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.