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Stepwise Unfolding Supports a Subunit Model for Vertebrate Kinetochores

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Stepwise Unfolding Supports a Subunit Model for Vertebrate Kinetochores Stepwise unfolding supports a subunit model for vertebrate kinetochores Giulia Vargiua, Alexandr A. Makarova, James Allanb, Tatsuo Fukagawac, Daniel G. Bootha,1, and William C. Earnshawa,1 aWellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, United Kingdom; bMRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XU, Scotland, United Kingdom; and cLaboratory of Chromosome Biology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan Edited by Don W. Cleveland, University of California at San Diego, La Jolla, CA, and approved February 8, 2017 (received for review August 25, 2016) During cell division, interactions between microtubules and chromo- substructure. CENP-C and CENP-S separately contribute to the somes are mediated by the kinetochore, a proteinaceous structure stability of the centrochromatin structure during mitosis. located at the primary constriction of chromosomes. In addition to the centromere histone centromere protein A (CENP-A), 15 other Results and Discussion members of the constitutive centromere associated network (CCAN) Step-Wise Unfolding of CENP-A Centrochromatin. To characterize participate in the formation of a chromatin-associated scaffold that the folding of centromeric chromatin, we exploited the ability of supports kinetochore structure. We performed a targeted screen low-salt TEEN buffer, which lacks divalent cations (Materials and analyzing unfolded centrochromatin from CENP-depleted chromo- Methods) to unravel highly compact kinetochore chromatin into somes. Our results revealed that CENP-C and CENP-S are critical for extended fibers (23) (Fig. 1A). To identify unfolded centromeric the stable folding of mitotic kinetochore chromatin. Multipeak regions, we generated cells expressing GFP:CENP-A from a fitting algorithms revealed the presence of an organized pattern DT40 wild-type cell line. Expression of exogenous GFP:CENP-A of centrochromatin packing consistent with arrangement of CENP- had no effect on endogenous CENP-A levels (Fig. S1A). Fur- A–containing nucleosomes into up to five chromatin “subunits”— thermore, GFP:CENP-A was not present on chromosome arms each containing roughly 20–30 nucleosomes. These subunits could (Fig. S1B), a potential artifact associated with CENP-A over- be either layers of a boustrophedon or small loops of centromeric expression. GFP:CENP-A was found exclusively at the kineto- chromatin. chore, colocalizing with CENP-T, at all cell-cycle stages (Fig. S1B)—even on unfolded centrochromatin (Fig. S1C). This cell centromere | kinetochore | CENP-A | centrochromatin | boustrophedon line was used to analyze centromere unfolding in both interphase and mitotic cells (Fig. 1B). he centromere is the genetic locus located at the primary We used correlative light and electron microscopy (CLEM) to Tconstriction of mitotic chromosomes that directs chromo- determine the extent of chromatin unfolding induced by TEEN some segregation. Biochemically, the centromere is defined by treatment. A line-scan analysis of correlative EM images con- ± the presence of the histone H3 variant centromere histone firmed the presence of fibers with a mean diameter of 12.6 centromere protein A (CENP-A) (1, 2) interspersed with ca- 2.19 nm, consistent with the diameter of a single chromatin fiber C nonical H3 nucleosomes carrying active chromatin marks (3, 4). (Fig. 1 ). Thus, TEEN treatment can unfold chromatin to the This specialized chromatin class has been termed “cen- level of single fibers. Collective analysis of >1,300 individual centrochromatin fibers trochromatin” (5). During cell division, an elaborate multisubunit revealed that interphase prekinetochores unfolded to a significantly protein superstructure, the kinetochore, assembles on the surface greater extent than mitotic kinetochores [unfolded length of of the centrochromatin to direct chromosome segregation. Kinetochores contain ≥100 different proteins, 16 of which Significance comprise the constitutive centromere associated network (CCAN). The CCAN remains associated with centromeric chromatin across the entire cell cycle (6–8). The CCAN includes CENP-A, During cell division, microtubules apply piconewton forces to CENP-C, and four multisubunit complexes: CENP-L/-N (9), segregate duplicated chromosomes into daughter cells. The kinetochore, located on the surface of the centromere chro- CENP-H/-I/-K/-M (10), CENP-O/-P/Q-/R/-U (11), and CENP- – matin, couples microtubules to the chromosomes. Little is T/-W/-S/-X (12 15). known about the folding of the centromeric chromatin and Although numerous immuno-electron microscopy (16–18) and – how this templates the functional ultrastructure of the kinet- superresolution microscopy (19 21) studies have mapped the ochore. To better understand this fundamental problem, we CELL BIOLOGY locations of CCAN components relative to one another, the used a microscopy technique that allowed the DNA associated packing of the chromatin fiber in centrochromatin remains un- with centromeric chromatin to be unfolded and accurately known. Early studies of stretched chromosomes suggested a re- measured in the presence and absence of several key kineto- peating “subunit” structure for the kinetochore (1, 22). One chore components. By combining this microscopy method with subsequent hypothesis was that centrochromatin is composed of statistical analysis of the unfolded chromatin fibers, we ac- “amphipathic” helices or loops, with CENP-A–containing nu- quired data that allowed a subunit model of the kinetochore cleosomes facing the outer kinetochore and H3 chromatin ori- chromatin to be proposed. ented toward the interior (5). A recent study proposed that centrochromatin is folded back and forth into a sinusoidal patch Author contributions: D.G.B. and W.C.E. designed research; G.V., A.A.M., J.A., and D.G.B. performed research; T.F. contributed new reagents/analytic tools; G.V., D.G.B., and W.C.E. or boustrophedon with a multilayered structure stabilized during analyzed data; and G.V., D.G.B., and W.C.E. wrote the paper. mitosis by CENP-C (4). The authors declare no conflict of interest. Here, we have dissected centrochromatin organization by This article is a PNAS Direct Submission. progressively unfolding the chromatin at low ionic strength in 1To whom correspondence may be addressed. Email: [email protected] or daniel. lysed interphase and mitotic cells. Measurement of the lengths [email protected]. of the resulting fibers revealed that centrochromatin unfolds in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. a series of discrete (∼0.5 μm) steps, consistent with a repeat 1073/pnas.1614145114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1614145114 PNAS | March 21, 2017 | vol. 114 | no. 12 | 3133–3138 Downloaded by guest on September 28, 2021 reveal periodicities in the data. Our comprehensive datasets (>650 measurements per sample) allowed us to generate high- resolution histograms (100 × 0.1 μm bins). This revealed the apparent presence of subpopulations of unfolded fibers, an ob- servation masked with coarser bin widths (Fig. S2A). We then defined the periodicities observed (Fig. S2B), using the multipeak fitting software Igor Pro-6.2 (WaveMetrics, Inc.) (Materials and Methods). Five distinct peaks were recognized in interphase un- folded centrochromatin (Fig. 2A) and only three peaks in its mitotic counterpart (Fig. 2B). Each peak represented a node of accumu- lation of subpopulations of fibers, each corresponding to a potential subunit of centrochromatin released from the kinetochore. To estimate the amount of DNA present in each unfolding subunit, we determined the density of nucleosome packing in mitotic chromatin fibers unfolded under these conditions by analyzing TEM of chromosome spreads prepared in TEEN buffer. The average center-to-center distance between adjacent nucleosomes was 20.43 ± 0.68 nm (Fig. S3A). The comparable distance from chicken erythrocyte interphase chromatin in low- salt buffer was 38.71 ± 1.6 nm (Fig. S3B). These numbers could not be measured specifically at centromeres and thus provide only baseline values for estimating the chromatin packing. Having measured peak locations and an approximate inter- nucleosome distance, we could estimate the amount of DNA present within the kinetochore. The distances between the fiber origin, the first peak, and pairs of adjacent peaks (steps) were interpreted as measures of chromatin length per unfolding sub- unit. Interphase fibers unfolded in five steps with a mean step Fig. 1. Unfolding of centromere chromatin in interphase versus mitotic samples. (A) Schematic explaining the method used to unfold chromatin using TEEN buffer. TEEN has a low-salt concentration and contains EDTA as a divalent cation chelator. The excess of negative charges on the DNA and the hypotonic environment together cause cells to burst and the chromatin to unfold. (B) Representative fluorescence micrographs of unfolded cen- trochromatin fibers, detected using GFP:CENP-A and DAPI. (Scale bar, 1 μm.) (C) CLEM analysis of unfolded chromatin from asynchronous cells. DAPI and GFP:CENPA were used to identify typical unfolded fibers. The same regions were revisited using TEM. (Scale bar, 50 nm.) Fibers visualized by TEM were analyzed using multiple line scans (see representative line scan in black; scale bar, 50 nm) and pixel density measurements. The data were plotted in a line graph where
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