DNA Sequence-Specific Binding of CENP-B Enhances the Fidelity Of

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DNA Sequence-Speciﬁc Binding of CENP-B Enhances the Fidelity of Human Centromere Function

Graphical Abstract Authors Daniele Fachinetti, Joo Seok Han, ..., Alex J. Wong, Don W. Cleveland

Correspondence [email protected] (D.F.), [email protected] (D.W.C.)

In Brief Fachinetti et al. uncover the functional importance of CENP-B, the only human centromere protein known to bind in a DNA sequence-dependent manner. The authors show that by directly enhancing CENP-C recruitment and CENP-C- dependent nucleation of kinetochore assembly, CENP-B increases the ﬁdelity of epigenetically deﬁned human centromere function.

Highlights d CENP-B binding to alphoid DNA repeats stabilizes CENP-C and kinetochore nucleation d Centromere function is enhanced by mutual dependencies of CENP-A, CENP-B, and CENP-C d The CENP-B free Y and neocentromere chromosomes mis- segregate at elevated frequencies d CENP-B binding to alphoid DNA enhances ﬁdelity of epigenetically deﬁned centromeres

Fachinetti et al., 2015, Developmental Cell 33, 314–327 May 4, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.devcel.2015.03.020 Developmental Cell Article

DNA Sequence-Speciﬁc Binding of CENP-B Enhances the Fidelity of Human Centromere Function

Daniele Fachinetti,1,* Joo Seok Han,1 Moira A. McMahon,1 Peter Ly,1 Amira Abdullah,1 Alex J. Wong,1 and Don W. Cleveland1,* 1Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093, USA *Correspondence: [email protected] (D.F.), [email protected] (D.W.C.) http://dx.doi.org/10.1016/j.devcel.2015.03.020

SUMMARY sequence-specific binding protein (Verdaasdonk and Bloom, 2011). Human centromeres are specified by a stably in- Nevertheless, human centromere position is epigenetically herited epigenetic mark that maintains centromere specified (Ekwall, 2007; Karpen and Allshire, 1997). Among the position and function through a two-step mecha- strongest evidence for an epigenetically defined centromere nism relying on self-templating centromeric chro- was the discovery of neocentromeres in humans (Amor et al., matin assembled with the histone H3 variant 2004; du Sart et al., 1997; Ventura et al., 2004; Warburton, CENP-A, followed by CENP-A-dependent nucle- 2004) in which the initial functional centromere has moved from its previous location to a new site in formerly euchromatic ation of kinetochore assembly. Nevertheless, natu- DNA without a-satellite repeats or binding of CENP-B (Depinet ral human centromeres are positioned within spe- et al., 1997; du Sart et al., 1997; Warburton et al., 1997). Often cific megabase chromosomal regions containing associated with chromosomal rearrangements and found in a-satellite DNA repeats, which contain binding sites some types of cancer, each neocentromere is marked with chro- for the DNA sequence-specific binding protein matin stably assembled with CENP-A (Amor et al., 2004), the CENP-B. We now demonstrate that CENP-B centromere-specific histone H3 variant (Earnshaw and Rothfield, directly binds both CENP-A’s amino-terminal tail 1985; Palmer et al., 1987). and CENP-C, a key nucleator of kinetochore as- CENP-A is essential for centromere identity (Black et al., sembly. DNA sequence-dependent binding of 2007b). It marks and maintains centromere position (Black CENP-B within a-satellite repeats is required to sta- et al., 2004; Hori et al., 2013; Mendiburo et al., 2011) and recruits bilize optimal centromeric levels of CENP-C. Chro- additional centromere and kinetochore components (Carroll et al., 2009, 2010; Foltz et al., 2006; Liu et al., 2006) to both mosomes bearing centromeres without bound normal and ectopic centromere locations (Barnhart et al., CENP-B, including the human Y chromosome, are 2011; Guse et al., 2011; Hori et al., 2013; Mendiburo et al., shown to mis-segregate in cells at rates several- 2011). Indeed, use of gene targeting in both human cells and fold higher than chromosomes with CENP-B-con- fission yeast has demonstrated that CENP-A-containing chro- taining centromeres. These data demonstrate a matin is the primary epigenetic mark of centromere identity (Fa- DNA sequence-specific enhancement by CENP-B chinetti et al., 2013). of the fidelity of epigenetically defined human Centromere identity and function is achieved through a two- centromere function. step mechanism. First, new CENP-A assembly to centromeric chromatin is mediated by its CENP-A targeting domain (CATD) (Black et al., 2004, 2007a; Shelby et al., 1997) in conjunction INTRODUCTION with the CENP-A selective chaperone HJURP (Dunleavy et al., 2009; Foltz et al., 2009; Shuaib et al., 2010) whose activity is The centromere is the fundamental unit for ensuring chromo- tightly controlled across the cell cycle (Mu¨ ller and Almouzni, some inheritance. Correct centromere function is required for 2014). In the second step, either the amino- or carboxy-terminal preventing errors in chromosome delivery that would lead to tail of CENP-A is required for nucleation of assembly of a kinet- genomic instability and aneuploidy, both hallmarks of many hu- ochore that mediates high fidelity chromosome segregation man cancers. Although not conserved across species, centro- indefinitely (Fachinetti et al., 2013). Curiously, amino-terminal mere regions from fission yeast to man have arrays of repetitive tail-dependent kinetochore nucleation requires the presence of sequences (Fukagawa and Earnshaw, 2014). Human centro- CENP-B (Fachinetti et al., 2013). Starting from this suggestion meres carry extensive (1,500 to >30,000) copies of imperfectly of a CENP-B-dependent role in kinetochore function, we now repeated arrays of a 171 bp element, termed a-satellite DNA. use a combination of gene targeting and replacement in human In all but the centromere of the Y chromosome (Earnshaw and mouse cells, coupled with in vitro approaches, to identify a et al., 1987, 1989; Miga et al., 2014), this array contains a DNA sequence-dependent contribution to fidelity of human 17-base pair (bp) consensus motif that is the binding site of centromeric function that is mediated by CENP-B binding to CENP-B, the only known mammalian centromeric DNA centromeric a-satellite repeats.

314 Developmental Cell 33, 314–327, May 4, 2015 ª2015 Elsevier Inc. RESULTS decrease of centromere-bound CENP-C [Fachinetti et al., 2013]). CENP-A’s Amino-Terminal Tail Directly Binds the To determine if short-term reduction of CENP-B also had con- Alphoid DNA-Binding Protein CENP-B sequences on CENP-C maintenance at centromeres, we inte- To test the effect that complete loss of the CENP-A amino-termi- grated (at a unique genomic locus using the Flp-In system in nal tail has on centromere-bound CENP-B and on overall cell DLD-1 cells) an siRNA-resistant, doxycycline-inducible gene en- viability, we stably expressed (by retroviral integration) a full- coding CENP-B that was dually tagged with EYFP and AID length CENP-A or a CENP-A variant lacking its amino-terminal (auxin-inducible degron), the latter to enable rapid degradation tail (DNH2CENP-A) in human cells containing one disrupted upon addition of the synthetic auxin indole-3-acetic acid endogenous CENP-A allele and one floxed allele (CENP-A/F) (IAA) (Holland et al., 2012). In agreement with our initial gene (Figure 1A). After Cre-recombinase mediated inactivation of the inactivation approach (Figure 2D), siRNA-mediated CENP-B floxed allele and subsequent loss of endogenous CENP-A pro- depletion led to decrease in centromeric CENP-C by half (Fig- tein (Figures S1A and S1B), long-term cell viability was rescued ures S2B–S2D). In contrast, doxycycline-induced expression of by DNH2CENP-A (Figure 1B), albeit with a 4-fold increase in chro- CENP-BAID-EYFP maintained the initial level of CENP-C at each mosome mis-segregation and micronuclei formation (scored centromere (Figures S2B–S2D), while IAA-induced rapid degra- by live cell imaging in cells stably expressing H2B-mRFP to dation of CENP-BAID-EYFP was accompanied by reduction (again label chromosomes) (Figures 1A and 1C). Furthermore, loss of by half) of CENP-C bound at centromeres (Figure S2D). the CENP-A amino-terminal tail was accompanied by reduced To directly measure the level of CENP-C after siRNA-medi- CENP-B binding at centromeres (Figures 1A and 1D), as ated CENP-B depletion, we EYFP-tagged one or both alleles measured by quantifying centromeric CENP-B intensity by of the endogenous human CENP-C gene using TALEN-medi- immunofluorescence. ated gene targeting (Figures 2E–2G). As expected, quantifica- To determine if this CENP-A-dependent binding of CENP-B at tion of centromere fluorescence intensity of single centromeres centromeres could result from a direct interaction, recombinant in live cells revealed that CENP-C in CENP-C+/EYFP cells was CENP-B was incubated with GST or GST-tagged CENP-A frag- half that of centromeres in which both alleles were targeted ments and GST-containing proteins were affinity purified on (CENP-CEYFP/EYFP)(Figure 2H). Importantly, in agreement with glutathione-immobilized beads (Figures 1E and 1F). CENP-B our indirect immunofluorescence analysis, siRNA-mediated bound directly to the amino-terminal tail of CENP-A (GST- depletion of CENP-B led to loss of half of centromeric EYFP in-

CENP-A1–44), but not to GST alone or to a CENP-A mutant lack- tensity (Figure 2H). ing its amino-terminal tail (GST-CENP-AD1–44)(Figure 1F). The first 29 amino acids of the CENP-A tail were sufficient for this Direct Binding of CENP-B to CENP-C interaction (Figure S1C), in agreement with the observation that To determine whether the CENP-B influence on the level of the first 29 amino acids of CENP-A’s amino terminal tail stabilize centromere-bound CENP-C in cells could be mediated by CENP-B binding at centromeres (Fachinetti et al., 2013). physical interaction between CENP-B and CENP-C, we tested direct binding of the proteins to each other in vitro (Figure 3A). CENP-B Supports CENP-C Maintenance at Centromeres After co-incubation of GST-CENP-C and His-CENP-B, either Deletion of the CENP-A amino-terminal tail not only affected protein was affinity purified (Figure S2E), followed by immuno- CENP-B binding, but also reduced by half centromere-bound blot. GST pull-down for CENP-C co-purified a portion of CENP-C (Figure 1D), a major centromere component required CENP-B and vice versa (Figure 3B), indicative of direct binding for kinetochore assembly (Fukagawa et al., 1999). In vitro (Carroll and in agreement with a previous two-hybrid study (Suzuki et al., 2010; Guse et al., 2011) and in vivo (Fachinetti et al., 2013) et al., 2004). findings have reported that the small (six amino acid) carboxy- terminal tail of CENP-A is one element for CENP-C recruitment CENP-B Is Not Required for Initial Loading of New to centromeres. Complete loss of the CENP-A carboxy-terminal CENP-C at Centromeres tail did not, however, abolish centromeric CENP-C binding (Fa- We then tested if CENP-B was also responsible for CENP-C chinetti et al., 2013), indicating the existence of another pathway deposition at centromeres besides its role in CENP-C stabiliza- for its recruitment. tion. To test the initial loading of CENP-C at centromeres and Because the CENP-A amino-terminal tail binds to CENP-B its dependency on CENP-B, a tetracycline-inducible CENP-C and its deletion reduced both CENP-B and CENP-C bound to tagged with EYFP and AID was stably integrated at a specific centromeres (Figure 1), we tested if CENP-B was required for chromosomal locus. After culturing in IAA to induce degradation the maintenance of a fraction of centromeric CENP-C. Long- of the accumulated tagged CENP-C assembled at centromeres, term dependency of centromere recruitment of CENP-C on IAA was withdrawn and immunofluorescence was used to CENP-B was tested by disrupting both CENP-B alleles in hu- monitor CENP-CAID-EYFP re-accumulation and its assembly at man diploid RPE-1 cells using a CRISPR/Cas9 nuclease (Fig- centromeres (Figures S3A and S3B). Interestingly, siRNA-medi- ure 2A and Figure S2A). Complete loss of CENP-B (Figures ated reduction in CENP-B by more than 90% (Figure S3C) did 2B–2D) resulted in a 50% reduction of CENP-C at centromeres not significantly change the percentage of cells that efficiently but not of its total cellular levels (Figures 2B and 2D), with only a loaded new CENP-CAID-EYFP (Figure S3D), but did reduce the in- slight decrease of centromeric CENP-A levels (Figure 2D), a tensity of centromeric CENP-CAID-EYFP (Figure S3E). This result reduction insufficient to explain the observed CENP-C reduc- indicates that CENP-B is not required for CENP-C deposition tion (CENP-A must be depleted >75% to produce 2-fold at centromeres, but only for its retention.

Developmental Cell 33, 314–327, May 4, 2015 ª2015 Elsevier Inc. 315 A

B C D

Figure 1. CENP-A Amino-Terminal Tail Interacts with CENP-B (A) Schematic representing the different CENP-A rescue constructs amino terminally tagged with EYFP (enhanced yellow ﬂuorescent protein), the CENP-A/F cell line, and the experiments described in (B)–(D). (B) Images of representative crystal violet-stained colonies from the colony formation assay in (A). (C) Frequency of mitotic errors in the indicated cell lines. Bars represent the mean of >50 cells per condition. Error bars represent the SEM of three independent experiments. (D) Bar graph showing centromere intensities of EYFP-rescue constructs, CENP-B and CENP-C for the indicated cell lines. Values represent the mean of six independent experiments (>30 cells per experiment, average of 30 centromeres per cell). Error bars represent the SEM. Unpaired t test: ***p < 0.0001. (E) Schematic of the experiment performed in (F). (F) GST pull down and subsequent immunoblot for GST-tagged CENP-A co-incubated with His-tagged CENP-B. See also Figure S1.

316 Developmental Cell 33, 314–327, May 4, 2015 ª2015 Elsevier Inc. AB

FG H

Figure 2. CENP-B Is Required for Full CENP-C Maintenance at Centromeres (A) Schematic of the experiments described in (B)–(D) with the use of CRISPR/Cas9-mediated gene deletion. (B) Immunoblot for accumulated CENP-B and CENP-C after identification of cells with both CENP-B alleles disrupted by action of the CRISPR/Cas9; a-tubulin was used as a loading control. (C) Representative immunofluorescence images of centromere-bound CENP-B following CRISPR-mediated disruption of both CENP-B alleles in RPE1 cells. ACA was used to mark centromere positions. Scale bar represents 5 mm. (D) Bar graphs of centromere intensities for CENP-A (red), CENP-B (blue), and CENP-C (green) in the indicated cell lines quantified with specific antibodies as described in (A). Bars represent the mean of three independent experiments (>30 cells per experiment). Error bars represent the SEM. Unpaired t test: **p < 0.006. (E) Schematic of the experiments described in (F)–(H) with the use of TALEN-mediated gene targeting. (F) CENP-C genotypes validated in the indicated cell lines using PCR to distinguish normal (+/+), double tagged (EYFP/EYFP), and single tagged (+/EYFP). (G) Immunoblot for CENP-C to distinguish non-tagged, single, or double allele-tagged CENP-C with EYFP. (H) Bar graphs represent CENP-C-EYFP centromere intensity in the indicated cell lines measured by live cell imaging with or without siRNA treatment of CENP-B. Bars represent the mean of three independent experiments (>30 cells per experiment, average of 30 centromeres for cell). Error bars represent the SEM. Unpaired t test: ***p < 0.0001. See also Figure S2.

Developmental Cell 33, 314–327, May 4, 2015 ª2015 Elsevier Inc. 317 AB Figure 3. CENP-B Supports Faithful Chro- mosome Segregation via Direct CENP-C Binding (A) Schematic of a GST afﬁnity pull-down assay for testing CENP-B binding to GST-tagged CENP-C. (B) GST (top) or His (bottom)-pull-down using the assay detailed in (A). (C) Schematic of the experiments described in (D)–(F). (D) Immunoblot of cell extracts to measure total CENP-B level 48 hr after siRNA treatment; a-tubulin was used as a loading control. (E) Bar graph of centromere intensities for CENP-C in the indicated cell lines. Bars represent the mean C of three independent experiments (>30 cells per experiment). Error bars represent the SEM. Un- paired t test: *p < 0.04. (F) Bar graph of frequency of micronuclei formation and mis-aligned mitotic chromosomes identiﬁed with live cell imaging. Bars represent the mean of >50 cells per condition. Error bars represent the SEM of three independent experiments. Unpaired D t test: *p < 0.04, **p < 0.006. See also Figure S3.

siRNA-mediated depletion of CENP-B (Figures 3C–3E). This CENP-B-depen- E F dent loss of CENP-C at centromeres was accompanied by more than half of mitoses with misaligned chromosomes and micronuclei found within 60% of interphase cells (Figures 3C and 3F). The KMN network (comprised of the Mis12, Knl1, and Ndc80 complexes) is essential for connection between the centromere and spindle microtubules (Hori and Fukagawa, 2012). Recognizing that CENP-C has been proposed to be required for kinetochore function through recruitment of the Mis12 complex (Przew- loka et al., 2011; Screpanti et al., 2011), we measured the centromeric levels of Dns1 and Hec1, subunits of the Mis12 and Ndc80 complexes, respectively, on metaphase centromeres in cells with CENP-B Acts to Enable Highest Fidelity of Chromosome normal or reduced centromere-bound CENP-C (Figures S3F Segregation and S3G). Reduction of centromeric CENP-C in CENP-B/ cells To determine the consequence of CENP-B loss on the ﬁdelity of or in CENP-A/ cells rescued by DNH2CENP-A lead to corre- centromere function, we measured chromosome mis-segrega- spondingly reduced levels of Dsn1 and slightly reduced Hec1, tion rates using live cell imaging (Figure 3C) in CENP-A/ cells similar to what was observed in CENP-A/ cells rescued by deleted in both endogenous CENP-A alleles and with centro- CENP-AH3-C (Figures S3H and S3I) (Fachinetti et al., 2013). mere function rescued by stable expression of full-length Thus, diminution of centromeric CENP-C drives chromosome CENP-A or a CENP-A variant (CENP-AH3-C) in which its CENP- mis-segregation at least in part from reduced recruitment of C binding site (CENP-A’s carboxy terminus) was replaced with the Mis12 complex. the corresponding region of histone H3 (Figures 3C and 3E). In We next tested if a role of CENP-B in supporting centromere CENP-A/ cells rescued with full-length CENP-A, reduction in function via maintenance of centromeric CENP-C in human cells CENP-B lowered centromeric CENP-C by 50% and produced was conserved in an additional mammalian species. We tested if a 2-fold increase in mitotic errors relative to the siRNA control chromosome mis-segregation rates and centromeric CENP-C (Figures 3C–3F). In CENP-A/ cells rescued by CENP-AH3-C, loading were affected in immortalized mouse embryonic ﬁbro- almost all centromere-bound CENP-C was lost following blasts (MEFs) in which both CENP-B alleles had been inactivated

318 Developmental Cell 33, 314–327, May 4, 2015 ª2015 Elsevier Inc. A B retrovirus H2B-mRFP Figure 4. CENP-B Is Required for Faithful ** Chromosome Segregation in Mouse Cells Live cell imaging (E) CENP-A by Supporting CENP-C Association with 100 CENP-C Score for micronuclei (C-D) Centromeres MEFs Score for 75 16h (A) Quantiﬁcations of CENP-A and CENP-C pro- CENP-B+/+ 6h micronuclei tein levels at centromeres in MEFs with or without or CENP-B-/- Remove (C-D) 50 CENP-B. Bars represent the mean of three inde- +Nocodazole Nocodazole pendent experiments (>30 cells per experiment). 25 C +/+ -/- CENP-B CENP-B Error bars represent the SEM. Unpaired t test: **p <

Centromere bound 0 +/+ -- 0.0016.

CENP-A or CENP-C (%) CENP-A / (B) Schematic of the experimental design to test CENP-B for frequency of micronuclei formation in CENP- B+/+ or CENP-B/ MEFs before and after recov- CENP-B E Micronuclei Lagging chromosomes ery from nocodazole-induced mitotic arrest or by 25 * live cell imaging. D 50 * (C) CENP-B deletion increases chances of chro- 20 mosome mis-segregation leading to micronuclei 40 15 formation. Representative images of nuclei in 30 * CENP-B-containing and CENP-B-depleted MEFs 10 quantiﬁed in (D). Scale bar represents 5 mm. An 20 5 arrow marks a micronucleus in the CENP-B/ micronuclei percentage of

observed events (%) 10 0 cell. +/+ -/- (D) Quantiﬁcation of micronuclei frequency in 0 +/+ / CENP-B + or - Nocodazole, - + - + CENP-B and CENP-B MEFs measured as in -/- then release CENP-B+/+ CENP-B (B). Bars represent the mean of >100 cells per condition. Error bars represent the SEM of three independent experiments. Unpaired t test: *p < 0.04. (E) Bar graphs quantifying lagging chromosomes in mitosis or micronuclei in interphase in the indicated cell lines. Bars represent the mean of >50 cells per condition scored by live cell imaging. Error bars represent the SEM of three independent experiments. Unpaired t test: *p < 0.04.

(Kapoor et al., 1998; Okada et al., 2007). As in the human RPE-1 was half of that of CENP-B-containing centromeres (49% or DLD-1 cells following CENP-B reduction, CENP-B null MEFs reduction; Figure 5B, bottom). Also, in agreement with a role displayed a 50% reduction in centromeric CENP-C, relative to of CENP-A in stabilizing CENP-B via its amino tail, CENP-B level wild-type MEFs, despite unchanged levels of CENP-A (Fig- at the inactive original centromere on the neocentromere-con- ure 4A). This was accompanied by an increased mitotic error fre- taining chromosome 4 was also 57% compared to other centro- quency (measured by imaging of fixed cells or by live imaging of meres (Figure 5B, middle). cells expressing H2B-mRFP), with accumulation of micronuclei We next tested the influence of the presence or absence of at more than twice the rate seen in CENP-B wild-type cells (Fig- cellular CENP-B on CENP-C binding at the Neo4 centromere ures 4B–4E). Using release from nocodazole arrest to enrich for and the fidelity of segregation of the Neo4 chromosome. To do mitotic errors (Thompson and Compton, 2011), higher error fre- this, we measured the frequency of Neo4 incorporation into mi- quencies were again observed in CENP-B deleted MEFs relative cronuclei by scoring for (1) centromeric CENP-A (or CENP-C) to wild-type MEFs (Figures 4B–4D). without CENP-B (representing the functional Neo4 centromere) and (2) an additional CENP-B spot (representing the inactive Elevated Mis-segregation of a Neocentromere without original centromere 4). In cells with normal CENP-B levels, Bound CENP-B Neo4 was found to be encapsulated in a micronucleus with a We then tested whether it was the presence of CENP-B per se 3-fold higher frequency than other chromosomes. Neo4 was or the sequence-dependent CENP-B binding at centromeres present in 6% of observed micronuclei, a rate three times higher that was required to reinforce both CENP-C binding at centro- than the 2.1% level expected for mis-segregation by chance, meres and fidelity of chromosome segregation. To address assuming that each chromosome has an equal chance to mis- this point, we examined a patient-derived cell line (PD-NC4) segregate (Figures 5C–5E). containing an alphoid DNA-free neocentromere on chromo- Because Neo4 centromere function relies only on CENP-A, some 4 (hereafter Neo4) (Amor et al., 2004)(Figure 5A). The but not on CENP-B, for CENP-C recruitment and consequently intensities of CENP-A/B/C were measured by immunofluores- kinetochore nucleation, we then tested whether reduction of cence on chromosome spreads. The neocentromere was iden- CENP-A preferentially increased mis-segregation of the Neo4 tified by scoring for a chromosome in which CENP-A (at the chromosome. We decreased centromere-assembled CENP-A Neo4 centromere) and CENP-B (at the inactive, original chromo- levels (by simultaneous siRNA-mediated reduction of CENP-A some 4 centromere DNA locus containing CENP-B boxes in its and its chaperone HJURP—Figures 5C and 5F). Remarkably, alphoid DNA repeats) did not colocalize (Figure 5A). CENP-A this CENP-A reduction selectively and significantly enhanced level at the Neo4 centromere was nearly normal (83% of the mis-segregation frequency of the Neo4 containing chromosome, average level at the other centromeres; Figure 5B, top), in agree- yielding a 5- to 6-fold (by release from nocodazole arrest) ment with previous reports (Bassett et al., 2010; Bodor et al., increased error frequency compared to all other chromosomes 2014). In contrast, CENP-C intensity at the CENP-B-free Neo4 (Figure 5E).

Developmental Cell 33, 314–327, May 4, 2015 ª2015 Elsevier Inc. 319 ABFigure 5. CENP-B Is Required for Faithful Chromosome Segregation of the Neocen- tromere Chromosome (A) (Left) Schematic of the neocentromere-containing Neo4 chromosome from PD-NC4 cells. (Right) Representative immunofluorescence images of the neocentromere chromosome in interphase or mitosis (yellow arrows). (B) Box-and-whisker graphs of CENP-A, CENP-B, or CENP-C intensities at the neocentromere; other C chromosomes; and the original chromosome 4 measured on metaphase spreads (see Experi- mental Procedures for details). Error bars represent the SEM. Bar in the box represents the median; the whiskers represent cells distribution. Unpaired t test: ***p < 0.0001. (C) Schematic of the experimental design for testing frequency of micronuclei formation after D siRNA treatment to reduce CENP-B or HJURP in PD-NC4 cells with the Neo4 neocentromere, followed by nocodazole-induced mitotic arrest and subsequent recovery. (D and E) Chromosome missegregation rates of the Neo4 chromosome or other chromosomes determined by immunofluorescence. (D) Representative immunofluorescence images of centromere-bound CENP-A and CENP-B after siRNA treatment against GAPDH. Yellow arrows, green and orange circles/ squares indicate the neocentromere chromosome, red circles/squares a normal chromosome encapsulated in micronuclei. Scale bar represents 5 mm. E (E) Quantitation of expected and experimentally determined mis-segregation rates from (D) for the Neo4 chromosome. The observed mis-segrega- F tion rate is the frequency with which the Neo4 was found inside micronuclei relative to the total number of micronuclei. The frequency of micronuclei formation was determined by immunofluorescence after the indicated siRNA treatment and, when indicated, following recovery from nocodazole-induced mitotic arrest. Unpaired t test: *p < 0.04, **p < 0.006. (F) Immunoblot of PD-NC4 cell extracts to measure total CENP-A and HJURP levels after siRNA treatment; a-tubulin was used as a loading control.

Elevated Mis-segregation of the CENP-B-free, Alphoid We next measured the rate of chromosome mis-segregation of DNA-Containing Y Chromosome the Y chromosome compared to the X chromosome or chromo- The Y chromosome is known to have repetitive alphoid DNA se- some 4, the latter two both having alphoid DNA repeats with quences but lacks functional CENP-B boxes (Earnshaw et al., functional CENP-B boxes. To do this, we used dual fluorescence 1987, 1989; Miga et al., 2014). To determine whether it is binding in situ hybridization (FISH) with centromere-specific probes to of CENP-B at each centromere rather than the presence of re- concomitantly depict two chromosomes. We then counted the petitive alphoid sequences that acts to maintain CENP-C level number of micronuclei containing chromosome 4, X or Y relative at centromeres and thereby support fidelity of chromosome to the total number of micronuclei (Figures 6A, 6D, and 6E). segregation, we measured the level of centromeric CENP-C on Remarkably, the CENP-B-free Y chromosome accumulated mitotic chromosomes from a male human cell line (DLD-1) (Fig- into micronuclei with 4-fold higher frequency (8.3%) compared ures 6A and 6B). CENP-A level on the Y was only slightly reduced to what would be expected from random mis-segregation of relative to other centromeres (82% of the average level on all haploid chromosomes into micronuclei (2.1%) or relative to the other centromeres; Figure 6C, left), in agreement with a previous measured X or 4 mis-segregation rate (2.8% and 4.8%, respec- report (Bodor et al., 2014). However, as was observed for the tively). In agreement with the dependency on CENP-A (but not Neo4 centromere, the intensity of centromeric CENP-C on the CENP-B) for chromosome segregation observed for the Neo4 CENP-B-free Y centromere (Figures 6B and 6C, middle) was neocentromere-containing chromosome (Figure 5E), siRNA- only about half (60%) of that of the other chromosomes (Figures mediated reduction of CENP-A by as little as four-fold (Figure 6F) 6B and 6C, right). selectively increased the frequency of Y-positive micronuclei to

320 Developmental Cell 33, 314–327, May 4, 2015 ª2015 Elsevier Inc. 18%, while only slightly increasing mis-segregation of the chro- tion that CENP-B strongly enhanced de novo centromere forma- mosome X or 4 (Figure 6E). In agreement with what was observed tion in artificial chromosomes (Ikeno et al., 1998; Ohzeki et al., earlier (Figures 3 and 4), an influence of CENP-B on fidelity of 2002; Okada et al., 2007; Okamoto et al., 2007), possibly by chromosome segregation was seen by more than a 2-fold eleva- altering modifications to centromere-associated histone H3 tion in the overall rate of micronuclei formation (Figure 6G) (Okada et al., 2007), thereby affecting HJURP and CENP-A following CENP-B depletion by siRNA (Figure 6F). Despite this, recognition/deposition (Bergmann et al., 2011). To these earlier reduction in CENP-B did not significantly affect the rate of mis- proposals, our effort has established that repetitive alphoid segregation of the CENP-B-free Y chromosome (Figure 6E). centromeric DNA sequences to which CENP-B binds do provide a contribution for binding of centromeric CENP-A and CENP-B Contributes to Cell-Cycle-Dependent CENP-A CENP-C on native centromeres. Indeed, we have now shown Loading at Centromeres that a CENP-A variant without its CENP-C binding domain With our evidence that CENP-B together with CENP-A is (NH2H3CATD), which fully supports long-term centromere function required to maintain a normal CENP-C level at each centromere, despite inability to directly recruit CENP-C (Fachinetti et al., we tested whether there is a contribution to CENP-A deposition 2013), supports incorporation or stabilization of new CENP-A provided by CENP-B. For this test (see schematic in Figure 7A), into centromeric chromatin in a CENP-B-dependent manner we took advantage of the SNAP-tag technique (Jansen et al., (Figure 7C). This CENP-B-dependent loading/stabilization of 2007) which permits in vivo covalent marking of proteins linked CENP-A occurs via the recruitment of CENP-C to centromeres to a 20 kD suicide enzyme (named SNAP). siRNA was used to in a parallel pathway to direct CENP-C recruitment by the deplete CENP-B (Figure S4A) from CENP-A/ cells in which CENP-A carboxy-terminal tail (Carroll et al., 2010; Guse et al., centromere function was rescued with a stably integrated full- 2011; Fachinetti et al., 2013; Kato et al., 2013). Our findings on length, SNAP-tagged CENP-A. CENP-A loading at centromeric this mutual dependency of CENP-A and CENP-C are in agree- chromatin was only slightly affected by loss of CENP-B (Figures ment with the demonstration that targeting of CENP-C to an 7A and 7C and Figures S4A and S4B). As expected, HJURP ectopic chromosomal site is sufficient for CENP-A recruitment depletion, on the other hand, eliminated new CENP-A deposition following removal of the endogenous centromere on that chro- (Figure 7C and Figures S4A and S4B) (Dunleavy et al., 2009; Foltz mosome (Hori et al., 2013) and with the CENP-C-dependent et al., 2009). Remarkably, CENP-A/ cells rescued with a his- recruitment of the methyl-transferase DNMT3B (Gopalakrishnan tone H3 variant carrying the CATD and amino-terminal tail of et al., 2009; Kim et al., 2012) or the Mis18 complex (Dambacher CENP-A, but not its carboxy-terminal tail (SNAP-NH2H3CATD) et al., 2012; Hori et al., 2013; Moree et al., 2011), which in turn is showed a marked reduction in new NH2H3CATD deposition recognized by the CENP-A chaperone HJURP (Barnhart et al., following CENP-B depletion (Figures 7A–7C and Figure S4B). 2011). Accordingly, total centromere-bound CENP-A levels decreased It is important to note that this CENP-B-dependent stabiliza- in cells supported by a variant that lacks the CENP-A/CENP-C tion of centromeric CENP-A and CENP-C requires sequence- interaction domain, but not in cells rescued with full-length dependent DNA binding, as it is found only on centromeres CENP-A (Figure S4C). carrying CENP-B boxes and indeed is absent from the Y centro- CENP-B depletion also reduced M18BP1 (a subunit of the mere and neocentromeres. In addition, our findings that CENP-B Mis18 complex) binding at centromeres assembled with a does not play a role in initial CENP-C recruitment (Figure S3), but CENP-C binding-deficient CENP-A variant (Figure S4D), in rather stabilizes it once centromere bound, reinforces the impor- agreement with a previously reported CENP-C/Mis18 interaction tance of a crucial epigenetically defined contribution to centro- (Dambacher et al., 2012; Moree et al., 2011) and consistent with mere function. CENP-A-containing chromatin is essential for the CENP-B-dependent portion of CENP-A loading at centro- the mutual reinforcement of CENP-A/B/C binding at centromeres mediated in part through the Mis18 complex. meres. Without it, the silenced centromere on the Neo4 chromosome cannot stabilize CENP-C despite a direct affinity for DISCUSSION CENP-B bound to the CENP-B boxes in its a-satellite repeats. The idea that CENP-B might play an important role in centro- CENP-A-containing chromatin has previously been demon- mere function had previously been dismissed by demonstration strated to be the epigenetic mark that is sufficient for long- that CENP-B null mice are viable (Hudson et al., 1998; Kapoor term maintenance of human centromere positioning and identity et al., 1998; Perez-Castro et al., 1998). In addition, the existence and for nucleation of a functional kinetochore (Fachinetti et al., of human neocentromeres and the Y chromosome lacking 2013). Adding to this, our current evidence has identified a par- CENP-B boxes had established that CENP-B is dispensable allel DNA sequence-specific role for direct stabilization of for human centromere function. CENP-C and recruitment of CENP-A that is provided by Nevertheless, we have shown here in both mouse and human CENP-B, the sole sequence-specific mammalian centromeric cells that depletion of CENP-B yields a higher rate of chromo- DNA binding protein identified so far (Muro et al., 1992; Stimpson some mis-segregation for chromosomes with centromeres to and Sullivan, 2010). This discovery offers an explanation for the which CENP-B had initially been bound (Figures 3 and 4). This existence and retention of CENP-B boxes and the high degree observation offers at least a partial explanation for the reproduc- of CENP-B conservation in divergent mammals (Sullivan and tive and developmental dysfunctions reported in CENP-B null Glass, 1991). mice due to disruption in the normal morphogenesis of the highly An initial suggestion of a functional role at centromeres for mitotically active uterine epithelial tissue (Fowler et al., 2000; CENP-B emerged from Masumoto and colleagues’ demonstra- 2004). When combined with evidence that widespread somatic

Developmental Cell 33, 314–327, May 4, 2015 ª2015 Elsevier Inc. 321 AB

DFG

Figure 6. The CENP-B-Free Alphoid DNA-Containing Y Chromosome Has Reduced Level of CENP-C and Increased Rate of Chromosome Mis-segregation (A) Schematic of the experiments shown in (B)–(F). (B) Representative image of Y chromosome in mitosis (yellow arrow) stained with CENP-C (green) and CENP-B (red) antibodies. (C) Box-and-whisker plots of CENP-A, CENP-B, or CENP-C intensities at the centromere of the Y chromosome compared to all other centromeres measured on metaphase spreads (see Experimental Procedures for details). Error bars represent the SEM. Bar in the box represents the median; the whiskers represent points distribution. Unpaired t test: ***p < 0.0001. (D) (Left) Representative immunofluorescence image of a FISH experiment on a metaphase spread using centromeric probes for the Y (red) and X (green) chromosomes. (Right) Representative immunofluorescence image of a FISH experiment on interphase cells using centromeric probes for the Y chromosome (red) or chromosome 4 (green). Yellow arrow indicates a nucleus in a cell that has undergone Y chromosome mis-segregation to accumulate two Y chromosomes. White arrow indicates a nucleus in which mis-segregation has led to loss of the Y chromosome. (E) Chromosome mis-segregation rates of chromosomes 4, Y, and X determined by FISH. (Left) Representative immunofluorescence image of a micronucleus- containing cell after siRNA treatment to lower CENP-A levels. Scale bar represents 5 mm. (Right) Bars represent the frequency of micronuclei formation

(legend continued on next page) 322 Developmental Cell 33, 314–327, May 4, 2015 ª2015 Elsevier Inc. A C

B D

Figure 7. CENP-B Supports Centromeric Chromatin Replication and Function (A) Schematic of SNAP-tagging experiment to measure new CENP-A deposition at centromeres after siRNA-mediated reduction of CENP-B or HJURP. (B) Representative immunofluorescence images show localization and intensity of SNAP-NH2H3CATD after siRNA of GAPDH or CENP-B; a-tubulin staining was used to identify telophase/early G1 cells. Scale bar represents 5 mm. (C) Quantitation of level of centromeric CENP-A deposition after depletion of CENP-B or HJURP. Bars represent the mean of four independent experiments (>30 cells per experiment). Error bars represent the SEM. Unpaired t test: ***p < 0.0001. (D) Model of CENP-B enhanced centromere function through its interaction with CENP-C. Centromere function is supported by a dual mechanism for CENP-C recruitment and subsequent CENP-C-dependent nucleation of kinetochore assembly, mediated both by CENP-A and by CENP-B. See also Figure S4. aneuploidy is surprisingly well tolerated in mice (e.g., in mice with additional contributor for neocentromere mis-segregation may reduced levels of the centromeric motor protein CENP-E be inefficient Aurora B function, as previously proposed [Bassett [Weaver et al., 2007], the mitotic checkpoint protein Mad2 [Mi- et al., 2010]). The reduced segregation fidelity, which had previ- chel et al., 2001], or mitotic checkpoint kinase Bub1 [Jeganathan ously been reported for the Y chromosome to be associated with et al., 2007]), we predict that mice deficient in CENP-B develop shorter survival and high risk of blood cancer in an age- and similar somatic aneuploidy. While this was not tested by any of smoke-dependent manner (Dumanski et al., 2015; Forsberg the three groups that had produced CENP-B null mice (none of et al., 2014; Nath et al., 1995), correlates with reduced CENP-C which have been maintained) (Hudson et al., 1998; Kapoor binding, which we argue is the probable cause of reduced et al., 1998; Perez-Castro et al., 1998), we demonstrated that centromere function. Whereas our evidence has only tested fibroblasts from those mice (Kapoor et al., 1998) are chromoso- CENP-B influence on mitotic chromosome segregation, if a mally unstable with a chronically elevated rate of mis-segrega- similar contribution is also present in meiosis the selective pres- tion (Figure 4). sure for maintaining males will act to maintain the Y chromosome We also demonstrated that two human chromosomes that despite increased mis-segregation frequency. Therefore, our lack DNA-containing CENP-B binding sites (the Y chromosome findings strongly implicate CENP-B as a key contributor to and the neocentromere) mis-segregate at higher frequency centromere strength and the rate of faithful chromosome segre- than do chromosomes with the alphoid DNA-containing, gation through stabilization of binding of other centromere/kinet- CENP-B-bound centromeres (Figures 5 and 6). (Note that an ochore proteins. experimentally determined for chromosome 4, Y and X after the indicated siRNA treatment. The observed mis-segregation rate is the frequency with which the chromosome 4, Y or X chromosomes, respectively, were found inside micronuclei relative to the total number of micronuclei. Unpaired t test: **p < 0.004 (F) Immunoblot of DLD-1 cell extracts to measure total CENP-A and CENP-B levels after siRNA treatment; a-tubulin was used as a loading control. (G) Bars represent the total number of micronuclei after the indicated siRNA treatment. Error bars represent the SEM of three independent experiments.

Developmental Cell 33, 314–327, May 4, 2015 ª2015 Elsevier Inc. 323 Finally, our evidence for a direct contribution of CENP-B to CENP-C Gene Targeting with TALENs CENP-C stabilization at centromeres together with the discovery TALENs were assembled using the Golden Gate cloning strategy and library as of a direct interaction between CENP-A and CENP-B (via the described previously (Cermak et al., 2011) and cloned into a modified version of pcDNA3.1 (Invitrogen) also containing the Fok I endonuclease domain as CENP-A amino-terminal tail) demonstrates a redundant, mutual previously described (Miller et al., 2011). TALENs were designed to the C-ter- dependency among CENP-A, CENP-B, and CENP-C for centro- minal region of CENP-C gene: GAGGAAAGTGTTCTTC and GGTTGATCTTT mere association and function. Unresolved is whether individual CATC. DLD-1 cells were co-transfected with the TALEN expression vectors CENP-C dimers bind simultaneously to both CENP-A and and the donor cassette (containing the two homology arms for CENP-C C-ter- CENP-B. Thus, whereas human centromeres are specified pri- minal region and the AID and EYFP gene) by nucleofection (Lonza) using pro- marily by an epigenetic mechanism mediated through CENP- gram T-020, and positive clones were selected with FACS. A-containing chromatin, we identified a sequence-dependent Protein Purification component to centromere function provided by the centromeric GST tagged human CENP-A (1–29 or 1–44 amino acids) and 6xHis tagged DNA binding protein CENP-B. We now propose that CENP-B CENP-B variants were expressed in Rosetta E. coli after induction with does this by supporting a parallel pathway for CENP-A stabiliza- IPTG. GST-CENP-C variants were overexpressed in Sf9/High-Five insect cells tion and kinetochore formation via CENP-C recruitment. This using the Bac-to-Bac expression system (Invitrogen). Briefly, genes encoding CENP-B-mediated enhancement in CENP-C binding at centro- GST tagged human CENP-C variants were cloned into pFastBac1. Bacmids meres acts as a backup mechanism to ensure chromosome were obtained from DH10Bac E. coli after transformation with the pFastBac1 containing CENP-C gene and used to transfect Sf9 cells (72 hr) to produce ba- segregation when CENP-A levels are compromised (Figure 7D). culovirus. Hi-Five insect cells were infected by CENP-C baculovirus (1:10 dilu- This might explain the observation that centromere function is tion) for 48 hr, collected, and frozen until required. Cells were resuspended in still partially retained even in cells with as little as 1% of the orig- PBS containing protease inhibitors and lysed by sonication. Soluble lysate was inal CENP-A level (Fachinetti et al., 2013). recovered after centrifugation and proteins were affinity purified over nickel-ni- Although other natural metoazoan centromeres had been trilotriacetic acid sepharose beads (QIAGEN) or glutathione sepharose beads identified to be without repetitive sequences including horses (GE Healthcare Life Sciences). (Wade et al., 2009), chickens (Shang et al., 2010), and orangu- In Vitro Binding Assay tans (Locke et al., 2011), the overall evidence implies that repet- In vitro binding assays were conducted in 50 mM Tris-HCl (pH 7.7), 100 mM itive alphoid sequences can provide increased fidelity to human KCl, 0.1% Triton X-100, and 10% glycerol. For the GST pull-down assay, centromere function, consistent with the suggestion that the glutathione sepharose-bound GST-tagged protein and other recombinant observed human neocentromeres are likely to be centromeres proteins were combined and incubated at room temperature for 1 hr. Bound ‘‘in progress’’ (Marshall et al., 2008). Incorporation of repetitive protein complexes were washed four times with 20 volumes of the binding sequences during evolution is therefore likely to have enhanced buffer, eluted from the beads in 15 mM glutathione buffer. For the His pull- down assay, nickel sepharose-bound 63His-tagged protein and other recom- full centromere maturation and fixation, as proposed in primates binant proteins were combined and incubated in the presence of 20 mM (Ventura et al., 2007). Additionally, it is tantalizing to speculate imidazole at 4C for 1 hr. Bound protein complexes were washed four times that a-satellites and CENP-B play a role in the meiotic drive for with 20 volumes of the binding buffer supplemented with 20 mM imidazole, egg specification (the preferential segregation of one chromo- and eluted from the beads in 200 mM imidazole buffer. Eluted proteins were some over another) (Dawe and Henikoff, 2006; Henikoff et al., analyzed by immunoblotting. 2001), not only by supporting CENP-A binding (Chma´ tal et al., FISH Experiment 2014; Marshall and Choo, 2012), but also by actively reinforcing Cells were fixed in Carnoy’s Fixative (Meth-Acetic Acid 3:1) for 15 min at room centromere function. temperature, rinsed in 80% ethanol, and air-dried for 5 min. Probe mixtures (MetaSystems) were applied and sealed with a coverslip. Slides were dena- EXPERIMENTAL PROCEDURES tured at 75C for 2 min and incubated at 37C overnight in a humidified cham- ber. Slides were washed with 0.43SCC at 72C for 2 min, 23SCC, 0.05% siRNA, SNAP-Tagging, Clonogenic Colony Assay, and Adeno-Cre Tween-20 at room temperature for 1 min, and rinsed with ddH2O. Slides Treatment were incubated with DAPI solution for 10 min before mounting in anti-fade siRNAs were introduced using Lipofectamine RNAiMax (Invitrogen). A pool of reagent. four siRNAs directed against CENP-C, CENP-B, HJURP, GAPDH (Fachinetti For information regarding DNA constructs, cell culture conditions, genera- et al., 2013), and CENP-A (GAGCACACACCUCUUGAUA, UUACAUGCAGG tion of stable cell lines, the surveyor nuclease assay, antibodies and experi- CCGAGUUA, AGACAAGGUUGGCUAAAGG, UAAAUUCACUCGUGGUGUG) mental conditions for immunoblotting and immunofluorescence, live-cell were purchased from Dharmacon. SNAP labeling was conducted as microscopy and procedures for centromere quantifications, see the Supple- described previously (Jansen et al., 2007). Clonogenic colony assays and mental Experimental Procedures. Adeno-Cre treatment were done as described (Fachinetti et al., 2013). SUPPLEMENTAL INFORMATION CENP-B Gene Deletion by the CRISPR/Cas9 System An expression vector (Addgene plasmid 42230) expressing cas9 and sgRNA Supplemental Information includes Supplemental Experimental Procedures (Cong et al., 2013) was digested with BbsI and the linearized vector was gel and four figures and can be found with this article online at http://dx.doi.org/ purified. A pair of complementary oligos to the targeting site (50 GAAGAA 10.1016/j.devcel.2015.03.020. CAAGCGCGCCATCC 30) were annealed and ligated into the vector. RPE-1 CENP-Af/- cells were co-transfected with the sgRNA expression vector and AUTHOR CONTRIBUTIONS a GFP expressing vector (to identify transfected cells) by nucleofection (Lonza) using program U-017 and nucleofector solution V (Lonza). Forty-eight hours J.S.H. performed in vitro protein purification. D.F. and J.S.H. performed the after transfection, high GFP-expressing cells were single cell sorted into 96- binding assay. D.F. and M.A.M. performed gene targeting of CENP-B and well plates using a fluorescence in situ hybridization (FACS)Aria II. Genomic CENP-C. D.F. and P.L. performed FISH experiments. D.F., A.A., and A.J.W. DNA sequencing, immunostaining, and immunoblotting were used to identify performed all the remaining experiments. D.F. and D.W.C. conceived the cells with disruption of both targeted alleles. experimental design, analyzed the data, and wrote the manuscript.

324 Developmental Cell 33, 314–327, May 4, 2015 ª2015 Elsevier Inc. ACKNOWLEDGMENTS Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., and Zhang, F. (2013). Multiplex genome engineering The authors thank William C. Earnshaw (Wellcome Trust Centre for Cell using CRISPR/Cas systems. Science 339, 819–823. Biology, University of Edinburgh), P. Kalitsis (Murdoch Childrens Research Dambacher, S., Deng, W., Hahn, M., Sadic, D., Fro¨ hlich, J., Nuber, A., Institute Victoria, Australia), A. Desai, R. Khaliullin, Neil Hattersley, and Hoischen, C., Diekmann, S., Leonhardt, H., and Schotta, G. (2012). CENP-C C. Bartocci (Ludwig, La Jolla, California), F. Dibazar, Y. Arbely, B. Vitre, facilitates the recruitment of M18BP1 to centromeric chromatin. Nucleus 3, and others members of the Cleveland lab for helpful suggestions. We thank 101–110. P. Maddox (University of North Carolina), I. Cheeseman (MIT, Boston), A. De- Dawe, R.K., and Henikoff, S. (2006). Centromeres put epigenetics in the sai (Ludwig, La Jolla, California), P. Kalitsis (Murdoch Children’s Research driver’s seat. Trends Biochem. Sci. 31, 662–669. Institute Victoria, Australia), H. Masumoto (Kazusa DNA Research Institute, Japan), and B.E. Black (University of Pennsylvania, Philadelphia) for Depinet, T.W., Zackowski, J.L., Earnshaw, W.C., Kaffe, S., Sekhon, G.S., providing reagents. We also thank the Neuroscience Microscopy Shared Fa- Stallard, R., Sullivan, B.A., Vance, G.H., Van Dyke, D.L., Willard, H.F., et al. cility (P30 NS047101, University of California, San Diego) and the FACS facil- (1997). Characterization of neo-centromeres in marker chromosomes lacking ity in the Sanford Consortium for Regenerative Medicine, La Jolla, California. detectable alpha-satellite DNA. Hum. Mol. Genet. 6, 1195–1204. This work was supported by a grant from the NIH (R01-GM 074150 to Dumanski, J.P., Rasi, C., Lo¨ nn, M., Davies, H., Ingelsson, M., Giedraitis, V., D.W.C.). D.W.C. receives salary support from the Ludwig Institute for Cancer Lannfelt, L., Magnusson, P.K.E., Lindgren, C.M., Morris, A.P., et al. (2015). Research. Mutagenesis. Smoking is associated with mosaic loss of chromosome Y. Science 347, 81–83. Received: December 21, 2014 Dunleavy, E.M., Roche, D., Tagami, H., Lacoste, N., Ray-Gallet, D., Nakamura, Revised: February 9, 2015 Y., Daigo, Y., Nakatani, Y., and Almouzni-Pettinotti, G. (2009). HJURP is a cell- Accepted: March 25, 2015 cycle-dependent maintenance and deposition factor of CENP-A at centro- Published: May 4, 2015 meres. Cell 137, 485–497. Earnshaw, W.C., and Rothfield, N. (1985). Identification of a family of human REFERENCES centromere proteins using autoimmune sera from patients with scleroderma. Chromosoma 91, 313–321. Amor, D.J., Bentley, K., Ryan, J., Perry, J., Wong, L., Slater, H., and Choo, K.H. (2004). Human centromere repositioning ‘‘in progress’’. Proc. Natl. Acad. Sci. Earnshaw, W.C., Sullivan, K.F., Machlin, P.S., Cooke, C.A., Kaiser, D.A., USA 101, 6542–6547. Pollard, T.D., Rothfield, N.F., and Cleveland, D.W. (1987). Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen. J. Cell Biol. Barnhart, M.C., Kuich, P.H.J.L., Stellfox, M.E., Ward, J.A., Bassett, E.A., Black, 104, 817–829. B.E., and Foltz, D.R. (2011). HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore. J. Cell Biol. 194, 229–243. Earnshaw, W.C., Ratrie, H., 3rd, and Stetten, G. (1989). Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in Bassett, E.A., Wood, S., Salimian, K.J., Ajith, S., Foltz, D.R., and Black, B.E. cytological spreads. Chromosoma 98, 1–12. (2010). Epigenetic centromere specification directs aurora B accumulation but is insufficient to efficiently correct mitotic errors. J. Cell Biol. 190, 177–185. Ekwall, K. (2007). Epigenetic control of centromere behavior. Annu. Rev. Genet. 41, 63–81. Bergmann, J.H., Rodrı´guez, M.G., Martins, N.M., Kimura, H., Kelly, D.A., Masumoto, H., Larionov, V., Jansen, L.E., and Earnshaw, W.C. (2011). Fachinetti, D., Folco, H.D., Nechemia-Arbely, Y., Valente, L.P., Nguyen, K., Epigenetic engineering shows H3K4me2 is required for HJURP targeting Wong, A.J., Zhu, Q., Holland, A.J., Desai, A., Jansen, L.E.T., and Cleveland, and CENP-A assembly on a synthetic human kinetochore. EMBO J. 30, D.W. (2013). A two-step mechanism for epigenetic specification of centromere 328–340. identity and function. Nat. Cell Biol. 15, 1056–1066. Black, B.E., Foltz, D.R., Chakravarthy, S., Luger, K., Woods, V.L., Jr., and Foltz, D.R., Jansen, L.E.T., Black, B.E., Bailey, A.O., Yates, J.R., 3rd, and Cleveland, D.W. (2004). Structural determinants for generating centromeric Cleveland, D.W. (2006). The human CENP-A centromeric nucleosome-associ- chromatin. Nature 430, 578–582. ated complex. Nat. Cell Biol. 8, 458–469. Black, B.E., Brock, M.A., Be´ dard, S., Woods, V.L., Jr., and Cleveland, D.W. Foltz, D.R., Jansen, L.E.T., Bailey, A.O., Yates, J.R., 3rd, Bassett, E.A., Wood, (2007a). An epigenetic mark generated by the incorporation of CENP-A into S., Black, B.E., Cleveland, D.W., and Cleveland, D.W. (2009). Centromere- centromeric nucleosomes. Proc. Natl. Acad. Sci. USA 104, 5008–5013. specific assembly of CENP-a nucleosomes is mediated by HJURP. Cell 137, 472–484. Black, B.E., Jansen, L.E.T., Maddox, P.S., Foltz, D.R., Desai, A.B., Shah, J.V., and Cleveland, D.W. (2007b). Centromere identity maintained by nucleosomes Forsberg, L.A., Rasi, C., Malmqvist, N., Davies, H., Pasupulati, S., Pakalapati, assembled with histone H3 containing the CENP-A targeting domain. Mol. Cell G., Sandgren, J., Diaz de Sta˚ hl, T., Zaghlool, A., Giedraitis, V., et al. (2014). 25, 309–322. Mosaic loss of chromosome Y in peripheral blood is associated with shorter Bodor, D.L., Mata, J.F., Sergeev, M., David, A.F., Salimian, K.J., Panchenko, survival and higher risk of cancer. Nat. Genet. 46, 624–628. T., Cleveland, D.W., Black, B.E., Shah, J.V., and Jansen, L.E. (2014). The quan- Fowler, K.J., Hudson, D.F., Salamonsen, L.A., Edmondson, S.R., Earle, E., titative architecture of centromeric chromatin. eLife 3, e02137. Sibson, M.C., and Choo, K.H. (2000). Uterine dysfunction and genetic modi- Carroll, C.W., Silva, M.C.C., Godek, K.M., Jansen, L.E.T., and Straight, A.F. fiers in centromere protein B-deficient mice. Genome Res. 10, 30–41. (2009). Centromere assembly requires the direct recognition of CENP-A nucle- Fowler, K.J., Wong, L.H., Griffiths, B.K., Sibson, M.C., Reed, S., and Choo, osomes by CENP-N. Nat. Cell Biol. 11, 896–902. K.H.A. (2004). Centromere protein b-null mice display decreasing reproductive Carroll, C.W., Milks, K.J., and Straight, A.F. (2010). Dual recognition of CENP-A performance through successive generations of breeding due to diminishing nucleosomes is required for centromere assembly. J. Cell Biol. 189, 1143– endometrial glands. Reproduction 127, 367–377. 1155. Fukagawa, T., and Earnshaw, W.C. (2014). The centromere: chromatin foun- Cermak, T., Doyle, E.L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, dation for the kinetochore machinery. Dev. Cell 30, 496–508. J.A., Somia, N.V., Bogdanove, A.J., and Voytas, D.F. (2011). Efficient design Fukagawa, T., Pendon, C., Morris, J., and Brown, W. (1999). CENP-C is neces- and assembly of custom TALEN and other TAL effector-based constructs sary but not sufficient to induce formation of a functional centromere. EMBO J. for DNA targeting. Nucleic Acids Res. 39, e82–e82. 18, 4196–4209. Chma´ tal, L., Gabriel, S.I., Mitsainas, G.P., Martıńez-Vargas, J., Ventura, J., Gopalakrishnan, S., Sullivan, B.A., Trazzi, S., Della Valle, G., and Robertson, Searle, J.B., Schultz, R.M., and Lampson, M.A. (2014). Centromere strength K.D. (2009). DNMT3B interacts with constitutive centromere protein CENP-C provides the cell biological basis for meiotic drive and karyotype evolution in to modulate DNA methylation and the histone code at centromeric regions. mice. Curr. Biol. 24, 2295–2300. Hum. Mol. Genet. 18, 3178–3193.

Developmental Cell 33, 314–327, May 4, 2015 ª2015 Elsevier Inc. 325 Guse, A., Carroll, C.W., Moree, B., Fuller, C.J., and Straight, A.F. (2011). In vitro Moree, B., Meyer, C.B., Fuller, C.J., and Straight, A.F. (2011). CENP-C recruits centromere and kinetochore assembly on defined chromatin templates. M18BP1 to centromeres to promote CENP-A chromatin assembly. J. Cell Biol. Nature 477, 354–358. 194, 855–871. Henikoff, S., Ahmad, K., and Malik, H.S. (2001). The centromere paradox: sta- Mu¨ ller, S., and Almouzni, G. (2014). A network of players in H3 histone variant ble inheritance with rapidly evolving DNA. Science 293, 1098–1102. deposition and maintenance at centromeres. Biochim. Biophys. Acta 1839, Holland, A.J., Fachinetti, D., Han, J.S., and Cleveland, D.W. (2012). Inducible, 241–250. reversible system for the rapid and complete degradation of proteins in Muro, Y., Masumoto, H., Yoda, K., Nozaki, N., Ohashi, M., and Okazaki, T. mammalian cells. Proc. Natl. Acad. Sci. USA 109, E3350–E3357. (1992). Centromere protein B assembles human centromeric alpha-satellite Hori, T., and Fukagawa, T. (2012). Establishment of the vertebrate kineto- DNA at the 17-bp sequence, CENP-B box. J. Cell Biol. 116, 585–596. chores. Chromosome Res. 20, 547–561. Nath, J., Tucker, J.D., and Hando, J.C. (1995). Y chromosome aneuploidy, mi- Hori, T., Shang, W.H., Takeuchi, K., and Fukagawa, T. (2013). The CCAN re- cronuclei, kinetochores and aging in men. Chromosoma 103, 725–731. cruits CENP-A to the centromere and forms the structural core for kinetochore Ohzeki, J., Nakano, M., Okada, T., and Masumoto, H. (2002). CENP-B box is assembly. J. Cell Biol. 200, 45–60. required for de novo centromere chromatin assembly on human alphoid DNA. Hudson, D.F., Fowler, K.J., Earle, E., Saffery, R., Kalitsis, P., Trowell, H., Hill, J., J. Cell Biol. 159, 765–775. Wreford, N.G., de Kretser, D.M., Cancilla, M.R., et al. (1998). Centromere pro- Okada, T., Ohzeki, J., Nakano, M., Yoda, K., Brinkley, W.R., Larionov, V., and tein B null mice are mitotically and meiotically normal but have lower body and Masumoto, H. (2007). CENP-B controls centromere formation depending on testis weights. J. Cell Biol. 141, 309–319. the chromatin context. Cell 131, 1287–1300. Ikeno, M., Grimes, B., Okazaki, T., Nakano, M., Saitoh, K., Hoshino, H., McGill, Okamoto, Y., Nakano, M., Ohzeki, J., Larionov, V., and Masumoto, H. (2007). A N.I., Cooke, H., and Masumoto, H. (1998). Construction of YAC-based minimal CENP-A core is required for nucleation and maintenance of a func- mammalian artificial chromosomes. Nat. Biotechnol. 16, 431–439. tional human centromere. EMBO J. 26, 1279–1291. Jansen, L.E.T., Black, B.E., Foltz, D.R., and Cleveland, D.W. (2007). Palmer, D.K., O’Day, K., Wener, M.H., Andrews, B.S., and Margolis, R.L. Propagation of centromeric chromatin requires exit from mitosis. J. Cell Biol. (1987). A 17-kD centromere protein (CENP-A) copurifies with nucleosome 176, 795–805. core particles and with histones. J. Cell Biol. 104, 805–815. Jeganathan, K., Malureanu, L., Baker, D.J., Abraham, S.C., and van Deursen, Perez-Castro, A.V., Shamanski, F.L., Meneses, J.J., Lovato, T.L., Vogel, K.G., J.M. (2007). Bub1 mediates cell death in response to chromosome missegre- Moyzis, R.K., and Pedersen, R. (1998). Centromeric protein B null mice are gation and acts to suppress spontaneous tumorigenesis. J. Cell Biol. 179, viable with no apparent abnormalities. Dev. Biol. 201, 135–143. 255–267. Przewloka, M.R., Venkei, Z., Bolanos-Garcia, V.M., Debski, J., Dadlez, M., and Kapoor, M., Montes de Oca Luna, R., Liu, G., Lozano, G., Cummings, C., Glover, D.M. (2011). CENP-C is a structural platform for kinetochore assembly. Mancini, M., Ouspenski, I., Brinkley, B.R., and May, G.S. (1998). The cenpB Curr. Biol. 21, 399–405. gene is not essential in mice. Chromosoma 107, 570–576. du Sart, D., Cancilla, M.R., Earle, E., Mao, J.I., Saffery, R., Tainton, K.M., Karpen, G.H., and Allshire, R.C. (1997). The case for epigenetic effects on Kalitsis, P., Martyn, J., Barry, A.E., and Choo, K.H. (1997). A functional neocentromere identity and function. Trends Genet. 13, 489–496. centromere formed through activation of a latent human centromere and con- Kato, H., Jiang, J., Zhou, B.R., Rozendaal, M., Feng, H., Ghirlando, R., Xiao, sisting of non-alpha-satellite DNA. Nat. Genet. 16, 144–153. T.S., Straight, A.F., and Bai, Y. (2013). A conserved mechanism for centro- Screpanti, E., De Antoni, A., Alushin, G.M., Petrovic, A., Melis, T., Nogales, E., meric nucleosome recognition by centromere protein CENP-C. Science 340, and Musacchio, A. (2011). Direct binding of Cenp-C to the Mis12 complex joins 1110–1113. the inner and outer kinetochore. Curr. Biol. 21, 391–398. Kim, I.S., Lee, M., Park, K.C., Jeon, Y., Park, J.H., Hwang, E.J., Jeon, T.I., Ko, Shang, W.-H., Hori, T., Toyoda, A., Kato, J., Popendorf, K., Sakakibara, Y., S., Lee, H., Baek, S.H., and Kim, K.I. (2012). Roles of Mis18a in epigenetic regu- Fujiyama, A., and Fukagawa, T. (2010). Chickens possess centromeres with lation of centromeric chromatin and CENP-A loading. Mol. Cell 46, 260–273. both extended tandem repeats and short non-tandem-repetitive sequences. Liu, S.T., Rattner, J.B., Jablonski, S.A., and Yen, T.J. (2006). Mapping the as- Genome Res. 20, 1219–1228. sembly pathways that specify formation of the trilaminar kinetochore plates in human cells. J. Cell Biol. 175, 41–53. Shelby, R.D., Vafa, O., and Sullivan, K.F. (1997). Assembly of CENP-A into centromeric chromatin requires a cooperative array of nucleosomal DNA con- Locke, D.P., Hillier, L.W., Warren, W.C., Worley, K.C., Nazareth, L.V., Muzny, tact sites. J. Cell Biol. 136, 501–513. D.M., Yang, S.-P., Wang, Z., Chinwalla, A.T., Minx, P., et al. (2011). Comparative and demographic analysis of orang-utan genomes. Nature Shuaib, M., Ouararhni, K., Dimitrov, S., and Hamiche, A. (2010). HJURP binds 469, 529–533. CENP-A via a highly conserved N-terminal domain and mediates its deposition at centromeres. Proc. Natl. Acad. Sci. USA 107, 1349–1354. Marshall, O.J., and Choo, K.H.A. (2012). Putative CENP-B paralogues are not present at mammalian centromeres. Chromosoma 121, 169–179. Stimpson, K.M., and Sullivan, B.A. (2010). Epigenomics of centromere assembly and function. Curr. Opin. Cell Biol. 22, 772–780. Marshall, O.J., Chueh, A.C., Wong, L.H., and Choo, K.H.A. (2008). Neocentromeres: new insights into centromere structure, disease develop- Sullivan, K.F., and Glass, C.A. (1991). CENP-B is a highly conserved mamma- ment, and karyotype evolution. Am. J. Hum. Genet. 82, 261–282. lian centromere protein with homology to the helix-loop-helix family of proteins. Chromosoma 100, 360–370. Mendiburo, M.J., Padeken, J., Fu¨ lo¨ p, S., Schepers, A., and Heun, P. (2011). Drosophila CENH3 is sufficient for centromere formation. Science 334, Suzuki, N., Nakano, M., Nozaki, N., Egashira, S., Okazaki, T., and 686–690. Masumoto, H. (2004). CENP-B interacts with CENP-C domains containing Michel, L.S., Liberal, V., Chatterjee, A., Kirchwegger, R., Pasche, B., Gerald, Mif2 regions responsible for centromere localization. J. Biol. Chem. 279, W., Dobles, M., Sorger, P.K., Murty, V.V.V.S., and Benezra, R. (2001). MAD2 5934–5946. haplo-insufficiency causes premature anaphase and chromosome instability Thompson, S.L., and Compton, D.A. (2011). Chromosome missegregation in in mammalian cells. Nature 409, 355–359. human cells arises through specific types of kinetochore-microtubule attach- Miga, K.H., Newton, Y., Jain, M., Altemose, N., Willard, H.F., and Kent, W.J. ment errors. Proc. Natl. Acad. Sci. USA 108, 17974–17978. (2014). Centromere reference models for human chromosomes X and Y satel- Ventura, M., Weigl, S., Carbone, L., Cardone, M.F., Misceo, D., Teti, M., lite arrays. Genome Res. 24, 697–707. D’Addabbo, P., Wandall, A., Bjo¨ rck, E., de Jong, P.J., et al. (2004). Miller, J.C., Tan, S., Qiao, G., Barlow, K.A., Wang, J., Xia, D.F., Meng, X., Recurrent sites for new centromere seeding. Genome Res. 14, 1696–1703. Paschon, D.E., Leung, E., Hinkley, S.J., et al. (2011). A TALE nuclease architec- Ventura, M., Antonacci, F., Cardone, M.F., Stanyon, R., D’Addabbo, P., ture for efficient genome editing. Nat. Biotechnol. 29, 143–148. Cellamare, A., Sprague, L.J., Eichler, E.E., Archidiacono, N., and Rocchi, M.

326 Developmental Cell 33, 314–327, May 4, 2015 ª2015 Elsevier Inc. (2007). Evolutionary formation of new centromeres in macaque. Science 316, Warburton, P.E. (2004). Chromosomal dynamics of human neocentromere for- 243–246. mation. Chromosome Res. 12, 617–626. Verdaasdonk, J.S., and Bloom, K. (2011). Centromeres: unique chromatin Warburton, P.E., Cooke, C.A., Bourassa, S., Vafa, O., Sullivan, B.A., structures that drive chromosome segregation. Nat. Rev. Mol. Cell Biol. 12, Stetten, G., Gimelli, G., Warburton, D., Tyler-Smith, C., Sullivan, K.F., 320–332. et al. (1997). Immunolocalization of CENP-A suggests a distinct nucleosome Wade, C.M., Giulotto, E., Sigurdsson, S., Zoli, M., Gnerre, S., Imsland, F., Lear, structure at the inner kinetochore plate of active centromeres. Curr. Biol. 7, T.L., Adelson, D.L., Bailey, E., Bellone, R.R., et al.; Broad Institute Genome 901–904. Sequencing Platform; Broad Institute Whole Genome Assembly Team Weaver, B.A.A., Silk, A.D., Montagna, C., Verdier-Pinard, P., and Cleveland, (2009). Genome sequence, comparative analysis, and population genetics of D.W. (2007). Aneuploidy acts both oncogenically and as a tumor suppressor. the domestic horse. Science 326, 865–867. Cancer Cell 11, 25–36.

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