Supplemental Figure 1 from The Pluripotency Regulator PRDM14 Requires Hematopoietic Regulator CBFA2T3 to Initiate Leukemia in Mice

Western blot results of 2.5% input from transfected HA-CBFA2T3 constructs shown at two exposures.</p

Supplemental Table 1 from The Pluripotency Regulator PRDM14 Requires Hematopoietic Regulator CBFA2T3 to Initiate Leukemia in Mice

Mass spectrometry results listing LFQs, significance values and gene names.</p

CCDC71 is a chromatin-bound protein that associates with HP1.

A: Hierarchical catalogue of orthologs (OrthoDB v10.1) [56] diagram showing 262 putative genes within the CCDC71/71L family. B: Graphical depiction of the CCDC71 primary structure and features identified using Pfam [110] and Prosite [111]. Putative PxVxL-like motifs are also shown. The CCDC71/71L domain is shown in light grey and putative disordered regions in dark red. C: Sequence analysis of 254 CCDC71 orthologs aligned around the two PxVxL-like motifs. The sequence alignment was generated using COBALT [112] and visualized using WebLogo [113]. D: Subcellular fractionation of MO3.13 cells expressing FLAG-CCDC71. E: Immunolabeling of exogenous CCDC71 in HEK293 Flp-In and MO3.13 cells. F: Fold enrichment of CCDC71 BioID prey (BFDR ≤ 5%, SAINT [103]) over negative controls. Data were obtained from biological duplicates. G: Gene ontology terms represented in the CCDC71 BioID. Scale bars = 4 μm.</p

Data for plots other than BioID.

(XLSX)</p

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).</p

Changes at telomeres caused by a loss of SLF2 and ATRX.

Fold change of prey proteins identified by RAP1 BioID (BFDR ≤ 5%, SAINT [103]) in A: SLF2-null, and B: ATRX/SLF2-null vs. ATRX- and SLF2-expressing 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).</p

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.</p

Co-localization of SLF2 and telomeres, SLF2-null HEK293 Flp-In T-REx cells, and telomere exchanges in U2OS cells.

A: Western blot showing exogenous myc-SLF2 expression in lentivirus-infected HEK293 Flp-In T-REx (with inducible RAP1-BirA*, but the latter is not induced). B: Immunolabeling of myc-SLF2 (yellow) and SMC5 (red), and fluorescence in situ hybridization (IF-FISH) using a telomeric (green) probe in ATRX-expressing and -null HEK293 Flp-In cells. C: CRISPR-Cas9-mediated SLF2 gene disruptions in HEK293 Flp-In T-REx (RAP1-BirA*), detected by DNA sequencing, and matching qRT-PCR (D). E: Telomere exchange rates observed in U2OS cells. At least 30 mitotic spreads per condition were counted and the percent of telomeric exchanges plotted. F: Full western blots for Fig 4A. The red boxes indicate the areas shown in the main figure. (PDF)</p

FAM207A is a nucleolar protein involved in ribosome biogenesis.

A: Subcellular fractionation of MO3.13 cells expressing FLAG-FAM207A. B: Immunolabeling of exogenous FAM207A in HEK293 Flp-In and MO3.13 cells. C: Fold enrichment of FAM207A BioID prey (BFDR ≤ 5%, SAINT [103]) over negative controls. Data were obtained from biological duplicates. D: Gene ontology terms represented in the FAM207A BioID. Cy—cytoplasm; Nu—nucleus; Ch—chromatin. Scale bars = 4 μm.</p

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.</p