Kyriacou and Heun Epigenetics & (2018) 11:68 https://doi.org/10.1186/s13072-018-0237-6 Epigenetics & Chromatin

METHODOLOGY Open Access High‑resolution mapping of centromeric association using APEX‑chromatin fbers Eftychia Kyriacou1,2,3 and Patrick Heun3*

Abstract Background: The is a specialized chromosomal that forms the basis for the assembly of a multi- protein complex, the kinetochore and ensures faithful segregation during every cell division. The repetitive nature of the underlying centromeric sequence represents a major obstacle for high-resolution mapping of protein binding using methods that rely on annotated . Here, we present a novel microscopy-based approach called “APEX-chromatin fbers” for localizing protein binding over the repetitive centromeric sequences at kilobase resolution. Results: By fusing centromere factors of interest to ascorbate peroxidase, we were able to label their binding profles on extended chromatin fbers with biotin marks. We applied APEX-chromatin fbers to at least one member of each CCAN complex, most of which show a localization pattern diferent from CENP-A but within the CENP-A deline- ated centromeric domain. Interestingly, we describe here a novel characteristic of CENP-I and CENP-B that display extended localization beyond the CENP-A boundaries. Conclusions: Our approach was successfully applied for mapping protein association over centromeric chroma- tin, revealing previously undescribed localization patterns. In this study, we focused on centromeric factors, but we believe that this approach could be useful for mapping protein binding patterns in other repetitive regions. Keywords: Centromere, Chromatin fbers, CENP-A, CCAN, Kinetochore, APEX

Introduction CENP-A is a H3-variant and considered to Te centromere is a specialized chromosomal locus that be the epigenetic mark for centromere identity as it has serves as the platform for the assembly of a multi-protein been shown to be sufcient for the specifcation and the complex known as the kinetochore [1]. Te kinetochore epigenetic propagation of the centromere [4–8]. Cen- constitutes the structural element that mediates the tromeric chromatin is composed of interspersed blocks interaction with microtubules, in order to ensure faith- of CENP-A and H3 [9] and is decorated ful chromosome segregation during every cell division by both euchromatic and heterochromatic chromatin [2]. At its base lies centromeric chromatin containing the marks, while it is fanked by pericentric centromere-specifc histone CENP-A and the constitu- [10–12]. tive centromere-associated network of (CCAN), The CCAN consists of 16 proteins which can be and both together form the inner kinetochore. In , grouped into five different sub-complexes: the CENP- the CCAN serves as a binding platform for outer kine- H/I/K/M, the CENP-L/N, CENP-O/P/Q/U/R, CENP- tochore proteins that mediate direct interactions with T/W/S/X complexes, and CENP-C [2, 3]. CENP-C microtubules [2, 3]. plays a central role in the CCAN since it can directly bind both the CENP-A and members of the outer kinetochore complex [7, 13–19]. CENP- *Correspondence: [email protected] N was also shown to directly bind the CENP-A-con- 3 Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, taining nucleosome, while all the CCAN members UK Full list of author information is available at the end of the article together are thought to create a complex meshwork of

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kyriacou and Heun Epigenetics & Chromatin (2018) 11:68 Page 2 of 17

interactions important both for sustaining the kine- Results tochore structure and function but also involved in the A novel approach for mapping protein association propagation of the epigenetic mark by recruiting fac- at repetitive sequences tors responsible for the incorporation of new CENP-A Te extended chromatin fbers method was proven ben- [2, 3]. efcial for elucidating the organization of centromeric Even though in most organisms are chromatin in interspersed blocks of CENP-A and post- epigenetically defined, and no specific DNA sequence translationally modifed H3 nucleosomes [9, 10]. We seems to be sufficient to drive centromere forma- decided to take advantage of this established method and tion, CENP-B is a protein constitutively present at all develop it further in order to decipher the overall spa- human and mouse centromeres, except for the Y-chro- tial organization of centromere-associated proteins over mosome, by binding a specific 17-bp DNA motif, centromeric chromatin. For the preparation of chroma- termed the CENP-B box [20–23]. Recent reports high- tin fbers, a lysis bufer containing high concentration light the requirement of CENP-B for the stabilization of salt and detergent (salt-detergent lysis bufer) [31] or of both CENP-A and CENP-C at centromeres [5, 24, the less disruptive low-ionic strength TEEN bufer has 25]. Notably, CENP-B was also shown to be required been used to allow for breaking up nuclei and the sub- for the successful assembly of human artificial chro- sequent stretching of chromatin fbers [30, 32–34]. In mosomes [26, 27], pointing toward an important role this study, we further introduced an extra lysis step in in centromere establishment. the salt-detergent bufer prior to fxation and achieved Despite the fact that remarkable advances have been better stretching of the centromeric fber, since longer made in understanding the interactions of CCAN stretches of CENP-A were obtained (Fig. 1a). Each of the members and centromeric chromatin, these have been three methods (TEEN bufer, single and double lysis with mainly restricted to the nucleosomal level, namely a salt-detergent lysis bufer) was analyzed for their extent view of a dinucleosome H3-CENP-A particle and its of stretching and preservation of centromere factor stain- interactions with different CCAN members [2]. Other ing. While TEEN bufer allows for diferent centromeric studies have focused on the super-resolution organiza- proteins like CENP-B and CENP-C to be retained on tion of the kinetochore at the highly compacted meta- the chromatin fber, it achieves only relatively short fb- phase [28, 29] or extended kinetochore ers, as shown by the domain size occupied by CENP-A fibers [30]. However, how the CCAN members are (Fig. 1a, b). In turn, the higher degree of stretching and organized over the centromeric chromatin domain rel- resolution of the double lysis approach comes at the cost ative to CENP-A remains unclear. This is mainly due of stripping most non-nucleosomal centromere proteins, to the repetitive nature of centromeric sequences that including the members of the CCAN complexes, from does not allow the high-resolution mapping of CCAN the chromatin fber, similar to the single lysis using the and other centromeric proteins at endogenous cen- salt-detergent bufer (Fig. 1b). tromeres using conventional methods, such as chro- To overcome this problem and inspired by the DamID matin immunoprecipitation (ChIP) followed by DNA method [35], we took advantage of an existing proxim- sequencing, that rely on annotated genomes. ity-dependent protein labeling method that employs Here, we developed a novel microscopy-based the enzyme Ascorbate Peroxidase 2 (APEX) and devel- approach, which we call “APEX-chromatin fibers”, for oped a novel microscopy-based approach to map high-resolution mapping of centromeric proteins onto protein–chromatin association, which we call “APEX- centromeric chromatin. Using proximity-mediated chromatin fbers”. More specifcally, we fused our pro- ligation of biotin, this method allowed us to indirectly teins of interest to the engineered APEX which oxidizes map the association sites of these proteins on chro- phenol derivatives, like biotin-phenol, in the presence matin fibers in relation to CENP-A. We systematically of ­H2O2 [36, 37] and produces highly reactive molecules mapped members of all the CCAN complexes and that can attack electron-rich amino acids of proteins in describe novel localization patterns at centromeres. very close proximity (< 20 nm) [38]. Combined with Importantly, we demonstrate that both CENP-B and the extended chromatin fber preparation, this method CENP-I, unlike all the other tested CCAN mem- should allow to indirectly visualize the localization of bers, expand beyond the CENP-A-bound centromere proteins of interest fused to APEX by detecting bioti- domain. While here we focused on centromeric fac- nylation of salt-resistant chromatin proteins (Fig. 2a). tors, we believe that our approach could be a useful Terefore, we transiently expressed centromeric pro- tool for mapping protein association in other repeti- teins fused to myc-APEX (about 28 kDa) in U2OS cells tive genomic regions. for three days, induced biotinylation through addition of biotin-phenol and ­H2O2, and prepared double lysis Kyriacou and Heun Epigenetics & Chromatin (2018) 11:68 Page 3 of 17

tag does not infuence CENP-A localization. Next, we a CENP-A domain( m) 0510 15 20 prepared extended chromatin fbers and compared the

CENP-A CENP-A and biotin profle over the centromere region TEEN buffer (Fig. 2c). As expected, no biotinylation was observed in the DMSO-treated preparations, while the bioti-

r CENP-A Single lysis nylation profle in the biotin-phenol-treated sample

CENP-A revealed a large overlap with the CENP-A staining. For

lysis buffe Double lysis

Salt-detergen t more detailed analysis of the biotinylation profle, we focused on the intensity profle of the staining along the fber (Additional fle 1: Figure S1C, Materials and Meth- b ods). We found that about 70% of total CENP-A peaks r CENP-A CENP-A co-localize with biotin, while 86% of the total biotin peaks co-localize with CENP-A (Fig. 2d). We also ana- CENP-B CENP-C

EEN buffe lyzed the distribution of the CENP-A-mediated biotin

MERGE MERGE peaks and found that only 12.7% do not co-localize with CENP-A, and a minor fraction (1.3%) are found outside

sT CENP-A CENP-A the centromere reference domain (Additional fle 1: si r Figure S1D). Together, the above results show that the CENP-B CENP-C biotinylation profle on the chromatin fbers from cells

Single ly expressing APEX-myc-CENP-A is very similar to the MERGE MERGE pattern of endogenous CENP-A, despite a noticeable amount (14%) of biotin signals not perfectly co-local- s CENP-A CENP-A si izing with CENP-A, which could be considered as the

CENP-B CENP-C error of the method. Hence, for each APEX fusion pro- Salt-detergent lysis buffe tein that we subsequently analyzed, we used the overlap Double ly MERGE MERGE of APEX-CENP-A-mediated biotinylation relative to Fig. 1 Comparison of diferent methods for chromatin fber endogenous CENP-A as the reference for comparison. preparation. a Scatter plot depicting the size of CENP-A domain on In order to address how the CCAN proteins are organ- chromatin fbers prepared with three diferent methods (left panel). ized overall over centromeric chromatin, we applied Each dot represents one fber. Error bars represent mean with SD. n 32, 28 and 37 fbers for TEEN bufer, single lysis step and double APEX-chromatin fbers and analyzed the resulting bioti- = lysis , respectively, from one experiment. Representative pictures of nylation profles. For each CCAN complex (CENP-C, fbers (right panel). Scale bar: 2.5 μm. b Representative pictures of CENP-H/I/K/M, CENP-L/N, CENP-O/P/Q/U/R and chromatin fbers prepared with three diferent methods and stained CENP-T/W/S/X), we mapped at least one member to for CENP-A and either CENP-B or CENP-C. Scale bar: 2.5 μm analyze the number of peaks and how they are distrib- uted over the centromere domain defned by CENP-A, allowing us to group them in diferent categories of dis- tribution patterns. extended chromatin fbers. Centromeres were marked by staining for CENP-A, and biotinylation was detected with Streptavidin conjugated to Alexa555 (Fig. 2a). CENP‑C, CENP‑N and CENP‑T APEX fusions are confned As a proof-of-principle, we started by analyzing inside the CENP‑A‑bound domain but are diferently APEX-myc-CENP-A and compared its biotinylation organized than APEX‑CENP‑A pattern to endogenous CENP-A, which itself is retained First, we mapped CENP-C, which lies at the foundation on chromatin fbers and can be used as a direct refer- of the kinetochore and is a key component of the CCAN. ence. We frst confrmed that the fusion protein is Following the confrmation that the APEX-CENP-C expressed and correctly targeted to the centromere fusion protein is expressed and correctly targeted to by IF on interphase cells and mitotic chromosomes, centromeres (Additional fle 1: Figure S2A), we analyzed and immunoblotting (Fig. 2b, Additional fle 1: Figure the biotinylation profle on the chromatin fbers in rela- S1A,B). Biotinylation was observed specifcally at the tion to CENP-A (Fig. 3a, b, Additional fle 1: Figure S2D). centromeres only in the presence of biotin-phenol and Interestingly, signifcantly fewer CENP-C-mediated bio- not DMSO (Fig. 2b, Additional fle 1: Figure S1A), indi- tin peaks co-localize with CENP-A when compared to cating that the labeling reaction is spatially restricted APEX-CENP-A (72% vs. 86%, Fig. 2b). Tis suggests that to the centromeric region, and that the myc-APEX a fraction of CENP-C localizes at places where CENP-A is absent. In addition, we found that CENP-C is organized Kyriacou and Heun Epigenetics & Chromatin (2018) 11:68 Page 4 of 17

a Immunodetection EX

APEX AP +biotin-phenol X

Transfections Extended chromatin fibers X /DMSO preparation

+H O 1min U2OS cells 2 2 centromere X: centromeric protein CENP-A phenoxy l-biotin Biotin

Streptavidin-Alexa555

b d DAPI myc Biotin ACA MERGE 125

100 +DMSO

75

APEX-myc-CENP-A 50 +biotin phenol 25

c Percentage of total peaks 0 CENP-A Biotin CENP-A 8000 CENP-A Biotin 6000 not co-localizing Biotin 4000 co-localizing +DMS O

Grey value 2000 MERGE

0 02468

CENP-A 10000 CENP-A 6000 Biotin 5000 8000 4000 APEX-myc-CENP-A 3000 Biotin 6000 2000 1500 4000 1000 MERGE Grey value 2000 +biotin phenol 500

0 0 0246810

Fig. 2 A novel approach for mapping protein binding over centromeric chromatin. a Schematic representation of the APEX-chromatin fbers

workfow. b Representative images of settled U2OS cells expressing APEX-myc-CENP-A, following induction with H­ 2O2, stained for myc, biotin and centromere marker. Insets represent threefold magnifcations of the boxed regions. Scale bar: 5 μm. c Representative images of chromatin fbers prepared from cells in b, stained for CENP-A and biotin. Scale bar: 5 μm. Intensity plots for CENP-A and biotin gray values along the length of the fber (in μm) are shown on the right. d Mean percentages of total CENP-A or biotin peaks showing co-localization with each other or not. Error bar: SEM. n 42 fbers from four experiments = in a similar number of peaks as APEX-CENP-A (Addi- (Fig. 3c, Additional fle 1: Figure S2B), we found that like tional fle 1: Figure S2D), which are confned inside the CENP-C, CENP-N is organized in a similar number of centromere domain. peaks as APEX-CENP-A (Additional fle 1: Figure S2E). We then mapped CENP-N, a member of the CENP- In addition, similar to CENP-C, the CENP-N-mediated L/N complex, which similarly to CENP-C was also found biotin peaks do not perfectly co-localize with CENP-A to be a direct binder of the CENP-A nucleosome [13, (only 66% of the total peaks shows co-localization with 39]. After analyzing the biotinylation profle on chroma- CENP-A) but are also all confned inside the centromere tin fbers prepared from cells expressing CENP-N-APEX domain (Fig. 3d). Kyriacou and Heun Epigenetics & Chromatin (2018) 11:68 Page 5 of 17

a b ns 10000 CENP-A 125 ns CENP-A 9000

8000 CENP-C-Biotin s 7000 * 6000 100 5000 4000 3000 Biotin 2000 75 2000 1500 50

Grey value 1000 MERGE 500 25 % of total biotin peak APEX-myc-CENP-C 0 012345 0 CENP-A CENP-C APEX fusion

c 30000 d CENP-A ns 125 25000 CENP-N-Biotin * CENP-A s ** 20000 100

15000 75 Biotin 10000 Grey value 50 5000

CENP-N-myc-APEX 0 25 MERGE 02468 % of total biotin peak 0 CENP-A CENP-N APEX fusion

f ns e 12000 CENP-A CENP-A 125 ns CENP-T-Biotin T 10000 * 100 8000 Biotin 6000 75

4000

Grey value 50 MERGE 2000 25 APEX-myc-CENP-

0 % of total biotin peaks 01234567891011 0 CENP-A CENP-T APEX fusion

outside CENP-A domain not co-localizing with CENP-A inside CENP-A domain co-localizing with CENP-A Fig. 3 CENP-C-, CENP-N- and CENP-T-APEX are organized diferently from APEX-CENP-A but are confned inside the centromere domain. Representative images of chromatin fbers prepared from cells expressing APEX-CENP-C (a), CENP-N-APEX (c) or APEX-CENP-T (e), stained for CENP-A and biotin. Scale bar: 2.5 μm. Intensity plots for CENP-A and biotin gray values along the length of the fber (in μm) are shown on the right. For presentation purposes, CENP-A gray values in APEX-CENP-T plot were subjected to a fvefold increase. Distribution of biotin peaks compared to APEX-CENP-A (mean with SEM), for CENP-C (b), CENP-N (d) and CENP-T (f). 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. FDR adjusted Kolmogorov–Smirnov p values are represented as follows: ns for p values > 0.05, * for p values 0.05 and ** for p ≤ values < 0.001. n 42 fbers for CENP-A (from four experiments), 27 for CENP-C (from four experiments), 28 for CENP-N (from three experiments) and = 15 for CENP-T (from two experiments) Kyriacou and Heun Epigenetics & Chromatin (2018) 11:68 Page 6 of 17

Localizing CENP-T to the centromere has produced CENP‑I and CENP‑B extend outside the centromere domain diferent results in diferent studies. While super-reso- We next focused on the localization of CENP-I on chro- lution imaging of stretched kinetochore fbers places it matin fbers. After confrming the expression of the closer to the H3 blocks composing centromeric chro- APEX-tagged protein and its specifc localization at matin [30], more recent fndings using ChIP followed centromeres (Fig. 5a, Additional fle 1: Figure S4A-C), by sequencing of young α-satellites place CENP-T in we analyzed its biotinylation pattern. Unlike CENP-K the center of the CENP-B box, between two CENP-A and CENP-M of the CENP-H/I/K/M complex, CENP-I nucleosomes [40]. It was also shown that it interacts exhibited a diferent distribution profle that falls in nei- both with CENP-B and CENP-C [40]. Our analysis of ther of the frst two categories. First, it is organized in the biotinylation profles shows that CENP-T is organ- more peaks than APEX-CENP-A over the centromere ized in similar number of peaks as APEX-CENP-A (Additional fle 1: Figure S4D) and a large proportion of (Fig. 3e, Additional fle 1: Figure S2C,F) but a smaller biotin peaks does not co-localize with CENP-A inside portion of those co-localize with CENP-A (72% vs. the centromere (Fig. 5b). Surprisingly, a signifcant per- 86%) (Fig. 3f). Similar to CENP-C and CENP-N, we centage of peaks (11.7%) are also located outside the also fnd CENP-T to be confned inside the centromere centromere domain and a large proportion of the fbers domain but not exclusively where CENP-A is (Fig. 3f). analyzed (> 50%) show this “spreading” pattern (Fig. 5b, Tis defnes our frst category of association pat- e). terns, in which proteins are confned within the CENP- In addition to the CCAN members, we also explored A domain, but reveal a pattern that is distinct from the high-resolution localization of CENP-B, a DNA-bind- APEX-CENP-A, and includes CENP-C, CENP-N and ing protein present at all human and mouse centromeres CENP-T. except for the Y-chromosome. It binds a motif termed the CENP-B box, which is present in the α-satellite sequences [21]. After confrming that the CENP-B-APEX CENP‑P, CENP‑K and CENP‑M APEX fusions are organized fusion protein is expressed, targeted and is specifcally similarly to APEX‑CENP‑A active at centromeres (Additional fle 1: Figure S4E,F), Other CCAN members presented diferent distribution we analyzed its biotinylation profle on chromatin fb- profles than the one discussed above. For the CENP-O/ ers (Fig. 5c). Similar to CENP-I, we found that CENP-B P/Q/U/R complex we focused only on CENP-P. A CENP- is organized in more peaks over centromeric chromatin P-APEX fusion was transiently expressed in U2OS cells when compared to APEX-CENP-A (Additional fle 1: (Additional fle 1: Figure S3A) and chromatin fbers were Figure S4G) and inside the centromere domain not all prepared (Fig. 4a). We then analyzed the biotinylation biotin peaks co-localize with CENP-A (Fig. 5d). Inter- profle of CENP-P and found that it is not signifcantly estingly, on the majority of fbers (> 70%), we fnd that a diferent from the biotinylation profle mediated by signifcant proportion of CENP-B-APEX-mediated biotin APEX-CENP-A (Fig. 4a, b, Additional fle 1: Figure S3D). peaks (16.2%) are located outside the centromere domain In particular, the ratio of biotin to CENP-A peaks is not (Fig. 5d, e). signifcantly diferent between the CENP-P and CENP- Our measurements revealed that both CENP-B- and A-APEX fusions (Additional fle 1: Figure S3D). Similarly, CENP-I-mediated biotin peaks spread signifcantly fur- the distribution of the biotin peaks over the centromere ther away from the CENP-A domain as compared to domain does not difer signifcantly (Fig. 4b). APEX-CENP-A (Fig. 5f). In agreement with this, the For the CENP-H/I/K/M complex, we mapped CENP- domain that is occupied by the CENP-I and CENP-B- I, CENP-K and CENP-M. CENP-K and CENP-M were mediated biotin is signifcantly larger than the respective also found to be organized in a similar pattern to APEX- domain corresponding to APEX-CENP-A (Fig. 5g). CENP-A (Fig. 4c–f, Additional fle 1: Figure S3B, C, E, In summary, by applying APEX-chromatin fbers for F). In particular, we fnd that neither the number of bio- mapping protein organization over centromeric chro- tin peaks mediated by CENP-K and CENP-M nor their matin, three diferent association patterns of centromere distribution is signifcantly diferent from the respective factors over centromeric chromatin were revealed number in cells expressing APEX-CENP-A (Additional (Fig. 5h). In the frst category, in which CENP-C, CENP- fle 1: Figure S3E-F, Fig. 4d, f). N and CENP-T fall, the centromeric factors are confned Tis defnes a second category of distribution pat- inside the CENP-A-bound centromere domain, but are terns, in which proteins are confned within the CENP-A organized diferently than the biotin marks deposited by domain and show no signifcantly diferent organization APEX-CENP-A. In the second category, CENP-P, CENP- from APEX-CENP-A, and includes CENP-P, CENP-M M and CENP-K are also confned inside the centromere and CENP-K. domain but their pattern is similar to the biotinylation Kyriacou and Heun Epigenetics & Chromatin (2018) 11:68 Page 7 of 17

a b 6000 CENP-A ns CENP-P-Biotin 125 ns 5000 ns CENP-A 4000 100

3000 75 Biotin 2000 Grey value 50 1000

APEX-myc-CENP-P 0 25 MERGE 02468% of total biotin peaks 0 CENP-A CENP-P APEX fusion

c 12000 CENP-A d ns CENP-K-Biotin 125 ns 10000 ns CENP-A 100 8000

6000 75 Biotin 4000

Grey value 50 2000 25 CENPK-myc-APEX

0 % of total biotin peaks MERGE 0 1234567 0 CENP-A CENP-K APEX fusion

e f 25000 CENP-A ns 125 CENP-M-Biotin ns ns 20000 CENP-A 100 15000 75 10000 Biotin Grey value 50 5000

CENP-M-myc-APEX 25 0 MERGE 012345 % of total biotin peaks 0 CENP-A CENP-M APEX fusion

outside CENP-A domain not co-localizing with CENP-A inside CENP-A domain co-localizing with CENP-A Fig. 4 CENP-P-, CENP-K and CENP-M-APEX are organized similarly to APEX-CENP-A. Representative images of chromatin fbers prepared from cells expressing APEX-CENP-P (a), CENP-K (c) or CENPM-APEX (e), stained for CENP-A and biotin. Scale bar: 2.5 μm. Intensity plots for CENP-A and biotin gray values along the length of the fber (in μm) are shown on the right. For presentation purposes, gray values for CENP-P-biotin were subjected to a twofold increase. Distribution of biotin peaks compared to APEX-CENP-A (mean with SEM), for CENP-P (b), CENP-K (d) and CENP-M (f). 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. Not signifcant FDR adjusted Kolmogorov–Smirnov p values > 0.05 are represented by ns. n 42 fbers for CENP-A (from four experiments), 16 for CENP-P (from two experiments), 13 for CENP-K (from three experiments) and 10 for = CENP-M (from two experiments) Kyriacou and Heun Epigenetics & Chromatin (2018) 11:68 Page 8 of 17

(See fgure on next page.) Fig. 5 CENP-B- and CENP-I-APEX extend further than the CENP-A-bound centromere domain. Representative images of chromatin fbers prepared from cells expressing CENP-I-APEX (a) or APEX-CENP-B (c), stained for CENP-A and biotin. Scale bar: 2.5 μm. Intensity plots for CENP-A and biotin gray values along the length of the fber (in μm) are shown on the right. For presentation purposes, CENP-A gray values in APEX-CENP-B plot were subjected to a fourfold increase. Distribution of biotin peaks compared to APEX-CENP-A (mean with SEM), for CENP-I (b) and CENP-B (d). 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. e Mean percentages of total fbers analyzed showing spreading for CENP-A, CENP-B and CENP-I APEX fusion proteins. FDR adjusted unpaired Student’s t test p values 0.05 are represented by *. n four experiments for CENP-A ≤ = and fve experiments for CENP-B and CENP-I APEX fusion proteins. Error bars: SD. f Scatter plot depicting the distance of spreading of biotin peaks from the CENP-A domain. Each fber is represented by two dots, for the distance of spreading left and right of the centromere domain. Error bars represent mean with SD. g Mean ratio of biotin to CENP-A domain size. Error bars: SD. FDR adjusted Kolmogorov–Smirnov p values are represented as follows: ns for p values > 0.05, * for p values 0.05 and ** for p values < 0.001. n 42 fbers for CENP-A (from four experiments), 24 for CENP-B ≤ = (from fve experiments) and 30 for CENP-I (from fve experiments). (H) Summary of the three association pattern categories. The frst category includes CENP-C, -N and -T, which are confned inside the centromere domain but are diferently organized than APEX-CENP-A. The second category includes CENP-P, -M and -K which are organized similarly to APEX-CENP-A. The last category includes CENP-I and CENP-B, which extend further than the centromere domain pattern deposited by APEX-CENP-A. In the last cate- spreading outside the centromere domain (Fig. 7a). In gory, CENP-I and CENP-B were found to extend beyond fact, most fbers show either CENP-B or CENP-I spread- the centromere domain. ing alone (29 and 42%, respectively) (Fig. 7a). Interest- ingly, in the vast majority of fbers with both proteins Endogenous CENP‑B and CENP‑I spread further spreading outside the centromere domain, CENP-B outside the CENP‑A‑bound domain and CENP-I are found together on the same side of the To confrm that the “spreading” pattern observed for centromere domain (70%) (Fig. 7b, c). Only in few cases CENP-B and CENP-I APEX fusions was not a result of CENP-B and CENP-I are located at the opposite sides of overexpression of the transgenic APEX constructs, we the centromere domain (12%), while for some (18%) the prepared fbers using the TEEN bufer that allows the two proteins are located together on one side and one retention of diferent centromeric proteins (Fig. 1b). By of the two (or both) is also found on the opposite side of using antibodies against the endogenous proteins, we the centromere domain (Fig. 7b). Tis suggests that when visualized CENP-B, CENP-I, CENP-C, CENP-H and both CENP-B and CENP-I spread outside the centromere CENP-T on TEEN fbers (Fig. 6a, b, Additional fle 1: Fig- domain, they tend to localize on the same side. ure S5A-C). In agreement with the “spreading” pattern observed using our APEX approach, we fnd that the dis- Discussion tance that endogenous CENP-B or CENP-I spread out- Te extended chromatin fbers method has proven to be side the CENP-A domain is signifcantly higher than the very useful to study the structural organization of cen- equivalent distance for CENP-C, CENP-H and CENP- tromeric chromatin [9, 10, 41]. While some protocols T (Fig. 6c). To test whether transient expression of the favor stretching of the chromatin fber and achieve bet- APEX-tagged proteins might exacerbate their spreading ter spatial resolution, their harsher lysis conditions come outside the CENP-A domain, we prepared fbers using at the expense of many non-nucleosomal proteins being the TEEN bufer from cells expressing either CENP-I or stripped from the chromatin. Accordingly, gentler lysis CENP-B APEX fusions (Additional fle 1: Figure S5D-G). conditions (e.g., TEEN bufer) allow the retention of chro- We did not observe a diference in the comparison for matin associated factors but result in shorter fbers. Tis CENP-I, indicating that expression of the transgene does led us to develop a method that achieves well-stretched not afect its spreading. For CENP-B, we noticed a slight chromatin fbers, yet maintains a read-out of protein increase in average domain size for CENP-B, suggesting association on the fber, using biotinylation of chromatin. that additional CENP-B has a small efect on expanding After successful establishment of the technique, we went further beyond the CENP-A domain than endogenous on to elucidate the overall spatial organization of mem- CENP-B does. bers of the CCAN over centromeric chromatin. To investigate whether CENP-B and CENP-I spread- Firstly, we were able to confrm the specifcity of our ing correlates, we used CENP-C as a marker for the cen- approach by directly comparing the binding patterns of tromere domain and simultaneously stained for both CENP-A and APEX-CENP-A-mediated biotin over cen- CENP-B and CENP-I. When we analyzed fbers that tromeric chromatin. As a control, we confrmed that in show spreading outside the centromere domain, we fxed interphase cells and mitotic chromosomes, bioti- found that only 29% show both CENP-B and CENP-I nylation is restricted to the centromere. As expected, we Kyriacou and Heun Epigenetics & Chromatin (2018) 11:68 Page 9 of 17

a b 125 CENP-A 32000 CENP-A * 22000 * CENP-I-Biotin 100 ** 12000 2000 Biotin 2000 75 1500 1000 50 Grey value MERGE 500

CENP-I-myc-APEX 25 0 0 2468101214161820 % of total biotin peaks 0 CENP-A CENP-I APEX fusion

c d 125 **ns s 100 ** CENP-A 15000 CENP-A 12500 CENP-B-Biotin 75

10000 50 Biotin 7500 25

5000 % of total biotin peak

Grey value 0 MERGE 2500 CENP-ACENP-B APEX-myc-CENP-B 0 APEX fusion 0246 outside CENP-A domain not co-localizing with CENP-A inside CENP-A domain co-localizing with CENP-A

efg g 6 100 * ** 2.0 ** 90 * 5 * 80 * 70 4 1.5 60 3 50 1.0 40 2 30 20 1 0.5 10 0 Biotin/CENP-A domai n 0 % of fibers showing spreadin 0.0 A I - CENP-A CENP-B CENP-I CENP-ACENP-B CENP-I

CENP CENP-B CENP- APEX fusion APEX fusion APEX fusion

h Category 1: CENP-C,- N and -T Confined in centromere domain but different distribution

Category 2: CENP-P, -K and -M Confined in centromere domain in a similar distribution

Category 3: CENP-I and -B Extend beyond the centromere domain

CENP-A centromeric protein Kyriacou and Heun Epigenetics & Chromatin (2018) 11:68 Page 10 of 17

Having confrmed the specifcity of our labeling a CENP-A 5000 CENP-A CENP-B approach, we mapped at least one member of each r 4000 CCAN complex. We found biotin deposited by CENP-P, CENP-B 3000 2000 -K and -M to be organized similarly to biotin deposited Grey value 1000 by APEX-CENP-A over centromeric chromatin. CENP-

TEEN buffe MERGE 0 P and the other members of the CENP-O/P/Q/U/R 012345 complex were described to be very close to centromeric chromatin in mitotic kinetochores [29], while all three proteins, CENP-P, CENP-K and CENP-M, were reported b CENP-A 30000 CENP-A 25000 CENP-I to be important for proper kinetochore function but r 20000 with no specialized role at the centromere. It was only CENP-I 15000 recently that the complex, and in particular CENP-Q, 10000 Grey value 5000 was shown to play an important role in microtubule

TEEN buffe MERGE 0 binding cooperatively with members of the outer kine- 01234 tochore [42]. Surprisingly, despite the fact that CENP-C and CENP-N c ** were reported to directly interact with CENP-A-contain- 3 ing nucleosomes [13, 17, 39, 43, 44], we found that nei- ther of the two overlaps perfectly with CENP-A inside the 2 centromere domain, but instead are also found in places

1 where CENP-A is not present. Our fnding might also point to the possibility that CENP-C or CENP-N interacts 0 with proteins other than CENP-A or the CCAN at centro- Distance of spreading from meric chromatin, which are yet to be identifed. CENP-C has been described to possess DNA-binding ability [45, CENP-C CENP-H CENP-T CENP-B CENP-I Fig. 6 Endogenous CENP-B and CENP-I extend further than the CENP-A 46]. It is, therefore, possible that following its targeting domain on TEEN fbers. Representative images of chromatin fbers to the centromere, it can associate with DNA in addition prepared from U2OS cells using the TEEN bufer and stained for CENP-A to its interaction with CENP-A nucleosomes, allowing and either CENP-B (a) or CENP-I (b). Scale bar: 2.5 μm. Intensity plots for it to be stably bound on centromeric chromatin even in CENP-A and CENP-B or CENP-I gray values along the length of the fber places where there is no CENP-A, e.g., after its distribu- (in μm) are shown on the right. c Scatter plot depicting the distance of spreading from the edges of the CENP-A domain for CENP-C, CENP-H, tion to the two sister following DNA replica- CENP-T, CENP-B and CENP-I. Each fber is represented by two dots, for tion. Likewise, CENP-N’s association with other CCAN the distance of spreading left and right of the centromere domain. Error members [44, 47] might confer stability for binding over bars represent mean with SD. FDR adjusted Kolmogorov–Smirnov p centromeric chromatin, despite the absence of CENP-A values < 0.001 are represented as **. Not signifcant p values > 0.05 are nucleosomes. not depicted. n 142 fbers for CENP-B (from three experiments), 160 = fbers for CENP-C (from two experiments), 189 fbers for CENP-H (from While super-resolution study of chicken kinetochore two experiments), 177 fbers for CENP-I (from two experiments) and 142 fbers [30], FRET analyses [48] and ChIP data [49] place fbers for CENP-T (from two experiments) CENP-T in close proximity to the H3-rich domains of the centromere domain, we fnd that a high percentage (72%) of CENP-T peaks co-localize with CENP-A. Tis could be explained by the fact that the CENP-A signals seen on the chromatin fbers contain several CENP-A nucleosomes observe strong overlap of the CENP-A and biotin staining [9] and CENP-T was found to co-immunoprecipitate in the chromatin fbers, with 86% of the CENP-A-medi- with CENP-A nucleosomes when chromatin was not fully ated biotin peaks overlapping with CENP-A. A minor digested [49–51]. In addition, it has recently been shown population (14%) of the biotin signals did not perfectly that in ChIP experiments carried out on young α-satellites, overlap with the CENP-A antibody staining, which could CENP-T constitutes a bridge between adjacent CENP-A be explained by higher sensitivity in the detection of bio- nucleosomes [40], suggesting that the two proteins are in tin as compared to CENP-A, and could be attributed as close proximity, in agreement with our fndings. the error of the method. We could further confrm that Interestingly, we found CENP-B and CENP-I to extend biotinylation was restricted inside the CENP-A-bound beyond the CENP-A-bound centromere domain. It is centromere domain, suggesting that unspecifc biotinyla- unclear what the role of these proteins is outside the tion is not introduced to the adjacent chromatin. CENP-A domain at centromeres, but their localization Kyriacou and Heun Epigenetics & Chromatin (2018) 11:68 Page 11 of 17

a 110 b CENP-B and CENP-I spreading on the same side 100 90 80 70 60 70% 18% 12% 50 40 30 CENP-B and CENP-I 20 spreading on opposite sides % of total fibers showing spreading 10 0

CENP-B+CENP-I CENP-I only CENP-B only

25000

CENP-C c CENP-C 20000 15000

10000 Grey valu e 5000

0 0.00.5 1.01.5 2.02.5 3.03.5 4.0 CENP-B 4000 r

3000 CENP-B

2000 Grey valu e CENP-I 1000

0 0.00.5 1.01.5 2.02.5 3.03.5 4.0 TEEN buffe 12500

10000 CENP-I MERGE 7500 5000 Grey valu e

2500

0 0.00.5 1.01.5 2.02.5 3.03.5 4.0

Fig. 7 CENP-B and CENP-I are found on the same side when they both spread. a Distribution of TEEN fbers showing spreading outside the centromere domain. Mean with SEM. Dark gray: fbers where only CENP-B spreads, light gray: fbers where only CENP-I spreads and black: fbers where both CENP-B and CENP-I spread. b Distribution of fbers where both CENP-B and CENP-I spread outside the centromere domain. Percentage is the mean of three experiments. A total of 182 fbers were analyzed. c Image of a fber stained for CENP-C, CENP-B and CENP-I, showing that CENP-B and CENP-I spread on the same side of the centromere domain. Scale bar: 2.5 μm. Intensity plots for each staining along the length of the fber (in μm) are shown on the right

could be implemented at the boundaries of the boustro- It has previously been proposed that CENP-B might phedon model [30], without necessarily afecting the be present outside the CENP-A-bound centromeric function of the kinetochore. domain, based on biochemical data that suggested that Kyriacou and Heun Epigenetics & Chromatin (2018) 11:68 Page 12 of 17

half the amount of CENP-B at centromeres is associated the CENP-A bound domain refects the chromosomes with canonical H3 and not CENP-A nucleosomes on that contain more than one adjacent α-satellite arrays the type-I α-satellite array [52]. CENP-B boxes are regu- containing CENP-B boxes which are not all bound by larly distributed across the α-satellite arrays and CREST CENP-A, like on [67]. antibodies, which recognize mostly CENP-B, displayed Despite the advantages of our approach, we also staining of an entire α-satellite array on low resolution acknowledge several limitations involved. Although chromatin stretches [20, 53, 54]. In contrast, CENP-A was the extended chromatin fbers method is the only avail- shown to bind only a fraction of the whole α-satellite array able method for higher resolution mapping of protein [55–57]. Tus, using an alternative approach, we confrm binding over repetitive regions, it undoubtedly does here the previous notion that CENP-B displays extended not achieve the resolution of ChIP coupled to sequenc- binding outside the centromere domain. Its localization ing methods. Here, we focused on proteins that exclu- beneath the kinetochore during mitosis further suggests sively localize to centromeres, which can be identifed that CENP-B has a specialized binding pattern over cen- and visualized by the specifc presence of CENP-A. tromeric chromatin [58]. CENP-B’s role is diverse and For proteins that display broad distribution across the among others, it is able to promote heterochromatin for- , the analysis of the binding patterns can be mation in ectopically integrated alphoid DNA in mouse challenging, in part due to the entanglement of sev- cells through interaction with Suv39h1 [27]. Its presence eral fbers. Moreover, our mapping was carried out in beyond the centromere domain might hint toward a role the presence of the endogenous protein, which possibly of CENP-B in regulating the boundary between centro- reduces the coverage of biotinylation. Indeed, we fnd meric chromatin and pericentric heterochromatin. How- that for APEX-CENP-A, a small proportion of CENP-A ever, it was unexpected that CENP-I extends further than does not co-localize with biotin, suggesting that there the CENP-A domain. Similar to CENP-B [27], CENP-I are endogenous CENP-A binding sites not labeled with has additional roles as compared to the other members biotin (Fig. 2d). Tis can be solved by endogenously of the CENP-H/I/K/M complex, especially in promot- tagging the of interest with APEX. Additionally, ing CENP-A assembly [59, 60]. Given a recent report that as biotinylation through APEX takes place in a radius suggests that new CENP-A is loaded preferentially at the of 20 nm, the localization of fusion proteins on chro- boundaries of the centromere domain, to ensure main- matin can only be detected if they are sufciently close tenance of the CENP-A-bound domain size [57], it is to proteins which can be retained on the chromatin intriguing to speculate that CENP-B and CENP-I might fber. A longer fexible linker between the protein of be involved in the regulation of this process, since they interest and APEX might help circumventing this limi- localize to the boundaries of centromeric domain. tation. Tus, our approach can be tailored for diferent While in this study we focused on understanding the experimental contexts and combined with other meth- organization of CCAN proteins in relation to CENP- ods such as FISH, and provide a powerful tool for map- A, it would be very interesting for our approach to ping protein distribution in other repetitive genomic be combined with FISH in future experiments. Great regions. eforts are being made in order to assemble human cen- tromeric sequences [61–65], and specifc centromere probes have been used in the past to study particular Conclusions centromeres [56, 66]. Tese studies could be extended In summary, we developed a novel approach that with our protein-mapping approach combined with allowed us to map the overall association pattern of at FISH, to elucidate the organization of each CCAN on least one member of each CCAN complex over cen- centromeres of specifc chromosomes. Such a novel tromeric chromatin. We fnd that the CCAN is organ- combination of methods could also allow exploring ized in three categories of distribution profles, in whether the asymmetric spreading pattern of CENP-B which some present a localization similar to CENP-A, and CENP-I is chromosome specifc, especially since in another proteins are confned inside the centromere it is not observed in all fbers analyzed. It is notewor- domain but are diferently distributed than CENP- thy that CENP-A is also asymmetrically distributed on A, and lastly two proteins were found to extend fur- some human chromosomes, especially the centromere ther than the centromere domain. In this study, we of the [55, 57]. It would be therefore focused on centromeric factors, but we believe that our interesting to address whether the asymmetric distribu- approach could be applicable for mapping protein dis- tion of CENP-B and CENP-I correlates with that. Alter- tribution in other repetitive regions. natively, it would be interesting to understand that this asymmetric extension of CENP-B and CENP-I outside Kyriacou and Heun Epigenetics & Chromatin (2018) 11:68 Page 13 of 17

Materials and methods slowly pulled out the bufer and fxed in 3.7% Formal- Cell culture and transfections dehyde in TEEN bufer. Slides were then washed three U2OS cells were grown in DMEM supplemented with times in 1XPBS and processed for indirect immuno- 10% FBS and 1% Penicillin–Streptomycin at 37 °C in a 5% fuorescence. For extended chromatin fbers preparation CO­ 2 incubator. Cells were seeded in 6-well plates, a day using salt-detergent lysis bufer, the slides were quickly 5 prior to transfection at a density of 2.5 − 4 × 10 cells per transferred into a Coplin jar containing freshly prepared well. Transfections were performed with Lipofectamine salt-detergent lysis bufer (25 mM Tris, pH 9.5, 500 mM 3000 (Life Technologies) according to the manufacturer’s NaCl, 500 mM Urea, 1% Triton X-100, 1 mM PMSF), instructions, using 2.5 μg of DNA and Opti- incubated for 20 min at RT and were then slowly pulled MEM I reduced serum medium (Life Technologies). out of the bufer. For the double lysis protocol, slides were Downstream experiments were performed three days then washed in PBST for 15 min at RT and incubated post-transfection. again in fresh salt-detergent lysis bufer for 15 min at RT and were slowly pulled out the bufer. Chromatin fbers APEX induction were fxed 3.7% Formaldehyde in PBST for 10 min at RT. APEX induction was carried out according to published Indirect immunofuorescence methods [37]. Briefy, the cell medium was replaced by complete DMEM containing 500 μΜ fnal biotin-phe- Following fxation as described in each relevant appli- nol (Iris biotech) or DMSO (control) and incubated for cation, the slides were washed once in PBST and then blocked in Image-iT® FX signal enhancer in a humidifed 30 min at 37 °C in a 5% CO­ 2 incubator. Te APEX enzyme chamber at RT for at least 30 min. All antibodies were was induced by addition of 1 mM fnal ­H2O2 diluted in 1XDPBS, in the medium, for 1 min at room temperature incubated in a 1:1 mix of PBST and 10% normal goat (RT). Following the induction, the medium was aspirated serum (Life Technologies) overnight at 4 °C in a humidi- and cells were washed three times with quencher solu- fed chamber and were used in 1:50 dilution unless other- tion (10 mM sodium azide, 10 mM sodium ascorbate, wise stated: CENP-A (Abcam-ab13939, 1:100), CENP-B 5 mM Trolox in 1XDPBS). Next, cells were washed once (Abcam-ab25734, Santacruz-sc376283), myc (Abcam- with 1XDPBS, trypsinized, counted and used for down- ab9106, 1:100), CREST (Europa Bioproducts, 1:200), stream applications. CENP-I (Abcam-ab118796), CENP-C and CENP-H (Tat- suo Fukagawa), CENP-T (Ben Black). All antibodies were Cytological preparations used in 1:100 dilution on mitotic chromosome prepara- tions. Secondary antibodies coupled to Alexa Fluor 488, Cells were allowed to settle on Poly-lysine coated slides 555 and 647 (Invitrogen) or Streptavidin conjugated to in a humidifed chamber at 37 °C, 5% CO­ 2 for at least 1 h Alexa 555 were used at 1:100 dilutions (except Strepta- and were fxed with 3.7% Formaldehyde in 0.1% Triton vidin Alexa 555 on chromatin fbers, which was used in X-100 in 1XPBS (PBST) for 10 min at RT. For preparation 1:25 dilution). Counterstaining of DNA was performed of mitotic chromosomes, following trypsinization and 5 with DAPI (5 µg/ml), and coverslips were mounted on before fxation as described above, 1.5 × 10 cells were the slides with 25 μl of ­SlowFade® Gold antifade reagent. treated with Colcemid (1 μg/ml) for 30 min at 37 °C, gen- tly shaking. Cells were then pelleted by centrifugation at Immunoblots 1000g for 5 min at RT, and the pellet was resuspended in For preparation of protein extracts, cell pellets were 500 μl of hypotonic bufer (75 mM KCl) and incubated washed once in warm 1XDPBS, counted and then for 10 min at RT, before it was cytospun into a single- resuspended directly in 2X Tris–Glycine SDS sample chamber cytospin funnel for 10 min at 900 rpm on high bufer (Novex, Life Technologies) in a concentration acceleration in a Shandon Cytospin 4 onto a poly-lysine of 2.5 104 cells/μl, sonicated and boiled for 5 min at coated glass slide. For preparation of chromatin fbers, × 95 °C. Te samples were loaded on 10 or 15% self-casted 5 104 cells were pelleted by centrifugation at 1000g for × SDS–polyacrylamide gels, and blotted on Nitrocellulose 5 min at RT. Te cell pellet was resuspended in 500 μl of membranes. Immunoblotting was conducted with the hypotonic bufer (75 mM KCl) and incubated for 10 min following primary antibodies: anti-CENP-A (Abcam- at RT. Ten, they were transferred into a single-chamber ab13939) or CENP-I (Abcam-ab118796), both used in a cytospin funnel and spun for 4 min at 800 rpm on high dilution of 1:1000. acceleration in a Shandon Cytospin 4 onto a poly-lysine coated glass slide. For TEEN fbers, the slides were incu- Microscopy bated in TEEN bufer (1 mM Triethanolamine-HCl pH 8.0, 1 mM NaCl, 0.5 mM EDTA) for 30 min, they were All IF images were taken as 20–50 z-stacks of 0.2 μm increments, using a 100× oil immersion objective on a Kyriacou and Heun Epigenetics & Chromatin (2018) 11:68 Page 14 of 17

Deltavision RT Elite Microscope and a CoolSNAP HQ Additional fle Monochrome camera. All images were deconvolved using the aggressive deconvolution mode on a SoftWorx Additional fle 1. Figure S1. (A) Representative images of mitotic Explorer Suite (Applied Precision) and are shown as chromosomes from untransfected U2OS cells or cells expressing APEX- CENP-A following induction with ­H2O2, stained for myc, biotin and quick projections of maximum intensity. CENP-A. Insets represent threefold magnifcations of the boxed regions. Scale bar: 5 μm. (B) Immunoblot of protein extracts from cells transiently Image analysis and quantifcations expressing APEX-CENP-A and untransfected U2OS cells, using an antibody against CENP-A. The bottom panel shows Ponceau staining of the blot. All images were analyzed on Fiji software [68]. Quick (C) Schematic for the analysis of plot profles of extended chromatin projections of maximum intensity of the deconvolved fbers prepared with salt-detergent lysis bufer. Hypothetical intensity plot images were imported in the software and analyzed as (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 follows: quantifcations were manually performed on accounted. The= number of peaks was calculated for each staining.≥ To cor- the intensity profle plots of each staining as described rect for the size of diferent centromeres, the ratio of the number of biotin in Additional fle 1: Figure S1C. For the generation of to endogenous CENP-A peaks was always calculated. (ii) Measurement of distances between closest peaks. The green arrow-headed line (dCA) the intensity profle plots, a segmented line was manu- depicts the distance between a CENP-A peak and its closest biotin peak ally drawn above the identifed fber. Te intensity profle while the red arrow-headed line (db) depicts the distance between a bio- was calculated for each staining separately, and graphs tin 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 plotted in Excel. Each peak with a gray value ≥ 300 were marked as “co-localizing” peaks. These data allowed the calculation was marked as a peak for all experiments, except experi- of the percentage of total biotin or CENP-A peaks co-localizing. (iii) The ments with TEEN fbers where the threshold for antibody size of the domain covered by CENP-A (centromere domain) or biotin was determined by measuring the distance between the frst and last peak of signals was set to 200 (except Streptavidin, for which each staining. To correct for the size of diferent centromeres, the ratio of the signal threshold was 300, as above). Te distance biotin to centromere domain size was always calculated. (iv) For calculat- between peaks was measured by calculating the difer- ing the distance of spreading of biotin peaks outside the centromere domain (ds) the distance of the furthest biotin peak from the frst CENP-A ence between the X-axis positions of each marked peak peak (left and right) was measured. If ds was 0.192 μm (co-localizing involved in the quantifcation. A “threshold for co-local- with CENP-A) or if no peaks were found outside≤ the centromere domain, ization” was set to 0.192 μm (3 pixels) since the average ds was set to zero. (D) Distribution of biotin peaks from cells express- ing APEX-CENP-A (mean with SEM) which were used as a reference for width of dots on the fbers was measured as ~ 4 to 5 pixels downstream analyses. Dark gray: percentage of peaks co-localizing with and based on the optical resolution limit. For quantifca- CENP-A, light gray: percentage of peaks not co-localizing with CENP-A tions of the size of fbers in Fig. 1a, a segmented line was inside the CENP-A-bound domain, black: percentage of peaks found outside the CENP-A domain. n 42 fbers from 4 experiments. Figure S2. manually drawn above the identifed fber and using the (A-C) Representative images of= settled U2OS cells expressing APEX-CENP- “measure” tool on Fiji, the size was determined. At least C, CENP-N-APEX or APEX-CENP-T, respectively following induction with 10 fbers were analyzed for each condition, pooled from ­H2O2, stained for myc, biotin and centromere marker. Insets represent threefold magnifcations of the boxed regions. Scale bar: 5 μm. (D-F) Mean at least 2 experiments, except the experiment in Fig. 1, ratios of the number of biotin peaks to CENP-A peaks on chromatin fbers which was performed only once. from cells expressing CENP-C, CENP-N and CENP-T APEX fusion proteins, as compared to APEX-CENP-A. Not signifcant FDR adjusted Kolmogorov– Statistical analysis Smirnov p values > 0.05 are represented as ns. n 42 fbers for CENP-A (from 4 experiments), 27 for CENP-C (from 4 experiments),= 28 for CENP-N All statistical analyses and graphs construction were per- (from 3 experiments) and 15 for CENP-T (from 2 experiments). Error bars: formed in GraphPad Prism version 7.02 for Windows SD. Figure S3. (A-C) Representative images of settled U2OS cells express- ing APEX-CENP-P, CENPK-APEX or CENP-M-APEX, respectively following (GraphPad Software, La Jolla California USA, www.graph​ induction with ­H2O2, stained for myc, biotin and centromere marker. Insets pad.com). Before performing any analysis, datasets were represent threefold magnifcations of the boxed regions. Scale bar: 5 μm. checked for normality by performing a Shapiro–Wilkin- (D-F) Mean ratios of the number of biotin peaks to CENP-A peaks on chro- matin fbers from cells expressing CENP-P, CENP-K or CENP-M APEX fusion son normality test. Parametric or nonparametric tests proteins, as compared to APEX-CENP-A. Not signifcant FDR adjusted Kolmogorov–Smirnov p values > 0.05 are represented by ns. n 42 fbers were performed accordingly, as indicated in each fgure. = P values were adjusted in R using the FDR method and 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). are indicated in each fgure. Te Venn diagram (Fig. 7b) Error bars: SD. Figure S4. (A,E) Representative images of settled U2OS cells was generated on http://jura.wi.mit.edu/bioc/tools​/venn. expressing CENP-I-APEX or APEX-CENP-B, respectively following induction php. with ­H2O2, stained for myc, biotin and centromere marker. Insets represent threefold magnifcations of the boxed regions. Scale bar: 5 μm. (B) Repre- sentative 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 magnifcations 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 Kyriacou and Heun Epigenetics & Chromatin (2018) 11:68 Page 15 of 17

staining of the blot. (D,G) Mean ratios of the number of biotin peaks to Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- CENP-A peaks on chromatin fbers from cells expressing CENP-I or CENP-B lished maps and institutional afliations. APEX fusion proteins, as compared to APEX-CENP-A. FDR adjusted Kol- mogorov–Smirnov p values 0.05 are represented by *. n 42 fbers for Received: 7 August 2018 Accepted: 26 October 2018 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 fbers prepared from U2OS cells using the TEEN bufer 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 fber (in μm) are shown on the right. (D, F) References Representative images of chromatin fbers prepared using the TEEN bufer 1. Allshire RC, Karpen GH. Epigenetic regulation of centromeric chromatin: from untransfected U2OS cells and cells expressing CENP-I-APEX (D) or old dogs, new tricks? Nat Rev Genet. 2008;9:923–37. 2. Musacchio A, Desai A. A molecular view of kinetochore assembly and APEX-CENP-B (F) following induction with H­ 2O2 in the presence of biotin phenol, and stained for CENP-A, biotin and CENP-I or CENP-B, respectively. function. Biology (Basel). 2017;6:5. https​://doi.org/10.3390/biolo​gy601​ Scale bar: 2.5 μm. Intensity plots for CENP-A, biotin and CENP-I/B gray 0005. values along the length of the fber (in μm) are shown on the right. (E, 3. McKinley KL, Cheeseman IM. The molecular basis for centromere iden- G) Scatter plots depicting the size of biotin and CENP-I (E) or CENP-B (G) tity and function. Nat Rev Mol Cell Biol. 2016;17:16–29. domain in untransfected U2OS cells on chromatin fbers prepared with 4. Mendiburo MJ, Padeken J, Fülöp S, Schepers A, Heun P. Dros- TEEN bufer. 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