H1 restricts euchromatin-associated methylation pathways from heterochromatic encroachment

Reviewer #1 (Public Review):

In this study, the authors obtained multiple, novel and compelling datasets to better understand the relationship between histone H1 and RNA-directed DNA methylation in plants. Most of the authors' claims concerning H1 and RNA polymerase V (Pol V) are backed by convincing and independent lines of evidence. However, the authors also make some overly broad conclusions, for which additional experiments/data analyses should be explored to improve confidence in their findings. Furthermore, Pol V produces noncoding transcripts that act as scaffold RNAs, which AGO4-bound siRNAs recognize in plant chromatin to mediate RNA-directed DNA methylation. Detection of Pol V transcript products at sites of Pol V redistribution in h1 mutants would significantly enhance the impact of this manuscript. Below I have listed several strengths and weaknesses of the manuscript.

Strengths
1. The authors report high-quality NRPE1 ChIP-seq data, allowing them to directly test how and where Pol V occupancy depends on histone H1 function in Arabidopsis.
2. nrpe1 mutants generated via CRISPR/Cas9 in the h1 mutant background (nrpe1 h1.1-1 h1.2-1 triple mutants), allow the authors to study the role of Pol V in ectopic DNA methylation in H1-deficient plants.
3. Pol V recruitment via ZincFinger-DMS3 expression (a modified version of Pol V's DMS3 recruitment factor) sends Pol V to new genomic loci and thus provides the authors with an innovative dataset for understanding H1 function at these sites.

Weaknesses
1. The manuscript does not include detection or quantification of Pol V transcripts generated at ectopic sites in the h1 mutant background.
2. Statistical tests are missing throughout and are needed to support several of the authors' claims.
3. The SUVH1-3xFLAG ChIP-seq analyses in Fig. 6 require additional controls and are not fully explained in the results. The broad conclusions drawn (based on those experiments) are thus not convincing.

Previous studies have charted the relationship between H1 function and RNA-directed DNA methylation (RdDM) via analyses of Pol IV-dependent 24 nt siRNAs and factors that recruit Pol IV (Choi et al., 2021 and Papareddy et al., 2020). Harris and colleagues have extended this work and shown that histone H1 function also antagonizes Pol V occupancy in the context of constitutive heterochromatin. The authors thus provide important evidence to show that H1 limits the encroachment of both polymerases Pol IV and Pol V into plant heterochromatin.

Reviewer #2 (Public Review):

Summary:
The main conclusion of the manuscript is that the presence of linker Histone H1 protects Arabidopsis pericentromeric heterochromatic regions and longer transposable elements via chromatin compaction from encroachment by other repressive pathways. The manuscript focuses on the RNA-dependent DNA-methylation (RdDM) pathway but indirectly finds that other pathways must also be ectopically enriched.

Strengths:
The authors present diverse sets of genomic data comparing Arabidopsis wild-type and h1 mutant background allowing an analysis of differential recruitment of RdDM component NPRE1, which is related to changes in DNA methylation and H1 coverage. As an addendum, the manuscript also contains recruitment data for SUVH1 in wild-type and h1 mutant backgrounds.

Furthermore, the authors make use of a line that recruits NRPE1 ectopically to show that H1 occupancy is not altered because of this recruitment. These are negative data, but well supported.

Weaknesses:
The manuscript mostly confirms earlier observations but shows very limited novelty. It has already been reported that different classes of TEs show a differential response with respect to DNA methylation in absence of H1. Furthermore, the fact that loss of H1 affect global chromatin accessibility was recently published by Teano et al. in Cell reports (Volume 42, Issue 8, 29 August 2023). The authors have neither cited this report (that had been available since 2021 in BioRxiv), nor set their work in context to this study. The study by Teano showed that for some TEs, loss of H1 is related to a switch from DNA-methylation dependent repressive pathways to Polycomb Group-dependent pathways. The current manuscript could have looked at overlapping classes and integrated information from both studies, which would be particularly interesting for the examples illustrated in Figure 5b, showing examples of TEs that lose NRPE1 targeting and methylation in all contexts in H1 deletion mutants.

The proposed mechanism is that RdDM along with many other chromatin factors re-distribute to heterochromatic regions in h1 mutants because these regions are more accessible. There is a general problem with measuring the "difference in chromatin compaction" with methods that mostly resolve highly accessible chromatin in contrast to any other chromatin, such as ATAC-seq or DNAse-seq (employed in this manuscript). The changes in the regions of interest are so subtle that they are not easily detected at the level of individual genes, although they become usually more obvious in metagene plots. The general question is if this inadequate method is sufficient to draw strong conclusions on chromatin compaction, but to be fair, the current manuscript is not alone in using this method without pointing out certain caveats.

As a consequence of redistribution to heterochromatic sites, the authors postulate that there are also sites that lose RdDM coverage in h1, but these sites are not really evidenced in the report.
Unfortunately, another weakness is that it is not possible to make easy use of the analysis from the available material as the current manuscript does not contain supplemental data indicating which TEs were and DMRs were considered in classes such as "long", "short", "heterochromatic", "euchromatic", "Class A", "Class B", "CMT2 dependent hypo-CHH", "DRM2 dependent CHH", "dynamic RdDM" etc. Since the bioinformatics pipelines are poorly documented (absence of dedicated script archive), the analysis cannot be easily recapitulated.