Imaging analysis of six human histone H1 variants reveals universal enrichment of H1.2, H1.3, and H1.5 at the nuclear periphery and nucleolar H1X presence

Reviewer #1 (Public Review):

In this manuscript, the authors have performed extensive imaging analysis of six human histone H1 variants, their enrichment and localization, their differential dynamics during interphase and mitosis, and their association with lamina-associated domains (LADs) or nucleolus-associated domains. The manuscript is well-written with high-quality confocal and super-resolution images. Various interesting observations are made on distribution patterns of H1 variants. H1.2, H1.3, and H1.5 are shown to be universally enriched at the nuclear periphery whereas H1.4 and H1X are found to be distributed throughout the nucleus. Interestingly, H1X was the only H1 variant found to be abundant in nucleoli. Depletion of H1 variants has been shown to affect chromatin structure in a variant-specific manner, with H1.2 knock-down resulting in global chromatin decompaction. Overall, the study presents several interesting insights on H1 variants conducted in a large number of cell lines.

Major Comments:

1. Though the co-immunostaining of a nucleolar marker (NPM1) is performed with H1X, it would be interesting to explore the localization of H1 variants with respect to some of the proteins critically involved in chromatin organization such as PC4 or HP1alpha. Since the phosphorylated form of PC4 has been shown to interact with H1, which variants specifically interact with PC4 and how their dynamic changes in interphase and mitosis would be worth exploring.

2. The manuscript would be a complete study if any physiological significance with the H1 variant distribution could be shown.

Reviewer #2 (Public Review):

Summary:
The manuscript by Salinas-Pena et. al examines the distribution of a subgroup of histone H1 variants primarily with the use of high-resolution microscopy. The authors find that while some H1s have a universal distribution pattern, some display a preference for discrete regions within the nuclear landscape namely, the periphery, the center, or the nucleolus. They also show that using the various H1s within a cell did not colocalize significantly with each other, rather, they occupy discrete 'nanodomains' throughout the nucleus which is visualized as a punctate signal.

The authors present evidence relating to a long-standing question in the field regarding the spatial distribution of the different H1 variants. Since reliable, specific antibodies toward the variants were unavailable, this question was unable to elicit a definitive answer. This study uses more recently available antibodies against endogenous H1s to put together a systematic and comprehensive view of a group of H1 variant distribution inside a nucleus and ties it with previously generated genome-wide data to demonstrate localization and some functional heterogeneity.

Strengths of the study.
1. First systematic, high-resolution view of H1 variants providing a significant advance towards the long hypothesized functional differences between H1 variants.
2. The use of endogenous antibodies allows the authors to bypass the need to use tagged proteins or overexpression strategies to study H1 distribution.
3. The availability of genome-wide H1 distribution data for the variants using the endogenous H1 antibodies to strengthen the presented visual data.

Weakness of the study.
One of the major reasons for slow progress in deciphering variant-specific function has been the dearth of quality, specific, antibodies. This study is heavily dependent on the antibody function and its ability to accurately report on the distribution. However, appropriate controls to confirm the specificity were not included. Commercially available antibodies are equally susceptible to quality issues.

Impact:
This study sets the stage for an exciting avenue of H1 study where variant-specific cellular functions can be explored which has otherwise been severely understudied.

Reviewer #3 (Public Review):

Summary:
This paper uses indirect immunofluorescence, superresolution fluorescence microscopy, and X-ChIP to demonstrate radial distribution profiles of all histone H1 somatic variants with the exception of histone H1.1. The results support earlier work from chromatin immunoprecipitation experiments that revealed biases for active versus repressed states of chromatin. The previous studies provided some support for the subtle sequence variation found primarily within the C-terminus of histone H1 variants conferred preferences in the type of DNA (e.g. methylated DNA) or chromatin-bound. The current study significantly strengthens that argument. Importantly, this was shown across multiple cell lines and reveals conserved properties of localization of histone H1 variants.

Strengths:
The strength of the manuscript is the combined use of quantitative analysis of indirect immunofluorescence and X-ChIP. The results generally support the polar organization of the genome and a corresponding distribution of histone H1 variants that reflect this polar organization. AT-rich chromatin is positioned near the lamina and is found to be enriched in H1.2, H1.3, and H1.5. H1.4 and H1.X were more biased towards the GC-rich intranuclear chromatin.

There is emerging functional evidence for variant-specific properties to histone H1 subtypes. This work provides an important building block in understanding how different histone H1 variants may have specific functional consequences. The histone H1 variant that is most abundant in most cell types, H1.2, was found to decrease the area of the immunofluorescent slice that was chromatin-free when depleted, suggesting a more important role in global chromatin organization.

Weaknesses:
While histone H1 variants may show biases in their distributions, it is unlikely that these are more than biases. That is, it is unlikely that specific H1 variants are unable to bind to nucleosomes in regions where they are depleted. Fluorescence recovery after photobleaching experiments has demonstrated differences in binding affinity but the capacity to bind a range of chromatin structures, including highly acetylated chromatin, for histone H1 variants. Thus, it is critical in assessing this data to have accurate quantitative information on the relative abundance of the different histone variants amongst the cell lines tested here. The paper relies upon quantification by immunoblotting.

Another uncertainty in both the ChIP and immunofluorescence datasets is the accessibility of the epitope. This weakness is highlighted by the apparent loss of H1.2 and H1.4 in mitotic chromosomes which is revealed to be false by the detection of the phosphorylated species. The distributions relative to the surface of chromosomes in mitosis and the depletion of H1.2, H1.3, and H1.5 from the central regions of interphase nuclei reveal an unusual dissipation of the staining that is suggestive of antibody accessibility or potentially overstaining and quenching of the fluorescence in the center of highly stained structures. The overall image quality of the immunofluorescence images is poor.

Author Response

We appreciate the insightful feedback provided by the editors and reviewers who have recognized the novelty of our study. We have mapped the spatial distribution of six endogenous somatic histone H1 variants within the nuclei of several human cell lines using specific antibodies, which strongly suggest functional differences between variants. We will submit a reviewed version of the manuscript to accommodate the reviewers comments.

To answer the reviewers comments at this stage:

1. We do have investigated co-localization of H1 variants with HP1 proteins and we are eager to add some of this data in a revised version of this manuscript.
   

2. Respect to the functional significance of the results presented here, we want to stress that as a consequence of the differential distribution and abundance of H1 variants among cell types, depletion of different variants has different consequences. For example, H1.2 depletion but not others has a great impact on chromatin compaction. Besides, cell lines lacking H1.3/H1.5 expression present a basal up-regulation of some Interferon stimulated genes (ISGs) and particular repetive elements, as it was previously described upon induced depletion of H1.2/H1.4 in a breast cancer cell line or in pancreatic adenocarcinomas with lower levels of replication-dependent H1 variants (Izquierdo et al. 2017 NAR 45:11622). So, our results reinforce the existing link between H1 content and immune signature. We are eager to add this data in a revised version of this manuscript. Moreover, we also analyzed the chromatin structural changes upon combined depletion of H1.2 and H1.4. Combined H1.2/H1.4 depletion triggers a global chromatin decompaction, which supports previous observations from ATAC-Seq and Hi-C experiments in these cells (Izquierdo et al. 2017 NAR 45:11622; Serna-Pujol et al. 2022 NAR 50:3892). Although H1 content is more compromised in these cells (30% total H1 reduction) compared to single H1 KDs, the phenotype observed could not be recapitulated when other H1 KD combinations, in which total H1 content was reduced similarly, were investigated (Izquierdo et al. 2017 NAR 45:11622), supporting that the deleterious defects were due to the non-redundant role of H1.2 and H1.4 proteins. Indeed, this manuscript supports this notion, as H1.2 and H1.4 show a different genome-wide and nuclear distribution. 
   

3. We totally agree with the reviewers that the use of commercially available antibodies does not guarantee their quality and specificity. As this issue was crucial for our studies, we extensively assayed performance and specificity of the antibodies, using different approaches. The validations were shown in our previous publications where these antibodies where successfully used for ChIP-seq (Serna et al. 2022 NAR 50:3892; Salinas-Pena et al, under revision). In summary, performance of H1.0 (05-629l, Millipore), H1.2 (ab4086, abcam), H1.4 (702876; Invitrogen), H1.5 (711912, Invitrogen) and H1X (ab31972; abcam) antibodies was tested by Western-Blot, ChIP and proteomic analyses (all the results are included in Supplementary Figure 1 in Serna et al. 2022 NAR 50:3892). Concretely, we tested specificity using inducible KDs for the depletion of each of the somatic H1 variants in T47D. We also checked that the antibodies did not recognize additional H1 variants using recombinant proteins or cell lines naturally lacking some of the variants. All the experiments confirmed that antibodies were variant-specific. In addition, when the corresponding epitope was absent, the antibodies did not gain new cross-reactivity with other variants. More recently, validation of the specificicity of the H1.3 antibody (ab203948) was performed following the same experimental approaches described for the rest of antibodies (Salinas-Pena et al, under revision). 
   

4. Our immunofluorescence data, together with ChIP-seq data, do not discard binding of H1 variants to a great variety of chromatin, but show enrichment or preferential binding to certain regions or chromatin types. Our data on the interphase nuclei does not suggest at all any type of quenching or saturation. Obviously, detection with antibodies depends on epitope accessibility, just like all immunofluorescence data ever published, and we have acknowledged that post-translational modifications of H1 may occlude antibody accessibility as some phospho-H1 antibodies give distribution patterns different than total/unmodified H1 antibodies. Thus, we cannot exclude that specific modified-H1s exhibit particular distribution patterns that are not being recapitulated in our data. This represents another layer of complexity in H1 diversity and we agree that exploration of the repertoire of H1 PTMs and their functional roles are an interesting matter of study that needs to be addressed.  Still, our data is highly relevant as it demonstrates for the first time the unique distribution patterns of H1 variants among multiple cell lines and it does not use overexpression of tagged H1 variants that in our experience produces mislocalization of H1s.  
   

5. We will further explain how the relative quantification of H1 variants in different cell lines was performed if not clear enough. We agree that more sophisticated mass spectrometry-based quantification is desirable and we are collaborating to do this using internal H1 peptide controls, but this is out of the scope of this manuscript as the observed patterns of distribution of H1 variants do not depend on mild differences in variants abundance. Only the absence of H1.3 and H1.5 in some cell lines alters the distribution of other variants.
   

6. We have also studied the spatial distribution of H1 variants in non-tumorogenic cell lines and we are eager to add this in a revised version of the manuscript.