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, 11 (11), 1076-83

Centromeric Chromatin Exhibits a Histone Modification Pattern That Is Distinct From Both Euchromatin and Heterochromatin

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Centromeric Chromatin Exhibits a Histone Modification Pattern That Is Distinct From Both Euchromatin and Heterochromatin

Beth A Sullivan et al. Nat Struct Mol Biol.

Abstract

Post-translational histone modifications regulate epigenetic switching between different chromatin states. Distinct histone modifications, such as acetylation, methylation and phosphorylation, define different functional chromatin domains, and often do so in a combinatorial fashion. The centromere is a unique chromosomal locus that mediates multiple segregation functions, including kinetochore formation, spindle-mediated movements, sister cohesion and a mitotic checkpoint. Centromeric (CEN) chromatin is embedded in heterochromatin and contains blocks of histone H3 nucleosomes interspersed with blocks of CENP-A nucleosomes, the histone H3 variant that provides a structural and functional foundation for the kinetochore. Here, we demonstrate that the spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin. We speculate that this distinct modification pattern contributes to the unique domain organization and three-dimensional structure of centromeric regions, and/or to the epigenetic information that determines centromere identity.

Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests

Figures

Figure 1
Figure 1
H3 is not di- or trimethylated at Lys9 in CEN chromatin. (ad) Extended chromatin fibers from human (a,c) or D. melanogaster (b,d) interphase cells were stained with antibodies to CENP-A or CID (green), and H3 Lys9-diMe or H3 Lys9-triMe (red), detected with FITC- and Cy3-conjugated secondary antibodies. Merged images are to the right of the single-color images. Spaces between CENP-A/CID blocks denote H3-containing nucleosome blocks, and did not stain for H3 Lys9-diMe or H3 Lys9-triMe. H3 Lys9-diMe, typically found in heterochromatin, was present on one side (67%, n = 30) or both sides (33%; n = 30) of the human CENP-A region (a). Lys9 was not dimethylated on H3 within the CID domain on chromatin fibers from D. melanogaster third-instar larval neuroblasts and S2 cells, and always flanked the CID region on both sides (b). In contrast, H3 Lys9-triMe was not present in the regions flanking CENP-A/CID (c,d). To demonstrate that staining for H3 Lys9-triMe was present in noncentromeric regions in these preparations, two different fibers are shown in d, one that is CID-negative but H3 Lys9-triMe-positive (denoted by asterisk), and one that is CID-positive and H3 Lys9-triMe-negative. Quantitative evaluations of the overlaps are presented in Figure 2. Scale bars, 15 μm in a,c; 5 μm in b,d.
Figure 2
Figure 2
Quantification of overlap between histone modifications and CENP-A/CID on chromatin fibers from flies and humans. Extended chromatin fibers from human cells and flies were analyzed by fluorescence line plots to determine the amount of overlap between CENP-A/CID and modified histone antibody signals. Chromatin fibers from human and flies showed <40% overlap of regions containing H3 Lys9-diMe with areas containing CENP-A or CID. Conversely, >60% overlap was seen between H3 Lys4-diMe and CENP-A or CID regions. Acetylated residues in H3 and H4, and H3 Lys4-triMe, did not overlap with CENP-A or CID regions.
Figure 3
Figure 3
H3 is not di- or trimethylated at Lys9 in CEN chromatin at metaphase. (aj) Immunofluorescence patterns on human (a,g) and D. melanogaster (d,j) metaphase chromosomes show localization of H3 Lys9-diMe or H3 Lys9-triMe (red) relative to CENP-A and CID staining (green). Chromosomal DNA was stained with DAPI (blue). (b,e,h,k) Enlarged views of chromosomes marked with asterisks in a, d, g (human chromosome 1) and j (D. melanogaster chromosome 3), with merged images to the right of the single-color images. Arrows point to the areas on individual chromosomes where CENP-A/CID is located. (c,f,i,l) Three-dimensional modeling of antibody staining on the same human and D. melanogaster metaphase chromosomes from b, e, h, and k. Chromosome cartoons depict the degree to which each three-dimensional model was rotated around the x-, y- and/or z-axes. In all cases, CENP-A/CID shows minimal overlap with H3 Lys9-diMe or tri-Me staining. H3 Lys9-diMe was present in the spaces between the CENP-A/CID cylinders on the two sister chromatids, as well as on either side of the centromere region along the chromosome arms. In contrast, H3 Lys9-triMe was offset significantly from CENP-A/CID staining along the chromosome arms, and was not present in the spaces between the sister centromeres. Quantifications of overlaps are shown in Figure 4. Scale bars, 15 μm in a,g; 5 μm in b,j.
Figure 4
Figure 4
Quantification of overlap between histone modifications and CENP-A/CID on metaphase chromosomes from humans and flies. Metaphase chromosomes from human cells and flies were analyzed by fluorescence line plots to determine the amount of overlap between CENP-A/CID and modified histone antibody signals. Overlap between CENP-A or CID staining, and H3 Lys9-diMe, was less than that seen for H3 Lys4-diMe. More overlap was observed in metaphase chromosomes in comparison with fibers for Lys9 methylation and some of the acetylated H3 and H4 residues, reflecting the lower resolution obtained in condensed chromosomes versus extended fibers.
Figure 5
Figure 5
H3 and H4 are hypoacetylated in CEN chromatin. (a,b) Immunofluorescence patterns on human (a) and D. melanogaster (b) chromatin fibers stained with H4 Lys12-Ac antibodies. Chromatin fibers and metaphases from human and D. melanogaster cells did not show enrichment for H3 or H4 lysine acetylation in CEN chromatin. For example, CENP-A is located close to, but does not overlap with, H4 Lys12-Ac. Single-color images are shown separated from the merged image. (ch) Antibody localization on metaphase chromosomes (c,f) shows that CENP-A/CID and H4 Lys12-Ac staining do not colocalize, and that pericentromeric regions are also hypoacetylated at H4 Lys12. (d,g) Enlarged view of chromosomes marked with asterisks in c and f, with single-color images shown to the right of the merged images. Arrows point to the areas on individual chromosomes where CENP-A/CID, but not H4 Lys12-Ac, were located. (e,h) Three-dimensional modeling of individual metaphase chromosomes from d and g demonstrate the separation of CENP-A/CID and acetylated histones. The separation is particularly pronounced on D. melanogaster chromosomes (f), which contain large amounts of pericentromeric heterochromatin. Chromosome cartoons depict the degree to which each three-dimensional model was rotated around the x-, y- and/or z-axes in e and h. Similar results were obtained after staining fibers and metaphases with antibodies to five other H3 and H4 acetylated residues, which are quantified in Figures 2 and 4.
Figure 6
Figure 6
H3 is dimethylated at Lys4 in CEN chromatin. (a,b) Extended chromatin fibers from human cells (a) and D. melanogaster S2 cells (b) were stained with antibodies to CENP-A/CID and H3 Lys4-diMe, detected with secondary antibodies conjugated with FITC (green) and Cy3 (red), respectively. Single-color images are shown separated from the merged image. H3 nucleosomes interspersed with CENP-A/CID nucleosomes are dimethylated at Lys4 in both humans and flies. (ch) Immunofluorescence patterns on human (c) and D. melanogaster (f) metaphases stained with H3 Lys4-diMe antibodies (red). DNA was stained with DAPI (blue). Antibody localization on metaphase chromosomes shows that H3 Lys4-diMe is located to close to, and partially overlapping with, CENP-A/CID (green). (d,g) Enlarged view of chromosomes marked with asterisks in c and f, with single-color images to the right of the merged images. (e,h) Three-dimensional modeling of antibody staining on individual metaphase chromosomes from d and g shows that H3 Lys4-diMe is located in the region between sister kinetochores, partially overlapping with CENP-A/CID staining (arrows), consistent with the coiling or looping model proposed for CEN chromatin higher ordering packaging at metaphase. The asterisk in d denotes human chromosome 1, which is particularly under-represented for H3 Lys4-diMe in the 1q region. Chromosome cartoons depict the degree to which each three-dimensional model was rotated around the x-, y- and/or z-axes in e and h. Fiber and metaphase results are quantified in Figures 2 and 4. Bars, 15 μm in a,c; 5 μm in b,f.
Figure 7
Figure 7
H3 is not trimethylated at Lys4 in CEN chromatin. (af) Metaphases and extended chromatin fibers were stained with antibodies to CENP-A or CID (green) and H3 Lys4-triMe (red), detected with FITC and Cy3-conjugated antibodies, respectively. Single-color images are shown separated from the merged image. On chromatin fibers from human and D. melanogaster interphase cells (a,b), H3 Lys4-triMe did not overlap with CENP-A/CID. A gap of 1–20 μM between CENP-A and H3 Lys4-triMe staining was observed on most human chromatin fibers (80%, n = 18). In addition, H3 Lys4-triMe antibody staining was present on only one side of CENP-A antibody staining in 70% of fibers (n = 16), and was observed on both sides in 30% of fibers (n = 7). On D. melanogaster fibers, H3 Lys4-triMe antibody staining was observed on both sides of the CID antibody staining, but the gap between H3 Lys4-triMe and CID on every fiber was at least 5 μm (average = 15 μm; n = 15). The pericentromeric regions that were negative for H3 Lys4-triMe staining may correspond to H3 Lys9-diMe modified nucleosomes (Fig. 1). (c,f) Immunofluorescence patterns on human (c) and D. melanogaster (f) metaphases stained with H3 Lys4-diMe antibodies (red). DNA was stained with DAPI (blue). Antibody localization on metaphase chromosomes shows that H3 Lys4-triMe did not coincide with CENP-A/CID (green). (d,g) Enlarged view of chromosomes marked with asterisks in c (human chromosome 1) and f (D. melanogaster chromosome 2), with single-color images to the right of the merged images. Arrows point to the areas on individual chromosomes where CENP-A/CID, but not H3 Lys4-triMe, were located. (e,h) Three-dimensional modeling of individual metaphase chromosomes from d and g shows the separation of CENP-A/CID and H3 Lys4-triMe into distinct domains. Chromosome cartoons depict the degree to which each three-dimensional model was rotated around the x-, y- and/or z-axes in e and h. Fiber and metaphase results are quantified in Figures 2 and 4. Bars, 15 μm in a,c; 5 μm in b,f.
Figure 8
Figure 8
Model for three-dimensional organization of centromeric (CEN) chromatin in D. melanogaster and humans. Incorporation of the two-dimensional and three-dimensional histone modification patterns extends our understanding of the chromatin composition and organization of the CEN region, and suggests that interspersed CENP-A/CID and H3 Lys4-dimethyl nucleosome blocks comprise a unique chromatin state that is distinct from the flanking heterochromatin. Associations between similarly modified nucleosome blocks are proposed to contribute to the formation of distinct three-dimensional structures in CEN and flanking chromatin. Interspersed CENP-A/CID and distinctly modified H3 and H4 may mediate formation of the ‘cylindrical’ three-dimensional structures observed in metaphase chromosomes. H3 Lys9-diMe chromatin, which recruits heterochromatin proteins such as HP1 and cohesion proteins such as RAD21/SCC1, is present in the inner kinetochore space between mitotic sister chromatids and in regions that flank CEN chromatin. This arrangement may position CENP-A toward the poleward face of the mitotic chromosome and facilitate recruitment of outer kinetochore proteins, and promote HP1 self-interaction and proper chromosome condensation and cohesion. Cohesins are presented as ringed structures, in accord with recent models.

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