Linker histone H1.8 inhibits chromatin binding of condensins and DNA topoisomerase II to tune chromosome length and individualization
- Laboratory of Chromosome and Cell Biology, The Rockefeller University, United States
- Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, United States
- Division of Structural Biology, The Institute of Cancer Research, United Kingdom
- Fondazione Human Technopole, Structural Biology Research Centre, 20157, Italy
- Howard Hughes Medical Institute, United States
Figures
Figure 1 with 1 supplement
Linker histone H1.8 suppresses enrichment of condensins and TOP2A on mitotic chromatin.
(A) Experimental scheme to generate replicated chromosomes in Xenopus egg extracts. (B) Western blots of total extracts showing depletion of H1.8 from Xenopus egg extracts and rescue with …
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Figure 1—source data 1
Source data for all the figures in Figure 1 and its figure supplement.
- https://cdn.elifesciences.org/articles/68918/elife-68918-fig1-data1-v3.zip
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Figure 1—source data 2
Mass spectrometry data for chromatin purified from ΔIgG and ΔH1 metaphase sperm chromosomes.
- https://cdn.elifesciences.org/articles/68918/elife-68918-fig1-data2-v3.xlsx
Figure 1—figure supplement 1
H1.8 depletion does not lead to global accumulation of DNA-binding proteins.
(A) Experimental scheme to incorporate Cy3-labeled nucleotides to use normalization of immunofluorescence signals on chromosomes. (B) Quantification of Cy3-dUTP signals normalized to Hoechst 33342 …
Figure 2 with 4 supplements
Linker histone inhibits binding of condensins and TOP2A to nucleosome arrays.
(A) Experimental scheme for testing the effect of recombinant H1.8 (rH1.8) on binding of purified condensins and TOP2A to arrays of nucleosomes assembled on the Widom 601 nucleosome positioning …
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Figure 2—source data 1
Source data for all the figures in Figure 2 and its figure supplements.
- https://cdn.elifesciences.org/articles/68918/elife-68918-fig2-data1-v3.zip
Figure 2—figure supplement 1
Mass photometry of condensin complexes.
Recombinant condensin complexes were diluted to 50 nM in buffer (10 mM HEPES pH 8, 2.5 mM MgCl2, 1 mM DTT, 5 mM ATP) supplemented with the indicated sodium chloride concentration and the indicated …
Figure 2—figure supplement 2
Recombinant human condensin I and X. laevis TOP2A are functional.
(A) Representative Hoechst (DNA) and CENP-A immunofluorescence images of chromosomes in indicated metaphase egg extracts after dilution, which disperses individualized chromosomes (left). Extracts …
Figure 2—figure supplement 3
Condensin binding is inhibited by magnesium.
(A) Native PAGE gel analysis of nucleosome array beads loaded with or without H1.8 after digestion of the array with AvaI, which released monomers of nucleosome positioning sequence. A complete …
Figure 2—figure supplement 4
H1.8 inhibits condensin binding to mononucleosomes.
Alexa647-labeled 196 bp mononucleosomes with or without H1.8 were incubated with indicated concentrations of condensin I and electrophoresed on a 5% native PAGE. Alexa647-labeled DNAs are shown. …
Figure 3 with 2 supplements
Chromosome elongation by H1.8 depletion is due to enhanced condensin I loading on chromatin.
(A) Schematic of extract dilution to disperse individualized chromosomes. (B) Western blots of total egg extracts showing depletions of indicated proteins. (C) Representative images of mitotic …
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Figure 3—source data 1
Source data for all the figures in Figure 3 and its figure supplements.
- https://cdn.elifesciences.org/articles/68918/elife-68918-fig3-data1-v3.zip
Figure 3—figure supplement 1
Condensin I loading determines chromosome length.
(A) Length of mitotic chromosomes in the indicated conditions after extract dilution showing no effect of CAP-D3 (condensin II) depletion in both mock and H1.8 depletion background. Each dot …
Figure 3—figure supplement 2
TOP2A overloading is not responsible for chromosome elongation.
(A) Sperm nuclei were replicated in undepleted interphase extracts supplemented with 25 nM Cy3-dUTP. The nuclei were then cycled back by mixing with different ratios of ΔTOP2A extracts. …
Figure 4 with 2 supplements
Effects of H1 and/or condensin I and II depletion on mitotic genome folding.
(A) Hi-C maps of metaphase X. laevis chromosome 3S, binned to 250 kb, in the indicated condition. (B) Genome-wide average contact probability decay curves for the indicated conditions showing the …
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Figure 4—source data 1
Source data for all the figures in Figure 4 and its figure supplements.
- https://cdn.elifesciences.org/articles/68918/elife-68918-fig4-data1-v3.zip
Figure 4—figure supplement 1
Effects of H1, condensin I, and condensin II depletion on mitotic genome folding.
(A) Contact probability decay curves of mitotic chromosomes in mock (ΔIgG) and H1-depleted (ΔH1) extracts from two different experiments showing the replicability of the Hi-C features. The solid and …
Figure 4—figure supplement 2
H3-H4 depletion leads to even smaller loop sizes.
(A) Western blotting showing the depletion of H3-H4 in total egg extract using the H4K12ac antibody (Zierhut et al., 2014). (B) Quantification of condensin I (CAP-G) immunofluorescence levels on …
Figure 5 with 1 supplement
Substantial interchromosomal links remain in metaphase chromosomes.
(A) Schematic showing the dispersal protocol and the different stages for the two chromosome individualization measurements. (B) Representative images of metaphase chromosomes in extracts containing …
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Figure 5—source data 1
Source data for Figure 5 and its figure supplement.
- https://cdn.elifesciences.org/articles/68918/elife-68918-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
Regulation of chromosome individualization by topo II, condensins, and H1.8 in Xenopus egg extracts.
(A) Biological replicate of Figure 5C. (B) Schematic of ICRF-193 addition to check for requirement of topo II activity in individualizing chromosomes. Interphase nuclei were first formed in ∆IgG or …
Figure 6 with 1 supplement
H1.8 suppresses condensin to limit chromosome individualization.
(A) Western blots of total egg extracts showing depletion levels in extract of condensin I and condensin II using the CAP-G and CAP-D3 antibodies, respectively. * represents non-specific band. (B) …
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Figure 6—source data 1
Source data for all the figures in Figure 6 and its figure supplement.
- https://cdn.elifesciences.org/articles/68918/elife-68918-fig6-data1-v3.zip
Figure 6—figure supplement 1
Additional data for chromosome clustering.
(A) Distribution of CENP-A foci per DNA mass in the experiment shown in Figure 6A–C. Clusters of unresolved chromosomes are represented by higher numbers (>3) of CENP-A foci per DNA mass indicates …
Figure 7 with 1 supplement
H1.8 suppresses hyper-individualization through condensins and topo II.
(A) Schematic of partial TOP2A depletion to test sensitivity of chromosome individualization to TOP2A levels. (B) Quantification of chromosome-associated TOP2A upon partial TOP2A depletion. Each dot …
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Figure 7—source data 1
Source data for all the figures in Figure 7 and its figure supplement.
- https://cdn.elifesciences.org/articles/68918/elife-68918-fig7-data1-v3.zip
Figure 7—figure supplement 1
H1.8 suppresses over-individualization through condensins and topo II.
(A) Western blots of total egg extracts showing partial depletion of TOP2A. Left three lanes: dilution series of total mock-depleted extracts (∆IgG) for signal quantitation. Middle ∆IgG lane: …
Figure 8
A graphical model of how H1.8 controls mitotic chromosome length.
In the absence of H1.8, more condensins and topo II bind to more DNA loops of shorter length, resulting in longer and more individualized chromosomes (top). H1.8 limits chromatin levels of …
Tables
Table 1
Summary table of chromatin levels of condensins, topo II, and chromosome phenotypes.
| Condensin I | Condensin II | TOP2A | Chromosome length | Chromosome individualization | |
|---|---|---|---|---|---|
| ∆H1 | 2.5× | 2× | 3.5× | 1.5× | ++ |
| ∆CAP-G | 0.1× | 1× | 1× | NA | Defective |
| ∆CAP-D3 | 1× | 0.4× | 1× | 1× | Unchanged |
| ∆H1∆CAP-G | 0.2× | 2× | 3.5× | 0.5× | + |
| ∆H1∆CAP-D3 | 2.5× | 0.9× | 3.5× | 1.5× | ++ |
| ∆H1∆CAP-G ∆CAP-D3 | 0.2× | 0.9× | 3.5× | NA | Defective |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Spodoptera frugiperda) | SF9 insect cells | Gibco | 11496015 | |
| Cell line (Trichoplusia ni) | High Five insect cells | Gibco | B85502 | |
| Cell line(Pichia pastoris) | Yeast cells | Invitrogen/Thermo Fisher Scientific Ryu et al., 2010 | C18100 | GS115 |
| Biological sample (Xenopus laevis) | Xenopus | NASCO | LM00531RRID:XEP_Xla100 | Female, adult frogs |
| Biological sample (Xenopus laevis) | Xenopus | NASCO | LM00715RRID:XEP_Xla100 | Male, adult frogs |
| Antibody | Anti-H3 (rabbit polyclonal) | Abcam | RRID:AB_302613 | WB (1 µg/ml) |
| Antibody | Anti-H2B (rabbit polyclonal) | Abcam | RRID:AB_302612 | WB (1 µg/ml) |
| Antibody | Anti α-tubulin (mouse monoclonal) | Sigma-Aldrich | RRID:AB_477593 | WB (1:10000) |
| Antibody | Anti-H1.8 (rabbit polyclonal) | Jenness et al., 2018 | RU1974 | WB (1 µg/ml) |
| Antibody | Anti-TOP2A (rabbit polyclonal) | Ryu et al., 2010 | NA | WB (2 µg/ml)IF (1 µg/ml) |
| Antibody | Anti-CAP-G (rabbit polyclonal) | Zierhut et al., 2014 | RU1008 | WB (2 µg/ml)IF (2 µg/ml) |
| Antibody | Alexa 488-anti-CAP-G (rabbit polyclonal) | This study | NA | IF (4 µg/ml),refer to ‘Antibodies’ section in Methods |
| Antibody | Anti-CAP-D2 (rabbit polyclonal) | Hirano et al., 1997 | NA | WB (2 µg/ml) |
| Antibody | Anti-CAP-G2 (rabbit polyclonal) | Gift from S. Rankin | OMRF195 | WB (4 µg/ml)IF (4 µg/ml) |
| Antibody | Anti-CAP-D3 (rabbit polyclonal) | This study | RU2042 | WB (2 µg/ml),refer to ‘Antibody production’ section in Methods |
| Antibody | Anti- CENP-A (rabbit polyclonal) | Wynne and Funabiki, 2015 | NA | IF (4 µg/ml) |
| Antibody | IRDye 680 LT anti-mouse IgG(H + L) (goat polyclonal) | LI-COR Biosciences | RRID:AB_2687826 | WB (1:10,000) |
| Antibody | IRDye 680 LT anti-rabbit IgG(H + L) (goat polyclonal) | LI-COR Biosciences | RRID:AB_621841 | WB (1:10,000) |
| Antibody | IRDye 800 CW anti-mouse IgG(H + L) (goat polyclonal) | LI-COR Biosciences | RRID:AB_621842 | WB (1:10,000) |
| Antibody | IRDye 800 CW anti-rabbit IgG(H + L) (goat polyclonal) | LI-COR Biosciences | RRID:AB_2687826 | WB (1:10,000) |
| Antibody | IRDye 800 CW anti-mouse IgG(H + L) (goat polyclonal) | LI-COR Biosciences | RRID:AB_621843 | WB (1:10,000) |
| Antibody | Anti-rabbit Alexa 555 (goat polyclonal) | Thermo Fisher Scientific | RRID:AB_141784 | IF (1:1000) |
| Antibody | Anti-rabbit Alexa 555 (goat polyclonal) | Jackson Immunoresearch | RRID:AB_2338079 | IF (1:250) |
| Antibody | Anti-rabbit Alexa 488 F(ab’)2 fragment (goat polyclonal) | LifeScience Technologies | RRID:AB_142134 | IF (1:1000) |
| Chemical compound, drug | Nocodazole | Sigma-Aldrich | M1404 | 10 µg/ml |
| Chemical compound, drug | ICRF-193 | Santa Cruz Biotechnology | sc-200889 | 50/500 µM |
| Other | Dynabeads-Protein A | Thermo Fisher Scientific | 100-08D | 250 ng antibody/1 µl beads |
| Other | Dynabeads-M280 Streptavidin | Thermo Fisher Scientific | 11206D | NA |
| Software, algorithm | MATLAB | MathWorks | R2019A | NA |
| Peptide, recombinant protein | DpnII | NEB | R0543 | |
| Peptide, recombinant protein | DNA polymerase I, large (Klenow) fragment | NEB | M0210S | |
| Peptide, recombinant protein | T4 DNA ligase 1 U/µl | Invitrogen | 15224090 | |
| Peptide, recombinant protein | T4 DNA polymerase | NEB | M0203L | |
| Peptide, recombinant protein | T4 polynucleotide kinase | NEB | M0201 | |
| Peptide, recombinant protein | Biotin-14-dATP | Invitrogen | 19524016 | |
| Commercial assay or kit | TruSeq Nano DNA Sample Prep Kit | Illumina | 20015964 | |
| Peptide, recombinant protein | Klenow fragment (3′ → 5′ exo-) | NEB | M0212L |
