High-resolution mapping of centromeric protein association using APEX-chromatin fibers

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 > 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 > 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 graph

Structural basis for centromere maintenance by Drosophila CENP-A chaperone CAL1

Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of histone variant CENP‐A‐containing nucleosomes. To preserve centromere identity, CENP‐A must be escorted to centromeres by a CENP‐A‐specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance, highlighting the plasticity of this process. In humans, CENP‐A recognition and centromere targeting are achieved by HJURP and the Mis18 complex, respectively. Using X‐ray crystallography, we here show how Drosophila CAL1, an evolutionarily distinct CENP‐A histone chaperone, binds both CENP‐A and the centromere receptor CENP‐C without the requirement for the Mis18 complex. While an N‐terminal CAL1 fragment wraps around CENP‐A/H4 through multiple physical contacts, a C‐terminal CAL1 fragment directly binds a CENP‐C cupin domain dimer. Although divergent at the primary structure level, CAL1 thus binds CENP‐A/H4 using evolutionarily conserved and adaptive structural principles. The CAL1 binding site on CENP‐C is strategically positioned near the cupin dimerisation interface, restricting binding to just one CAL1 molecule per CENP‐C dimer. Overall, by demonstrating how CAL1 binds CENP‐A/H4 and CENP‐C, we provide key insights into the minimalistic principles underlying centromere maintenance

Drosophila SWR1 and NuA4 complexes are defined by DOMINO isoforms

Histone acetylation and deposition of H2A.Z variant are integral aspects of active transcription. In Drosophila, the single DOMINO chromatin regulator complex is thought to combine both activities via an unknown mechanism. Here we show that alternative isoforms of the DOMINO nucleosome remodeling ATPase, DOM-A and DOM-B, directly specify two distinct multi-subunit complexes. Both complexes are necessary for transcriptional regulation but through different mechanisms. The DOM-B complex incorporates H2A.V (the fly ortholog of H2A.Z) genome-wide in an ATP-dependent manner, like the yeast SWR1 complex. The DOM-A complex, instead, functions as an ATP-independent histone acetyltransferase complex similar to the yeast NuA4, targeting lysine 12 of histone H4. Our work provides an instructive example of how different evolutionary strategies lead to similar functional separation. In yeast and humans, nucleosome remodeling and histone acetyltransferase complexes originate from gene duplication and paralog specification. Drosophila generates the same diversity by alternative splicing of a single gene

The paracentric inversion In(2Rh)PL alters the centromeric organization of chromosome 2 in Drosophila melanogaster

Centromeres are complex structures involved in an evolutionarily conserved function, the correct segregation of chromosomes and chromatids during meiosis and mitosis. The centromere is determined by epigenetic processes that result in a particular nucleosome organization (CEN chromatin) that differs from the rest of the chromatin including the heterochromatin that normally surrounds the centromere in higher organisms. Many of the current models of centromere origin and organization rely on the molecular and cytological characterization of minichromosomes and their derivatives, and on studies on the origin and maintenance of neocentromeres. Here, we describe the peculiar centromere organization observed in In(2Rh)PL, a paracentric D. melanogaster inversion in which the centromere is maintained in its natural context but is directly flanked by a euchromatic domain as a result of the rearrangement. We have identified the breakpoints of the inversion and show that the proximal one is within the centromere region. The data presented suggest that, notwithstanding the loss of all the pericentric 2Rh heterochromatin, the centromere of the In(2Rh)PL chromosome is still active but presents a nucleosomal organization quite different from the organization usually observed in the centromeric region

Identification of Drosophila centromere associated proteins by quantitative affinity purification-mass spectrometry

AbstractCentromeres of higher eukaryotes are epigenetically defined by the centromere specific histone H3 variant CENP-ACID. CENP-ACID builds the foundation for the assembly of a large network of proteins. In contrast to mammalian systems, the protein composition of Drosophila centromeres has not been comprehensively investigated. Here we describe the proteome of Drosophila melanogaster centromeres as analyzed by quantitative affinity purification-mass spectrometry (AP-MS). The AP-MS input chromatin material was prepared from D. melanogaster cell lines expressing CENP-ACID or H3.3 fused to EGFP as baits. Centromere chromatin enriched proteins were identified based on their relative abundance in CENP-ACID–GFP compared to H3.3-GFP or mock affinity-purifications. The analysis yielded 86 proteins specifically enriched in centromere chromatin preparations.The data accompanying the manuscript on this approach (Barth et al., 2015, Proteomics 14:2167-78, DOI: 10.1002/pmic.201400052) has been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000758

CAL1 is the Drosophila CENP-A assembly factor

Centromeres are specified epigenetically by the incorporation of the histone H3 variant CENP-A. In humans, amphibians, and fungi, CENP-A is deposited at centromeres by the HJURP/Scm3 family of assembly factors, but homologues of these chaperones are absent from a number of major eukaryotic lineages such as insects, fish, nematodes, and plants. In Drosophila, centromeric deposition of CENP-A requires the fly-specific protein CAL1. Here, we show that targeting CAL1 to noncentromeric DNA in Drosophila cells is sufficient to heritably recruit CENP-A, kinetochore proteins, and microtubule attachments. CAL1 selectively interacts with CENP-A and is sufficient to assemble CENP-A nucleosomes that display properties consistent with left-handed octamers. The CENP-A assembly activity of CAL1 resides within an N-terminal domain, whereas the C terminus mediates centromere recognition through an interaction with CENP-C. Collectively, this work identifies the “missing” CENP-A chaperone in flies, revealing fundamental conservation between insect and vertebrate centromere-specification mechanisms