but provide little evidence to support this claim. It is not obvious whether the 26 states model is much more useful than a 9-states model. Removing variables naturally affects the definition of states that depend on these variables, but it is also hard to define the biological significance of that change. This sensitivity analysis is thus not very developed.

There are issues with the logical sequence of arguments in Fig1 and Fig3. Fig1A shows that nucleosomes often contain both H3.1 and H3.3. Therefore pulling-down H3.1-containing nucleosomes also pulls down H3.3 and whether specific H2A variants associated with H3.1 cannot be answered in this way (Fig1B). The same issue likely carries to the investigation of the association with H3 modifications if Fig1C and 1D, since the H3.1-HA pull-down also pulls down endogenous H3.1 (so presumably the rest of the nucleosome, with H3.3, as well).

In Fig3, the conclusion that it is the loss of H2A.Z -> H2A.W exchange in the ddm1 mutant that causes loss of constitutive heterochromatin is rushed. The fact that the h2a.w mutant does not recapitulate the loss of constitutive heterochromatin seen in ddm1 argues against this interpretation. It's also difficult to conclude about the importance of dynamic exchanges when the ddm1 mutation has been present for generations and the chromatin landscape has fully readapted. Further work is needed to support the authors' hypothesis.

The study also relies on a large number of custom (polyclonal) antibodies with no public validation data. Lack of specificity, a common issue with antibodies, would muddle the interpretation of the data.

Overall, this study nicely illustrates that, in Arabidopsis, histone variants (and H2A variants in particular) display specificity in modifications and genomic locations, and correlate with some chromatin sub-states. This encourages future work in epigenomics to consider histone variants with as much attention as histone modifications.

Reviewer #3 (Public Review):

How chromatin state is defined is an important question in the epigenetics field. Here, Jamge et al. proposed that the dynamics of histone variant exchange control the organization of histone modifications into chromatin states. They found 1) there is a tight association between H2A variants and histone modifications; 2) H2A variants are major factors that differentiate euchromatin, facultative heterochromatin, and constitutive heterochromatin; 3) the mutation in DDM1, a remodeler of H2A variants, causes the mis-assembly of chromatin states in TE region. The topic of this paper is of general interest and results are novel.

Overall, the paper is well-written and results are clearly presented. The biochemical analysis part is solid.

Reviewer #4 (Public Review):

This work aims at analyzing the impact of histone variants and histone modifications on chromatin states of the Arabidopsis genome. Authors claim that histone variants are as significant as histone modifications in determining chromatin states. They also study the effect of mutations in the DDM1 gene on the exchange of H2A.Z to H2A.W, which convert the silent state of transposons into a chromatin state normally found on protein coding genes.

This is an interesting and well done study on the organization of the Arabidopsis genome in different chromatin states, adding to the previous reports on this issue.