Maresca TJ et al. (2005), Histone H1 is essential for mitotic chromosome ...

XB-ART-1728

J Cell Biol 2005 Jun 20;1696:859-69. doi: 10.1083/jcb.200503031.

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Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts.

Maresca TJ , Freedman BS , Heald R .

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During cell division, condensation and resolution of chromosome arms and the assembly of a functional kinetochore at the centromere of each sister chromatid are essential steps for accurate segregation of the genome by the mitotic spindle, yet the contribution of individual chromatin proteins to these processes is poorly understood. We have investigated the role of embryonic linker histone H1 during mitosis in Xenopus laevis egg extracts. Immunodepletion of histone H1 caused the assembly of aberrant elongated chromosomes that extended off the metaphase plate and outside the perimeter of the spindle. Although functional kinetochores assembled, aligned, and exhibited poleward movement, long and tangled chromosome arms could not be segregated in anaphase. Histone H1 depletion did not significantly affect the recruitment of known structural or functional chromosomal components such as condensins or chromokinesins, suggesting that the loss of H1 affects chromosome architecture directly. Thus, our results indicate that linker histone H1 plays an important role in the structure and function of vertebrate chromosomes in mitosis.

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Species referenced: Xenopus laevis
Genes referenced: bub1b cenpa kidins220 kif11 kif22 ndc80 rcc1
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	Figure 1. Characterization of embryonic histone H1. (A) Immunofluorescence of individual sperm nuclei in X. laevis egg extract reactions in metaphase and interphase, stained with affinity-purified antibodies raised to embryonic histone H1 and costained with Hoechst DNA dye to show colocalization. Bar, 10 μm. (B) Western blot of four different X. laevis egg extract preparations probed with H1 antibodies that show variation in molecular mass. The three lines show relative positions of three different H1 isoforms. (C) Western blot of samples collected at 15-min time intervals from a CSF egg extract induced to enter interphase by calcium addition and then back into metaphase by the addition of fresh CSF extract. H1 migration does not change during interphase and metaphase. Phospho-H3 epitopes appear only in mitotic samples. (D) Embryonic histone H1 purified from extract that was pooled from the eggs of ∼20 frogs yielded at least four bands by SDS-PAGE and coomassie staining (left lane), all of which were recognized by Western blot analysis with affinity-purified H1 antibodies (right lane) and were confirmed by mass spectrometry to be embryonic H1 (not depicted). The destained gel was delivered to the Howard Hughes Medical Institute Mass Spectrometry Facility (University of California Berkeley) for protein band excision, digestion, and mass spectrometric analysis.
	Figure 2. CSF chromatids recruit reduced levels of histone H1. (A) Comparison of chromatin proteins that are associated with unreplicated chromatids formed in a CSF extract with cycled mitotic chromosomes that have undergone duplication. Silver-stained gel reveals a similar protein pattern, except in the region marked by an asterisk at ∼40 kD. By using Western blot analysis, we find that the proteins enriched in this region on duplicated chromosomes correspond to histone H1 bands, whereas XCAP-G levels appear similar. (B) Fluorescence images showing H1 and kinetochore marker BubR1 staining on CSF chromatids and duplicated mitotic chromosomes. Assembly reactions were fixed separately and were then pooled before isolating structures onto a coverslip and processing for immunofluorescence staining. Cycled chromosomes were discernible from CSF chromatids based on DNA morphology, BubR1staining intensity, and the presence of paired sister chromatids and kinetochores. The top panels are from one field of view containing the two types of assembled chromatin structures: CSF chromatids and cycled chromosomes, as indicated in the merged image. Each cluster contains approximately six to eight chromatids or cycled chromosomes. The bottom panels are enlarged images of another field of view showing one individual cycled chromosome next to two CSF chromatids, as indicated in the merged image. Enrichment of histone H1 on duplicated chromosomes is most clearly evident in the merged images. Bars, 10 μm. (C) Schematic diagram of the structures shown in the bottom panels of B. The top structure is a metacentric chromosome consisting of two sister chromatids, delineated by the black line and partially folded back on itself. The open arrows point to the two ends of the cycled chromosome. Based on BubR1 staining, the bottom structure likely consists of two tangled CSF chromatids. Closed arrows point to the kinetochores (K).
	Figure 3. Immunodepletion of H1 causes an elongated chromosome morphology. (A) Western blot of total, H1-depleted, and mock-depleted extract probed with H1 and RCC1 antibodies to show that >95% of H1 was removed, whereas chromatin protein RCC1 was unaffected by the procedure. (B) Fluorescence images of half spindles assembled around sperm nuclei incubated in CSF extracts. Structures contain a polarized array of microtubules oriented toward the DNA, from which H1 can be efficiently depleted. In merged images, microtubules are red, DNA is blue, and H1 is green. (C) Coomassie-stained gels showing recombinant H1 purified from insect cells and somatic H1 purified from calf thymus that were used in add-back experiments. (bottom) Western blot of mock-depleted, H1-depleted, and rescue conditions with 1.5 μM of embryonic H1 added to the depleted extract. (D) Immunofluorescence of individual replicated chromosomes from mock-depleted, H1-depleted extracts, and rescued reactions in which H1-depleted extracts were supplemented with endogenous levels (1.5 μM) of purified histone H1. The same results were obtained for embryonic H1 rescues. Chromosomes were costained with Hoechst DNA dye (blue) and BubR1 antibodies (red) to label kinetochores. (E) Quantification of chromosome lengths. (top) Distribution of chromosome lengths in mock-depleted and H1-depleted extracts (mock depleted, n = 43; H1 depleted, n = 35). (bottom) Box and whiskers plot from a representative rescue experiment (n = 62 for each condition). The middle line of each box is the median. Top and bottom lines are the third and first quartiles, and the whiskers indicate the maximum and minimum chromosome length measurements. The elongated conformation of H1-depleted chromosomes was largely rescued by the addition of either somatic or embryonic histone H1 to H1-depleted extracts. Bars, 10 μm.
	Figure 4. H1 depletion prevents proper chromosome alignment and segregation by the mitotic spindle. (A) Mock- and H1-depleted spindle assembly reactions containing rhodamine-labeled tubulin and stained with Hoechst DNA dye reveal that chromosomes cannot align properly in metaphase in the absence of H1 and are not effectively segregated during anaphase. (B) Fluorescence images of metaphase spindles showing that chromosome alignment defects caused by H1 depletion can be rescued by the readdition of somatic H1 to 1.5 μM. In merged images, microtubules are red and DNA is blue. (C) Quantification of chromosome alignment defects. The number of arms that had strayed from the metaphase plate were counted in mock- and H1-depleted spindles and in H1-depleted spindle reactions supplemented with purified H1 (mock depleted, n = 459; H1 depleted, n = 448; rescue, n = 165). The percentages of metaphase spindles with 0–2, 3–5, and >5 misaligned arms are shown. Error bars represent SD. Bars, 10 μm.
	Figure 5. Other chromosomal proteins bind at similar levels in the absence of histone H1. (A) Chromosome-associated protein (CAP) profiles of cycled chromosomes isolated from mock- and H1-depleted extracts and analyzed by SDS-PAGE and silver staining. (bottom) Core histones of the same sample run on a higher percentage gel. Note that the only apparent difference between the two samples is a band at the molecular mass of H1. (B) Western blot analysis of mock- and H1-depleted CAPs. Blots were probed with antibodies to topoismerase II, cohesin subunit SMC-1, condensin subunit XCAP-G, kinetochore component Ndc80, RanGEF RCC1, and histone H1. H1 is the only CAP observed to be absent after H1 depletion. (C) Immunofluorescence analysis of Kinesin-10 (Xkid) in mock- and H1-depleted spindles. In the merged image, microtubules are red, DNA is blue, and Xkid is green. Although H1 depletion impairs chromosome alignment, Xkid localization is not affected. Bar, 10 μm. (D) Immunofluorescence analysis showing similar localization of kinetochore proteins on mock- and H1-depleted chromosomes. Left panels show histone H3 variant CENP-A (green) and BubR1 (red), and right panels show condensin II subunit XCAP-G2 (green) and BubR1 (red). DNA is blue. Bar, 2.5 μm.
	Figure 6. Kinetochores appear to function normally in the absence of histone H1. (A) Kinetochores visualized by CENP-A immunofluorescence localize to the metaphase plate despite misalignment of chromosome arms in the absence of H1. In the merged image, microtubules are red, DNA is blue, and CENP-A is green. The DNA/CENP-A merged image is meant to further highlight the observed metaphase clustering of CENP-A despite arm misalignment. DNA is green and CENP-A is purple. Bar, 10 μm. (B) Kinetochores attach and orient similarly in mock- and H1-depleted spindles treated with Eg5 inhibitor monastrol. Note CENP-A staining (red) on discrete kinetochores that were attached to microtubules and were oriented toward the center of monoaster structures. Microtubules are green and DNA is blue. Bar, 10 μm. (C) Kinetochores segregate during anaphase in the absence of H1. Samples from mock- and H1-depleted spindle reactions induced to enter anaphase were fixed at 5-min intervals. Kinetochores visualized by the addition of directly labeled CENP-A antibodies segregate similarly in both reactions, whereas chromosome arms (blue) are elongated and tangled in the absence of H1. Bar, 10 μm. For time-lapse videos of kinetochore segregation in the presence and absence of H1, see Videos S1 and S2 (available at http://www.jcb.org/cgi/content/full/jcb.200503031/DC1). (D) Duplicated X. laevis sperm chromosomes stained with antibodies to histone H1 (green), CENP-A (red), and Hoechst DNA dye (blue). Linescan shows relative intensity of staining along the length of the chromosome. Note the lack of H1 signal on centromeric DNA nubbin (arrows). Bar, 2.5 μm.
	Figure 7. Schematics illustrating the potential role of histone H1 in chromosome condensation, alignment, and segregation. (A) Illustration comparing X. laevis spindles and chromosomes in metaphase and anaphase in the presence or absence of H1. Note that the kinetochores align and segregate normally in both mock- and H1-depleted conditions. H1-depleted chromosome arms do not align properly as a result of their elongated conformation. Inset highlights how elongated chromosomes would have productive microtubule–chromokinesin interactions within the confines of the spindle but not when dangling outside. H1-depleted chromosomes do not segregate properly as a result of their extended length and the presence of twisted and tangled chromatids that remain in the center of the spindle. (B) A closer analysis of H1-depleted interphase chromatin length and condensation factor levels could differentiate between two potential mechanisms of condensation factor deposition onto the chromatin template. Scenario A represents the compaction of a wild-type interphase fiber by condensation factors. Scenarios B and C assume the H1-depleted interphase chromatin template is physically longer than the wild-type template. If condensation factors are deposited at specific physical distances along the chromatin template (schematically represented by triangles and hatchmarks), then mitotic chromosome length might be normal (scenario B) in H1-depleted chromosomes. Regardless of the effect on metaphase length, scenario B would lead to increased levels of condensation factors on H1-depleted chromosomes. However, if the distribution of condensation machinery is defined at the level of the DNA template (∼10 kb), then compaction of H1-depleted interphase chromatin would generate longer metaphase chromosomes with the same level of condensation factors as wild-type chromosomes (scenario C). Our data support scenario C.

References [+] :

Black, Structural determinants for generating centromeric chromatin. 2004, Pubmed