Fig 1: SENP6, a SUMO protease, controls maintenance of centromeric CENP-A.a, b Schematics for high resolution SNAP pulse-chase (a) and quench-chase-pulse (b) assays. HeLa-CENP-A-SNAP cells were treated with SENP6 siRNA or a control scrambled siRNA. Pulse-chase experiment was performed for 48 h during RNAi to assay for CENP-A turnover (a). Quench-chase-pulse experiment was performed for the final 7 h of siRNA treatment to assay for CENP-A assembly (b). c, d shows typical image fields following the strategies in a, b, respectively. TMR-Star and Oregon Green SNAP labels visualize the maintenance or assembly of CENP-A-SNAP, respectively. CENP-B was used as a centromeric reference for quantification. Cells were counterstained for SENP6 to visualize its depletion in siRNA treated cells. Yellow arrowheads indicate nuclei that escaped SENP6 depletion which correlate with retention of ‘old’ CENP-A-SNAP. Bars, 10 µm. e Automated centromere recognition and quantification of c, d. Centromeric CENP-A-SNAP signal intensities were normalized to the control siRNA treated condition in each experiment. siRNA treatment; siSENP6 or scrambled (Ctrl). Three replicate experiments were performed. Bars indicate SEM. Parametric two-tailed Student’s t test were performed to calculate statistical significance. **p < 0.01, ***p < 0.001. f Centromeric levels of total, steady state CENP-A under siRNA mediated depletion of SENP6 in untagged HeLa cells. g Quantification of f as in c, d. h Centromeric levels of total, steady state CENP-A under siRNA mediated depletion of SENP6 in primary fibroblasts. i Quantification of h as in c and d. Source data are provided as a Source Data file.
Fig 2: The neocentromere adapts by accumulating INCENP and Borealin. (A) CPC component protein levels at Neo4p13 at indicated times points (0, 100, and 200 d of continuous culture) measured as in Fig. 2 A. Mean and SEM of five independent experiments (n = 47–54 spreads). P values are based on one-way ANOVA with Tukey’s multiple comparison test. (B) Pol II S2p levels at Neo4p13 at 0 and 200 d of continuous culture measured as in Fig. 3 E. Mean and SEM of five independent experiments (n = 50 spreads). P values were determined as in A. (C) Intercentromere and inter-kinetochore distance at Neo4p13 at indicated time points based on CENP-C and Hec1 immunostaining measured as in Fig. 2 C. Mean and SEM of three independent experiments (n = 20–32 spreads; P values are indicated in the figure, determined as in A). (D) Intercentromere distance based on CENP-C of different clones of Neo4p13 (C1–C4, four independent clones) after 200 d in continuous culture measured as in Fig. 2 C. Mean and SEM of three independent experiments (n = 27–30 spreads). P values are defined as in A. (E) Cell cycle profiles determined by flow cytometry using PI DNA staining of S40-RPE and Neo4p13 at 0, 100, and 200 d of continuous culture. Three independent populations for S40-RPE and four for Neo4p13 subjected to long-term culture experiments are shown. (F) Representative spreads of polyploid cells of Neo4p13 cell line after 100 or 200 d in continuous culture, stained for indicated proteins and DNA (as for Fig. 2 B). A neocentromere-containing chromosome is identified by lack of CENP-B staining. Scale bars, 2 µm.
Fig 3: Experimentally induced human neocentromere shows inner-centromere defects. (A) Centromeric protein levels in the neocentromere (Neo4p13) compared with random endogenous centromeres. Quantification of mitotic spreads coimmunostained for indicated proteins and CENP-B. Mean and SEM of five (n = 50 spreads, for CENP-C) or three (n = 21–34 spreads, for all other proteins) independent experiments. P values based on one-way ANOVA with Tukey’s multiple comparison test. (B) CPC protein levels at Neo4p13 compared with random endogenous centromeres (Random CEN). Representative mitotic spreads coimmunostained for indicated CPC proteins and CENP-B. Zw10 outer kinetochore protein is included as a comparison to an unchanged reference protein. Insets show Neo4p13 and a random centromere equally scaled for visual comparison. Scale bars, 2 µm. Mean and SEM of five independent experiments (n = 45–52 spreads) analyzed as in A. (C) Intercentromere distance measured by coimmunostaining mitotic spreads for CENP-C and CENP-B. Quantification of distance (in micrometers) between the peak intensities of each CENP-C dot pair in one plane compared with equivalent pairs of random centromeres. Mean and SEM (n = 30 spreads) of three independent experiments. Scale bars, 2 µm. P values determined as in A. (D) Intercentromere distance based on Hec1 staining measured and analyzed as in C. Mean and SEM (n = 29 spreads) of three independent experiments.
Fig 4: Strategy for human centromere deletion and neocentromere isolation. (A) A cassette carrying the 5′ portion of the eYFP was integrated on the 4q-arm in frame with CEP135 protein (light brown) and the 3′ portion on the 4p-arm, flanking the centromere. Cassettes carry a murine intron (dark brown) that acts as a gRNA target for Cas9. Upon Cas9 cleavage one possible outcome is the circularization of the centromere fragment (∼8 Mb) leading to reconstitution of spliced CEP135-eYFP. The acentric chromosome serves as template for neocentromere formation (see Fig. S1). (B) Experimental design for centromere deletion and neocentromere selection and detection. (C) Selection of CEP135-eYFP positive cells by flow cytometry (FL2, fluorescence channel 2 used for scatter and autofluorescence measurements). (D) Micrograph showing CEP135-eYFP foci indicative of centromere fragment circularization. Scale bar, 2 µm. (E) Micrograph of neocentromere detection by FISH-IF in mitotic spreads. The FISH probe identifies chromosome 4, ACA (CENP-A, CENP-B, and CENP-C) localizes to active centromeres, and CENP-B binds specifically to alphoid DNA, absent from neocentromeres. Insets display the two chromosome 4s from the image on the left. Scale bars, 2 µm. Diagram on the right represents the expected staining pattern to identify the neocentromere. (F) Karyotypes of the parental and neocentromere cell lines by mFISH. S40-RPE carries a preexisting translocation (X,10) and an extra copy of chromosome 12, present in all the clones analyzed. Chr4, chromosome 4.
Fig 5: Sgo1 localization at the neocentromere is kinetochore biased. (A) Sgo1 localization in Neo4p13 compared with random endogenous centromeres. Representative mitotic spreads coimmunostained for Sgo1, ACA, and CENP-B. Insets show Neo4p13 and random centromeres with Sgo1 localization toward the kinetochores (two dots) or the inner centromere (one dot) and the quantification of each localization pattern (%) from three independent experiments (n = 50 spreads). P < 0.0001 (t test). Scale bars, 2 µm. (B) Genomic snapshot of quantitative ChIP-seq reads (RPKM, reads per kilobase per million) for Rad21 (mitotic cells) plotted as in Fig. 3 B. (C) Estimated allele specific coverage of Rad21 on the 4p13 region on the neocentromere-containing chromosome in Neo4p13 cells inferred as in Fig. 3 C. chr, chromosome; IP, immunoprecipitation.
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