Fig 2: Proximity-dependent biotin identification (BioID) of ATRX-associating proteins.A: Experimental pipeline. The BirA* biotin ligase was fused to ATRX to biotinylate proximal proteins. Labeled proteins were captured on streptavidin beads and analyzed by mass spectrometry. B: Dot plot showing prey proteins identified with ATRX-BirA* that were enriched over endogenous biotinylation (untransfected) and unspecific pan-cellular biotinylation (BirA* alone—BFDR = 5%, SAINT [53]). Data represent two biological replicates. Proteins in boldface remained statistically enriched when the nuclear localization signal (NLS)-BirA* control was used to further filter the ATRX BioID data. C-D: Proximity-ligation assay (PLA) showing proximal associations between endogenous proteins in the MO3.13 human glial cell model. PLA data plots account for ~100 nuclei in three independent experiments, with the exact number of nuclei assessed indicated in brackets. The p-values were obtained using a 2-sided Student’s t-test with unequal variance. Scale bars = 4 µm.

Fig 3: ATRX proteome overview and proposed model.A: Protein associations with ATRX, FAM207A, and CCDC71. Pale grey circles denote previously reported associations, while dark red circles represent newly identified proximal associations. Blue circles represent previously reported associations also identified by our BioID. Image was built using ProHits Viz [104] and the BioGrid database [52], and manually organized. B: Model proposing new biological roles for ATRX (other than histone deposition), mediated through proximal associations with FAM207A, CCDC71, and SLF2.

Fig 4: Loss of ATRX changes the enrichment of a subset of proteins at telomeres.A: Correlation between BioID bait proteins. RAP1 BioID experiments strongly correlated with one another independently of ATRX expression. B: Relative ATRX detection by RAP1 BioID in ATRX-expressing and isogenic cells where ATRX expression was knocked-down (KD) using shRNA or knocked-out (KO) using CRISPR-Cas9. C: Fold change of prey proteins identified by RAP1 BioID (BFDR = 5%, SAINT [103]) in ATRX-expressing vs. ATRX-null HEK293 Flp-In cells. Labeled proteins had =2.5 average spectra and =1.5-fold change between conditions. Proteins labeled in the scatter plot remained significant when NLS-BirA* was used as a control. Inset box represents additional proteins identified with NLS-BirA* as a bait (less stringent).

Fig 5: Loss of ATRX and SLF2 enables telomere exchanges.A: DNA sequencing and western blotting confirming the disruption of ATRX expression in CRISPR-Cas9-edited HEK293 Flp-In T-REx cells inducibly expressing RAP1-BirA*. B: Relative signal overlap between immunolabeled SLF2 and telomere fluorescence in situ hybridization (IF-FISH), in ATRX-expressing or -null HEK293 Flp-In cells. Cells were exposed to 10 µM pyridostatin (PDS, G4 stabilizer) for 60 min. At least 40 cells per condition were analyzed using a CellProfiler colocalization pipeline [106]. ATRX loss decreased SLF2 recruitment to telomeres in the absence of pyridostatin. C: Schematic of dual-colour chromosome-orientation fluorescence in situ hybridization (CO-FISH) [64] using PNA probes to label C- and G-rich telomere strands (left). Examples of normal CO-FISH signals and of a chromosome end with a telomere exchange are shown (right). D: Relative amount of telomere exchanges in ATRX, SLF2, and ATRX/SLF2 KO HEK293 Flp-In cells. At least 30 mitotic spreads per condition were counted and the percent of telomeric exchanges plotted. p-values were obtained using a 2-sided Student’s t-test with unequal variance.