Linker histone H1.8 inhibits chromatin binding of condensins and DNA topoisomerase II to tune chromosome length and individualization
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Evaluation Summary:
This paper will be of interest to scientists within the field of chromosome biology. The authors take advantage of the Xenopus egg cell free system and combine classical morphological analyses by immunofluorescence with chromosome conformation (Hi-C) analyses to elucidate the contribution of linker histone H1 to mitotic chromosome organization. The authors find that linker histone H1 limits the association of condensin and topoisomerase II to control chromosome length.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) are thought to drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 antagonizes condensins and topo II to shape mitotic chromosome organization. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer through shortening the average loop size and reducing the DNA amount in each layer of mitotic loops. Furthermore, excess loading of condensins and topo II to chromosomes by H1.8 depletion causes hyper-chromosome individualization and dispersion. We propose that condensins and topo II are essential for chromosome individualization, but their functions are tuned by the linker histone to keep chromosomes together until anaphase.
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Author Response:
Evaluation Summary:
This paper will be of interest to scientists within the field of chromosome biology. The authors take advantage of the Xenopus egg cell free system and combine classical morphological analyses by immunofluorescence with chromosome conformation (Hi-C) analyses to elucidate the contribution of linker histone H1 to mitotic chromosome organization. The authors find that linker histone H1 limits the association of condensin and topoisomerase II to control chromosome length.
We would like to note that our study also demonstrates the importance of H1.8 in preventing chromosome hyper-individualization prior to anaphase chromosome segregation by limiting the chromosome association of condensin and topo II. In addition, while it has been widely accepted that the linker histone controls local chromatin …
Author Response:
Evaluation Summary:
This paper will be of interest to scientists within the field of chromosome biology. The authors take advantage of the Xenopus egg cell free system and combine classical morphological analyses by immunofluorescence with chromosome conformation (Hi-C) analyses to elucidate the contribution of linker histone H1 to mitotic chromosome organization. The authors find that linker histone H1 limits the association of condensin and topoisomerase II to control chromosome length.
We would like to note that our study also demonstrates the importance of H1.8 in preventing chromosome hyper-individualization prior to anaphase chromosome segregation by limiting the chromosome association of condensin and topo II. In addition, while it has been widely accepted that the linker histone controls local chromatin compaction by facilitating nucleosome-nucleosome interaction, we demonstrate that H1.8 can control a larger scale chromosome organization through regulating condensins. We believe that these points are conceptually novel and important.
Reviewer #1:
In this manuscript, Choppakatla et al. reconstitute chromatin assembly on sperm DNA in Xenopus meiosis II extracts to test the role of linker Histone H1. They find that depletion of embryonic isoform histone H1.8 increases the chromosomal levels of Topoisomerase 2A, as well as condensin I and II. Using in vitro-assembled nucleosome arrays, together with purified condensin and linker histone H1, they provide evidence that linker histone H1.8 competes with condensin and Top2A binding. They show that histone H1 depletion extends chromosome length, in a manner dependent on condensin I. Hi-C analysis suggested shorter chromatin loops in histone H1.8-depleted extracts, dependent on condensin I. This led the authors to conclude that histone H1.8 limits the association of condensin I with chromosomes to reduce the number, and thereby, increase the length of loops, shortening the chromosomes. The last part of the paper (Figures 5 and 6) explore the interplay between histone H1,8, condensin and Top2A in chromosome individualisation, arguing for a role of H1.8 in preventing chromosome dispersion, hypothesised to facilitate chromosome capture at metaphase.
Strengths:
• Draws together two important questions (role of linker histone H1, how chromosome size is controlled) into a potentially important mechanism.
• Experiments are carefully conducted and controlled.
• The paper is well written and presented.
Weaknesses:
• The in vivo significance is unclear. Although the in vitro studies are extremely informative, a more thorough discussion of the biological importance of the mechanisms proposed would be useful, particularly with relevance to the cell cycle.
• The latter part of the paper exploring chromosome individualization is partly contradictory and difficult to contextualise.
We would like to thank the reviewer for recognition of the importance of our work. Regarding the criticism against the idea that H1 limits chromosome individualization in metaphase, please see our response to Essential Revision point #7. Regarding the in vivo significance, it has been shown previously that elongated chromosomes in ∆H1 extracts result in chromosome segregation defects in anaphase (Maresca, Freedman, and Heald 2005).
Reviewer #2:
This is an interesting study performed using frog cycling extracts. The authors show that depletion of an embryonic histone, the H1.8 linker histone, leads to an increase in the binding of two important effectors of chromosome shape, topo II and Condensins. The increased loading of these two effectors leads to longer chromosomes that are less individualized in extracts. The major strength of this study is the elegant use of the frog extract/biochemistry to carefully dissect the contribution of chromatin-binding proteins to the overall shape and dimensions of chromosomes. The major caveat of this study is that it is based exclusively on in vitro observations, and validation experiments with purified Condensins were performed with nonstoichiometric complexes. The biological rationale justifying a role for an early embryonic histone in the reduction of chromosome length is also unclear. Cells in early embryos are typically very large and should be less dependent on size-reduction mechanisms provided by histone H1.8. One would assume that chromosome size should be maximally reduced in small somatic cells and promoted by somatic linker histone H1 subtypes. The authors did not provide an explanation for this apparent contradiction.
We would like to thank the reviewer for their constructive criticisms. The Xenopus egg extract system has a long extensive history to make a number of fundamental discoveries that shaped the cell biology field. While this is technically an in vitro system, it recapitulates most, if not all, chromosome-dependent physiological events inside the egg. In this system, DNA replication, sister chromatid cohesion, mitotic chromosome compaction, and spindle assembly are recapitulated. This manuscript presents a molecular mechanism behind chromosome length and individualization control by the linker histone H1, and thus the system perfectly serves its purpose. The Xenopus egg extract system is also ideal for studying the mitotic roles of linker histones since we can circumvent the common problem in cellular system where linker histone depletion would affect transcriptional profiles, which would make it difficult to link between the biochemical properties and mitotic chromosome morphology. Importantly, unlike somatic tissue culture cell system where multiple linker histone subtypes are expressed, H1.8 is by far the dominant linker histone in egg extracts, further simplifying our analysis for our goal. As we responded in the comment to Essential Point 1, by quantitatively monitoring the chromatin proteins that are affected by H1.8 depletion, and recapitulating the phenomenon by the reconstituted system, we were able to support our conclusion at the level that is difficult to achieve in studies based on tissue culture system. It is plausible that a mechanism applies to somatic cells where linker histones bind mitotic chromatin as well and changing linker histone stoichiometry may play a role in controlling chromosome length among cells of different sizes as well (Woodcock, Skoultchi, and Fan 2006).
The reviewer also pointed out an important possibility that we used nonstoichiometric condensin complex for our in vitro usages. In the revised version, we conducted mass photometry analysis to confirm that our complex indeed can maintain its subunit stoichiometry during our in vitro assay condition (Figure 2-figure supplement 1). Also, please note that we have shown that our complex was able to rescue condensin I depletion phenotypes in Xenopus egg extracts (Figure 2-figure supplement 2A).
Reviewer #3:
In the current manuscript, Choppakatla et al. address the contribution of the linker histone H1 to mitotic chromosome assembly in the Xenopus egg cell-free system. They show that the presence of this histone limits the binding of condensins I and II as well as topoisomerase II. Depletion of H1 from the egg extracts results in assembly of longer and thinner chromosomes, and increases dispersion of individualized chromosomes. Hi-C analyses indicate that average loop size is shortened and the DNA amount in each layer of mitotic loops is reduced in the absence of H1, a phenotype attributed to the increased presence of condensin I.
Strengths:
- Experiments are carefully designed and performed, the figures are clear and properly labeled and the manuscript is written with clarity.
- Combination of classical assays (immunodepletion+chromosome assembly followed by image analysis) with Hi-C analyses (to my knowledge, this is the first time that Hi-C is used for chromosomes assembled in Xenopus extracts) as well as in vitro reconstitution of topoII and condensin binding to nucleosomal arrays to test the effect of H1.
- The results support most of the conclusions of the manuscript, explain the previously reported effect of H1 depletion on chromosome assembly and are consistent with previous reports regarding the contribution of condensin I and condensin II to chromosome organization.
Weakness:
- The last part of the manuscript regarding "chromosome individualization" is a bit confusing, probably because it is unclear what this process entails in molecular terms. On the one hand, the authors mention the existence of entanglements between metaphase chromosomes that must be removed to allow complete individualization of chromosomes before segregation. On the other hand, chromosomes assembled in the egg extract cluster together (even in the absence of a spindle). The correlation between these two phenotypes and the actual contribution of the different factors (H1, condensins, topoII) is unclear.
We thank overall positive evaluation and constructive criticisms by the reviewer. Regarding the issue about regulation of chromosome individualization, please read our response to Essential Point #7.
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Evaluation Summary:
This paper will be of interest to scientists within the field of chromosome biology. The authors take advantage of the Xenopus egg cell free system and combine classical morphological analyses by immunofluorescence with chromosome conformation (Hi-C) analyses to elucidate the contribution of linker histone H1 to mitotic chromosome organization. The authors find that linker histone H1 limits the association of condensin and topoisomerase II to control chromosome length.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this manuscript, Choppakatla et al. reconstitute chromatin assembly on sperm DNA in Xenopus meiosis II extracts to test the role of linker Histone H1. They find that depletion of embryonic isoform histone H1.8 increases the chromosomal levels of Topoisomerase 2A, as well as condensin I and II. Using in vitro-assembled nucleosome arrays, together with purified condensin and linker histone H1, they provide evidence that linker histone H1.8 competes with condensin and Top2A binding. They show that histone H1 depletion extends chromosome length, in a manner dependent on condensin I. Hi-C analysis suggested shorter chromatin loops in histone H1.8-depleted extracts, dependent on condensin I. This led the authors to conclude that histone H1.8 limits the association of condensin I with chromosomes to reduce the …
Reviewer #1 (Public Review):
In this manuscript, Choppakatla et al. reconstitute chromatin assembly on sperm DNA in Xenopus meiosis II extracts to test the role of linker Histone H1. They find that depletion of embryonic isoform histone H1.8 increases the chromosomal levels of Topoisomerase 2A, as well as condensin I and II. Using in vitro-assembled nucleosome arrays, together with purified condensin and linker histone H1, they provide evidence that linker histone H1.8 competes with condensin and Top2A binding. They show that histone H1 depletion extends chromosome length, in a manner dependent on condensin I. Hi-C analysis suggested shorter chromatin loops in histone H1.8-depleted extracts, dependent on condensin I. This led the authors to conclude that histone H1.8 limits the association of condensin I with chromosomes to reduce the number, and thereby, increase the length of loops, shortening the chromosomes. The last part of the paper (Figures 5 and 6) explore the interplay between histone H1,8, condensin and Top2A in chromosome individualisation, arguing for a role of H1.8 in preventing chromosome dispersion, hypothesised to facilitate chromosome capture at metaphase.
Strengths:
• Draws together two important questions (role of linker histone H1, how chromosome size is controlled) into a potentially important mechanism.
• Experiments are carefully conducted and controlled.
• The paper is well written and presented.
Weaknesses:
• The in vivo significance is unclear. Although the in vitro studies are extremely informative, a more thorough discussion of the biological importance of the mechanisms proposed would be useful, particularly with relevance to the cell cycle.
• The latter part of the paper exploring chromosome individualization is partly contradictory and difficult to contextualise.
-
Reviewer #2 (Public Review):
This is an interesting study performed using frog cycling extracts. The authors show that depletion of an embryonic histone, the H1.8 linker histone, leads to an increase in the binding of two important effectors of chromosome shape, topo II and Condensins. The increased loading of these two effectors leads to longer chromosomes that are less individualized in extracts. The major strength of this study is the elegant use of the frog extract/biochemistry to carefully dissect the contribution of chromatin-binding proteins to the overall shape and dimensions of chromosomes. The major caveat of this study is that it is based exclusively on in vitro observations, and validation experiments with purified Condensins were performed with nonstoichiometric complexes. The biological rationale justifying a role for an …
Reviewer #2 (Public Review):
This is an interesting study performed using frog cycling extracts. The authors show that depletion of an embryonic histone, the H1.8 linker histone, leads to an increase in the binding of two important effectors of chromosome shape, topo II and Condensins. The increased loading of these two effectors leads to longer chromosomes that are less individualized in extracts. The major strength of this study is the elegant use of the frog extract/biochemistry to carefully dissect the contribution of chromatin-binding proteins to the overall shape and dimensions of chromosomes. The major caveat of this study is that it is based exclusively on in vitro observations, and validation experiments with purified Condensins were performed with nonstoichiometric complexes. The biological rationale justifying a role for an early embryonic histone in the reduction of chromosome length is also unclear. Cells in early embryos are typically very large and should be less dependent on size-reduction mechanisms provided by histone H1.8. One would assume that chromosome size should be maximally reduced in small somatic cells and promoted by somatic linker histone H1 subtypes. The authors did not provide an explanation for this apparent contradiction.
-
Reviewer #3 (Public Review):
In the current manuscript, Choppakatla et al. address the contribution of the linker histone H1 to mitotic chromosome assembly in the Xenopus egg cell-free system. They show that the presence of this histone limits the binding of condensins I and II as well as topoisomerase II. Depletion of H1 from the egg extracts results in assembly of longer and thinner chromosomes, and increases dispersion of individualized chromosomes. Hi-C analyses indicate that average loop size is shortened and the DNA amount in each layer of mitotic loops is reduced in the absence of H1, a phenotype attributed to the increased presence of condensin I.
Strengths:
- Experiments are carefully designed and performed, the figures are clear and properly labeled and the manuscript is written with clarity.
- Combination of classical assays …Reviewer #3 (Public Review):
In the current manuscript, Choppakatla et al. address the contribution of the linker histone H1 to mitotic chromosome assembly in the Xenopus egg cell-free system. They show that the presence of this histone limits the binding of condensins I and II as well as topoisomerase II. Depletion of H1 from the egg extracts results in assembly of longer and thinner chromosomes, and increases dispersion of individualized chromosomes. Hi-C analyses indicate that average loop size is shortened and the DNA amount in each layer of mitotic loops is reduced in the absence of H1, a phenotype attributed to the increased presence of condensin I.
Strengths:
- Experiments are carefully designed and performed, the figures are clear and properly labeled and the manuscript is written with clarity.
- Combination of classical assays (immunodepletion+chromosome assembly followed by image analysis) with Hi-C analyses (to my knowledge, this is the first time that Hi-C is used for chromosomes assembled in Xenopus extracts) as well as in vitro reconstitution of topoII and condensin binding to nucleosomal arrays to test the effect of H1.
- The results support most of the conclusions of the manuscript, explain the previously reported effect of H1 depletion on chromosome assembly and are consistent with previous reports regarding the contribution of condensin I and condensin II to chromosome organization.Weakness:
- The last part of the manuscript regarding "chromosome individualization" is a bit confusing, probably because it is unclear what this process entails in molecular terms. On the one hand, the authors mention the existence of entanglements between metaphase chromosomes that must be removed to allow complete individualization of chromosomes before segregation. On the other hand, chromosomes assembled in the egg extract cluster together (even in the absence of a spindle). The correlation between these two phenotypes and the actual contribution of the different factors (H1, condensins, topoII) is unclear.
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