Freedman BS et al. (2010), Xenopus egg extracts increase dynamics of histo...

XB-ART-42178

PLoS One 2010 Sep 07;59:. doi: 10.1371/journal.pone.0013111.

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Xenopus egg extracts increase dynamics of histone H1 on sperm chromatin.

Freedman BS , Miller KE , Heald R .

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Linker histone H1 has been studied in vivo and using reconstituted chromatin, but there have been few systematic studies of the effects of the cellular environment on its function. Due to the presence of many other chromatin factors and specific chaperones such as RanBP7/importin beta that regulate histone H1, linker histones likely function differently in vivo than in purified systems. We have directly compared H1 binding to sperm nuclei in buffer versus Xenopus egg extract cytoplasm, and monitored the effects of adding nuclear import chaperones. In buffer, RanBP7 decondenses sperm nuclei, while H1 binds tightly to the chromatin and rescues RanBP7-mediated decondensation. H1 binding is reduced in cytoplasm, and H1 exhibits rapid FRAP dynamics in cytoplasm but not in buffer. RanBP7 decreases H1 binding to chromatin in both buffer and extract but does not significantly affect H1 dynamics in either condition. Importin beta has a lesser effect than RanBP7 on sperm chromatin decondensation and H1 binding, while a combination of RanBP7/importin beta is no more effective than RanBP7 alone. In extracts supplemented with RanBP7, H1 localizes to chromosomal foci, which increase after DNA damage. Unlike somatic H1, the embryonic linker histone H1M binds equally well to chromatin in cytoplasm compared to buffer. Amino-globular and carboxyl terminal domains of H1M bind chromatin comparably to the full-length protein in buffer, but are inhibited ∼10-fold in cytoplasm. High levels of H1 or its truncations distort mitotic chromosomes and block their segregation during anaphase. RanBP7 can decondense sperm nuclei and decrease H1 binding, but the rapid dynamics of H1 on chromatin depend on other cytoplasmic factors. Cytoplasm greatly impairs the activity of individual H1 domains, and only the full-length protein can condense chromatin properly. Our findings begin to bridge the gap between purified and in vivo chromatin systems.

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Species referenced: Xenopus laevis
Genes referenced: btg3 h1-3 incenp ipo7 mtor tub

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	Figure 1. H1 and RanBP7 Have Opposing Activities in Buffer.(A) Fluorescence images of sperm nuclei in buffer with or without 4 µM RanBP7 and 1 µM H1A-GFP. Average H1:DNA fluorescence intensity is shown below for conditions supplemented with H1. Scale bar, 10 µm. (B) Average nuclear area of sperm in buffer (n>50) for conditions described in (A). (C) Averaged FRAP curves (n = 5) and corresponding timelapse images of H1A-GFP on sperm chromatin in buffer with or without RanBP7. The photobleach is plotted at time = 0. Scale bar, 2 µm. All quantification is shown ± standard error.
	Figure 2. Effects of RanBP7 on H1 in Cytoplasm.(A) Fluorescence images and (B) area quantification of chromatin assembled in extracts with or without 1 µM H1-GFP and 4 µM RanBP7. In cytoplasm, adding RanBP7 does not greatly affect morphology but causes dissociation of H1 as measured by a reduction in fluorescence intensity. Average H1:DNA fluorescence intensity is shown below for conditions supplemented with H1. Scale bar, 10 µm. (C) Average FRAP curves (n≥7) and corresonding timelapse images of H1-GFP on chromatin in cytoplasm. H1-GFP recovers very rapidly and is not greatly affected by the addition of 4 µM recombinant RanBP7. H1-GFP signal was brightened in samples with RanBP7 relative to controls for visualization of the photobleaching and recovery. Photobleach occurs at time = 0. Scale bar, 2 µm. All quantification is shown ± standard error.
	Figure 3. RanBP7 Reveals H1 Foci.(A) Identically-scaled fluorescence images of fixed metaphase spindles from CSF reactions supplemented with 1 µM H1A-GFP and 4 µM RanBP7 or buffer control (+buff). In the presence of RanBP7, H1A-GFP is reduced on chromatin and concentrates on chromatin in small foci (arrowhead). The number of foci per nucleus (average ± standard error, n>40) is shown in the H1A-GFP column. (B) H1A-GFP foci do not colocalize with the centromere marker INCENP (INC). INCENP localization was performed using replicated chromosomes, on which H1A-GFP foci were less obvious but still detectable (insets). (C) Immunofluorescence images of UV-irradiated or unirradiated sperm nuclei assembled into chromatin in metaphase extracts supplemented with H1A-GFP, RanBP7, and biotin-dUTP. Insets are provided and the number of foci per nucleus is shown below the column for each condition. Scale bars, 10 µm.
	Figure 4. Effect of Cytoplasm on H1M and Domain Truncation Mutants.(A) Coomassie-stained gel of full-length (FL) H1M, amino-globular domains (ΔC), and C-terminal domain (ΔNG), with schematic of the proteins shown at right of the corresponding band. (B) Quantification of H1:DNA fluorescence intensity (average ± standard error) and (C) representative immunofluorescence images of sperm chromatin in buffer or extract supplemented with 1 µM H1M full-length or domains. In extract, full-length H1 localizes as efficiently as it does in buffer, while a sharp drop in localization intensity is observed for the domains. H1 was immunolocalized using the 6XHistidine tag (6XHis) common to all three constructs. For visualization purposes, DNA and H1 signal were brightened in the extract condition relative to buffer. Scale bar, 10 µm.
	Figure 5. Overexpression Phenotypes of H1 Full Length and Domain Truncations in Extract.(A) Anti-6XHistidine immunofluorescence of chromosome clusters from reactions supplemented with increasing concentrations of amino-globular (ΔC) or C-terminal (ΔNG) H1M truncation mutants, or 0.4 µM H1M full-length (FL) as a control. Truncation mutants required much higher concentrations for efficient localization. (B) Squash fix of chromatin and rhodamine-labeled microtubules (TUB) in egg extracts supplemented with increasing concentrations of full-length H1M. Added at prophase, H1M compacts the condensing metaphase chromosomes (MET) into a single mass within aberrant spindles. Such hypercondensed chromosomes are unable to segregate during anaphase (ANA). (C) Metaphase reactions supplemented with amino-globular or C-terminal domains at high concentrations. H1MΔC causes chromosome fragmentation, while H1MΔNG causes mitotic chromosomes to compact into a single mass. (D) Representative, identically-scaled fluorescence images of sperm chromatin incubated in CSF egg extracts supplemented with biotin dUTP with or without 20 µM H1MΔC. Morphological chromatin fragmentation was less obvious using the immunofluorescence protocol, but was observed in squash samples from these reactions. Scale bars, 10 µM.

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