Fig 2: SENP6 is required for centromeric CENP-A maintenance throughout the cell cycle.a Schematic of the genotype of cell line constructed for auxin (IAA)-mediated depletion of SENP6. OsTIR1 and CENP-A-SNAP are expressed as transgenes, SENP6 is homozygously tagged at its endogenous locus. b Experimental scheme for long-term and short-term CENP-A-SNAP pulse-chase (PC) assays following auxin (IAA) mediated depletion of SENP6. c, d Quantification of long-term and short-term PC experiments, respectively following the experimental scheme detailed in b. c ‘Old’ centromeric CENP-A-SNAP intensities are normalized to the mean of the non-treated condition (-) for the 24 h time point and plotted as bar graphs against auxin (IAA) treated (+) and non-treated (-) conditions for 24 h and 48 h. Three replicate experiments were performed. Bar indicates SEM. Parametric two-tailed Student’s t test were performed to calculate statistical significance. ***p < 0.001 d ‘Old’ centromeric CENP-A-SNAP intensities are measured and median intensities are plotted against auxin (IAA) or non-treated (Ctrl) conditions. Bars indicate SEM of three replicate experiments. e Experimental scheme of cell cycle synchronization coupled to CENP-A-SNAP pulse-chase (TMR pulse) and auxin (IAA) mediated depletion of SENP6 at different stages of the cell cycle (RO: RO3306, Cdk1 inhibitor). f–h Quantification as in c but normalized to non-treated condition (-) of ‘old’ CENP-A-SNAP intensities after auxin (IAA) mediated depletion of SENP6 at different stages of the cell cycle following the experimental scheme of e. i Experimental scheme of cell cycle synchronization in G1 phase coupled to CENP-A-SNAP quench-chase-pulse and auxin (IAA) mediated depletion of SENP6. EG5 inhibitor Dimethylenastron was used for a mitotic arrest followed by release into G1. j Quantification as in f–h of ‘new’ CENP-A-SNAP intensities in G1 stage of the cell cycle after auxin (IAA) mediated depletion of SENP6 following the experimental scheme in i. Source data are provided as a Source Data file.

Fig 3: CENP-A overexpression promotes distinct cell fates depending on p53 status.CENP-A overexpression reprograms cell fate with distinct effects on cell state and cell identity that depend on p53 status. Perturbation (a) by CENP-A overexpression (in blue) induces mitotic defects in both wild-type p53 (p53-WT, top panel blue) and p53-defective cells (p53-DN, dominant negative, bottom panel green). These defects provoke distinct cell fate decisions according to p53 status, impacting cell state (b) or identity (c). When p53 is functional, cell state shifts toward acute cell cycle arrest and senescence, reducing self-renewal capacity. Additional stress, like DNA damage from X-irradiation, amplifies this response, resulting in radiosensitivity. Furthermore, functional p53 ensures the preservation of epithelial identity. In contrast, when p53 is defective, the cells evade arrest and continue cycling, allowing CENP-A overexpression to promote epithelial–mesenchymal transition (EMT). * symbol: reprogramming stimulated by CENP-A overexpression.

Fig 4: FACS-based strategy to generate a CENP-A knockout allele.(A) Schematic to scale outlining targeting strategy. Correct targeting of the rAAV transduced construct results in the in-frame fusion of EYFP after the first codon of exon 3 and the deletion of the remainder of exon 3 and exon 4. polyA: SV40 poly adenylation signal. (B) Targeting of CENP-A-EYFP knockout (CAtail-EYFP) construct into human hTERT-RPE cells. FACS profiles of the 1st (enrichment) and 2nd (purification) runs are shown. Gates used to collect EYFP positives are indicated. Number of cells collected per total number of cells analyzed is indicated for each run. (C) Live cell differential interference contrast (DIC) and epifluorescent (EYFP) image is shown for a representative monoclonal isolate. Note the loss of centromeric signal and resulting diffuse nuclear EYFP fluorescence. (D) PCR verification of CENP-A knockout. Left and right flanks were analyzed analogous to Figure 3B. Right panel (across): A PCR using two primers flanking the targeted domain resulting in the amplification of both the targeted knockout allele (KO) as well as the untargeted wild type allele (WT). Sizes of marker fragments (M) and of expected products are indicated. All primer numbers correspond to those listed in methods (E) Immunoblot showing targeted CENP-A knockout clone and parental wild type hTERT-RPE cells. Anti-CENP-A antibody reveals a novel high molecular weight species in which a 70 amino acid N-terminal fragment of CENP-A is fused to EYFP (CAtail-EYFP).

Fig 5: FACS-based strategy to isolate fluorescent knockin clones.(A) Targeting of CENP-A-EYFP knockin construct into human hTERT-RPE or HeLa cells. FACS profiles of the 1st (enrichment) and 2nd (purification) runs are shown. Gates used to collect EYFP positives are shown. Number of cells collected per total number of cells analyzed is indicated for each run. (B) Live cell differential interference contrast (DIC) and epifluorescent (EYFP) image is shown for representative monoclonal isolates for hTERT-RPE (top) and HeLa (bottom). (C) FACS profiles of parental (non-EYFP fluorescent) hTERT-RPE and HeLa cells with gate setting used for purification-mode sorting of rAAV infected cells. Positive hits detected under these conditions represent background fluorescence.