MOESM1 of High-resolution mapping of centromeric protein association using APEX-chromatin fibers
Creators: name: Kyriacou, Eftychia
name: Heun, Patrick
Publisher: name: figshare
Year: 2018
Subjects: subject: Biophysics
subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cell Biology
subject: Molecular Biology
subject: Evolutionary Biology
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: 111714 Mental Health
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: Hematology
subject: 60506 Virology

description: Additional file 1. Figure S1. (A) Representative images of mitotic chromosomes from untransfected U2OS cells or cells expressing APEX-CENP-A following induction with H2O2, stained for myc, biotin and CENP-A. Insets represent threefold magnifications of the boxed regions. Scale bar: 5 μm. (B) Immunoblot of protein extracts from cells transiently expressing APEX-CENP-A and untransfected U2OS cells, using an antibody against CENP-A. The bottom panel shows Ponceau staining of the blot. (C) Schematic for the analysis of plot profiles of extended chromatin fibers prepared with salt-detergent lysis buffer. Hypothetical intensity plot (endogenous CENP-A in green, biotin in red). The dashed gray line depicts the gray value = 300 threshold. (i) Each peak with a gray value ≥ 300 was accounted. The number of peaks was calculated for each staining. To correct for the size of different centromeres, the ratio of the number of biotin to endogenous CENP-A peaks was always calculated. (ii) Measurement of distances between closest peaks. The green arrow-headed line (dCA) depicts the distance between a CENP-A peak and its closest biotin peak while the red arrow-headed line (db) depicts the distance between a biotin peak and its closest CENP-A peak. The above distances were measured for each peak. If the distance between two peaks was ≤ 0.192 μm, they were marked as “co-localizing” peaks. These data allowed the calculation of the percentage of total biotin or CENP-A peaks co-localizing. (iii) The size of the domain covered by CENP-A (centromere domain) or biotin was determined by measuring the distance between the first and last peak of each staining. To correct for the size of different centromeres, the ratio of biotin to centromere domain size was always calculated. (iv) For calculating the distance of spreading of biotin peaks outside the centromere domain (ds) the distance of the furthest biotin peak from the first CENP-A peak (left and right) was measured. If ds was ≤ 0.192 μm (co-localizing with CENP-A) or if no peaks were found outside the centromere domain, ds was set to zero. (D) Distribution of biotin peaks from cells expressing APEX-CENP-A (mean with SEM) which were used as a reference for downstream analyses. Dark gray: percentage of peaks co-localizing with CENP-A, light gray: percentage of peaks not co-localizing with CENP-A inside the CENP-A-bound domain, black: percentage of peaks found outside the CENP-A domain. n = 42 fibers from 4 experiments. Figure S2. (A-C) Representative images of settled U2OS cells expressing APEX-CENP-C, CENP-N-APEX or APEX-CENP-T, respectively following induction with H2O2, stained for myc, biotin and centromere marker. Insets represent threefold magnifications of the boxed regions. Scale bar: 5 μm. (D-F) Mean ratios of the number of biotin peaks to CENP-A peaks on chromatin fibers from cells expressing CENP-C, CENP-N and CENP-T APEX fusion proteins, as compared to APEX-CENP-A. Not significant FDR adjusted Kolmogorov–Smirnov p values &gt; 0.05 are represented as ns. n = 42 fibers for CENP-A (from 4 experiments), 27 for CENP-C (from 4 experiments), 28 for CENP-N (from 3 experiments) and 15 for CENP-T (from 2 experiments). Error bars: SD. Figure S3. (A-C) Representative images of settled U2OS cells expressing APEX-CENP-P, CENPK-APEX or CENP-M-APEX, respectively following induction with H2O2, stained for myc, biotin and centromere marker. Insets represent threefold magnifications of the boxed regions. Scale bar: 5 μm. (D-F) Mean ratios of the number of biotin peaks to CENP-A peaks on chromatin fibers from cells expressing CENP-P, CENP-K or CENP-M APEX fusion proteins, as compared to APEX-CENP-A. Not significant FDR adjusted Kolmogorov–Smirnov p values &gt; 0.05 are represented by ns. n = 42 fibers for CENP-A (from 4 experiments), 16 for CENP-P (from 2 experiments), 13 for CENP-K (from 3 experiments) and 10 for CENP-M (from 2 experiments). Error bars: SD. Figure S4. (A,E) Representative images of settled U2OS cells expressing CENP-I-APEX or APEX-CENP-B, respectively following induction with H2O2, stained for myc, biotin and centromere marker. Insets represent threefold magnifications of the boxed regions. Scale bar: 5 μm. (B) Representative images of mitotic chromosomes from untransfected U2OS cells or cells expressing CENP-I-APEX following induction with H2O2, stained for CENP-I, biotin and CENP-A. Insets represent threefold magnifications of the boxed regions. Scale bar: 5 μm. (C) Immunoblot of protein extracts from cells transiently expressing CENP-I-APEX and untransfected U2OS cells, using an antibody against CENP-I. The bottom panel shows Ponceau staining of the blot. (D,G) Mean ratios of the number of biotin peaks to CENP-A peaks on chromatin fibers from cells expressing CENP-I or CENP-B APEX fusion proteins, as compared to APEX-CENP-A. FDR adjusted Kolmogorov–Smirnov p values ≤ 0.05 are represented by *. n = 42 fibers for CENP-A (from 4 experiments), 24 for CENP-B (from 5 experiments) and 30 for CENP-I (from 5 experiments). Figure S5. (A-C) Representative images of chromatin fibers prepared from U2OS cells using the TEEN buffer and stained for CENP-A and either CENP-C (A), CENP-H (B) or CENP-T (C). Scale bar: 2.5 μm. Intensity plots for CENP-A and CENP-C, CENP-H or CENP-T gray values along the length of the fiber (in μm) are shown on the right. (D, F) Representative images of chromatin fibers prepared using the TEEN buffer from untransfected U2OS cells and cells expressing CENP-I-APEX (D) or APEX-CENP-B (F) following induction with H2O2 in the presence of biotin phenol, and stained for CENP-A, biotin and CENP-I or CENP-B, respectively. Scale bar: 2.5 μm. Intensity plots for CENP-A, biotin and CENP-I/B gray values along the length of the fiber (in μm) are shown on the right. (E, G) Scatter plots depicting the size of biotin and CENP-I (E) or CENP-B (G) domain in untransfected U2OS cells on chromatin fibers prepared with TEEN buffer. Each dot represents one fiber. Error bars represent mean with SD. For (E): n = 24 fibers from untransfected U2OS cells (CENP-I) and 38 fibers from cells expressing CENP-I-APEX (biotin) from two experiments. For (G): n = 38 fibers from untransfected U2OS cells (CENP-B) and 40 fibers from cells expressing APEX-CENP-B (biotin) from two experiments. FDR adjusted Kolmogorov–Smirnov p values are displayed in the graphs