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CENP-A Ubiquitylation Is Inherited through Dimerization between Cell Divisions

SUMMARY The presence of chromatin containing the histone H3 variant CENP-A dictates the location of the centromere in a DNA sequence–independent manner. But the mechanism by which centromere inheritance occurs is largely unknown. We previously reported that CENP-A K124 ubiquitylation, mediated by CUL4A-RBX1-COPS8 E3 ligase activity, is required for CENP-A deposition at the centromere. Here we show that pre-existing ubiquitylated CENP-A is necessary for recruitment of newly synthesized CENP-A to the centromere and that CENP-A ubiquitylation is inherited between cell divisions. In vivo and in vitro analyses using dimerization mutants and dimerization domain fusion mutants revealed that the inheritance of CENP-A ubiquitylation requires CENP-A dimerization. Therefore, we propose models in which CENP-A ubiquitylation is inherited and, through dimerization, determines centromere location. Consistent with this model is our finding that overexpression of a monoubiquitin-fused CENP-A mutant induces neocentromeres at noncentromeric regions of chromosomes.


INTRODUCTION
CENP-A is a centromere-specific histone H3 variant that is required to ensure kinetochore assembly for proper chromosome segregation; defects in CENP-A function lead to aneuploidy and thereby cancer (Amato et al., 2009;Au et al., 2008;Heun et al., 2006; neocentromeres are important in clarifying the mechanism of epigenetic inheritance of centromeres. In our previous study, we showed that CENP-A K124 ubiquitylation serves as a signal for the deposition of CENP-A at centromeres (Niikura et al., 2015). Here we report that CENP-A K124 ubiquitylation is epigenetically inherited through dimerization. Based on this molecular mechanism, models in which the location of the centromere is inherited are proposed.

CENP-A Monoubiquitylation or Diubiquitylation Is Epigenetically Inherited between Cell Divisions
It has been suggested that the epigenetic centromere mark is generated through a "templategoverned" mechanism (Mendiburo et al., 2011;Raychaudhuri et al., 2012): the preassembled "old" CENP-A nucleosomes may act as a template, allowing the local stoichiometric loading of new CENP-A nucleosomes during each cell cycle. We have previously shown that CENP-A K124 ubiquitylation serves as a signal for the deposition of CENP-A at centromeres (Niikura et al., 2015). Therefore, we hypothesized a model in which CENP-A K124 ubiquitylation is epigenetically inherited ( Figure 1A). This model predicts that CENP-A K124 ubiquitylation depends on pre-existing K124-ubiquitylated CENP- A. In human cells, 10% of the normal level of CENP-A is sufficient to drive kinetochore assembly (Liu et al., 2006). We performed CENP-A siRNA knockdown, targeting the 5' and 3' untranslated regions of CENP-A mRNA to reduce the quantity of endogenous CENP-A to 7.2% of its normal level in HeLa cells ( Figure S1A and Table S2, CA-UTR #1). This severe loss of endogenous CENP-A prevented endogenous CENP-C localization at centromeres (Figures S1B and S1C); this effect confirmed the previous result (Liu et al., 2006). The severe loss of endogenous CENP-A also substantially abrogated ubiquitylation ( Figure 1A, panel [2]; Figures 1B and 1C,sample [2] in the histogram) and centromere localization of exogenously coexpressed CENP-A WT (wild type)-Flag (Figure 1A,panel [2]; Figures 1D and 1E,sample [2] in the histogram; representative images and histograms of other cell cycle stages are shown in Figures S1D and S1E). We further confirmed the localization of Flag-tagged CENP-A proteins by chromosome spreading (Figure S1F,sample [2]). These results suggest that the presence of endogenous CENP-A is required for ubiquitylation of newly synthesized CENP-A and for centromere localization. In this experiment, we confirmed that the maximum expression level of CENP-A WT-Flag was achieved when a substantial amount of endogenous CENP-A was already depleted from cells (reduced to 5.0% of the normal level) 48 h after cotransfection ( Figure 1F).
To confirm the result obtained in our experiment in which CENP-A was depleted, we used CENP-A KO (knock-out) cells (CENP-A −/F RPE1 cells) (Fachinetti et al., 2013) instead of siRNA. Fachinetti et al. reported that only 1% of the initial CENP-A level is detectable in 7 days (as expected for the 1/2 7 dilution, which results in a concentration of approximately 0.8%) after Ad-Cre infection and that no centromere-bound CENP-A was detected 9 days following the excision of CENP-A alleles (Fachinetti et al., 2013). Consistent with these findings, the endogenous CENP-A level was reduced to approximately 2% of the initial level in 6 days ( Figure S2A, endo. CENP-A; as expected for the 1/2 6 dilution, which results in a concentration of approximately 1.6%) and to less than 1% of the initial level in 7 days (data not shown) after transient expression of Cre recombinase (using retro-Cre, a retrovirus vector that induces excision of exons 3 and 4 of the floxed allele). In our experiment, the retrovirus expressed exogenous Flag-CENP-A WT 4 days after retro-Cre infection ( Figure  S2A, exo. Flag-CENP-A). Two days after infection by the Flag-CENP-A-expressing retrovirus, the severe loss of endogenous centromeric CENP-A substantially abrogated ubiquitylation ( Figure 1A ). We confirmed that exogenous "untagged" CENP-A WT ( Figure S2C, second lane from left) did not localize to the centromere when endogenous CENP-A was reduced to approximately to 2% of the initial level after 6 days of retro-Cre infection (Figures S2F and S2G,sample [2]); this result eliminated the possibility that "Flag-tagged" CENP-A WT did not localize to the centromere because it is dysfunctional. These results confirmed the finding that the presence of ubiquitylated CENP-A is required for ubiquitylation and centromere localization of newly synthesized CENP-A.
Previously, we showed that 6xHis-CENP-A WT is monoubiquitylated by the purified Cul4A-Rbx1-COPS8 complex in vitro (Niikura et al., 2015). Therefore, we examined whether the in vivo results are true in vitro. Addition of purified monoubiquitin-fused GST-CENP-A K124R (GST-CENP-A K124R-Ub (K48R]) into the reactions enhanced ubiquitylation of 6xHis-CENP-A WT (Figure 2A, compare lanes 3-5; see Figure S3A for verification of purified recombinant proteins). Such enhancement supports the in vivo results.
If the presence of ubiquitylated CENP-A is required for ubiquitylation of newly synthesized CENP-A, why can purified 6xHis-CENP-A WT be ubiquitylated in vitro? We assumed that 6xHis-CENP-A WT expressed in insect cells contained some ubiquitylated 6xHis-CENP-A, but the levels were undetectable. Therefore, we depleted presumably existing ubiquitylated CENP-A from purified 6xHis-CENP-A WT by using Agarose-TUBE 2 (LifeSensors). In vitro ubiquitylation was then performed with the remaining nonubiquitylated 6xHis-CENP- We also tested bacterially expressed and purified CENP-A, which is not ubiquitylated ( Figure S3C), as substrate for in vitro ubiquitylation assay, and consistent results were obtained ( Figure S3D, compare lanes 2 and 3).
Taken together, our results indicate that pre-existing ubiquitylated CENP-A is required for ubiquitylation of newly synthesized CENP-A and for centromere localization. Thus, CENP-A ubiquitylation appears to be inherited epigenetically between cell divisions.

CENP-A Dimerization through H115/L128 Is Required for CENP-A K124 Ubiquitylation
If our "epigenetic" model is correct ( Figure 1A), then the CUL4A E3 complex should recognize a heterodimer of nonubiquitylated CENP-A and K124-ubiquitylated CENP-A (hemiubiquitylation) but not a homodimer of nonubiquitylated CENP-A ( Figure 1A). In this case, heterodimerization would be required for K124 ubiquitylation of nonubiquitylated CENP-A. Thus, CENP-A K124 ubiquitylation would depend on pre-existing K124ubiquitylated CENP-A. In Drosophila melanogaster, disruption of the dimerization interface of CENP-A/CID reduces its centromere localization in vivo . In human cells, SDS-resistant CENP-A dimers have been reported (Niikura et al., 2015;Shelby et al., 1997;Yoda et al., 2000). Bassett et al. reported that a human CENP-A dimerization mutant (CENP-A H115A/L128A ) cannot stably assemble into chromatin (Bassett et al., 2012). Consistent with this finding, the CENP-A H115A/L128A mutation reduced dimerization of CENP-A in cell lysates ( Figure S3F) and abrogated dimerization in immunoprecipitation analysis ( Figure 3A). Moreover, the CENP-A H115A/L128A mutation abrogated ubiquitylation of CENP-A in vivo ( Figure 3B) and localization of CENP-A to the centromeres ( Figures 3C and 3D; chromosome spreads are shown in Figures S3G). It should be noted that monoubiqutin-fused CENP-A H115A/L128A was not able to interact with CENP-A WT properly ( Figure S3H, lanes 4 and 5). These results suggested that dimerization is required for CENP-A localization to the centromere.
We could not conclude that dimerization is directly required for CENP-A K124 ubiquitylation, because the dimerization mutant may disturb proper nucleosome formation. Therefore, we examined ubiquitylation of CENP-A H115A/L128A in vitro. Indeed, CENP-A H115A/L128A protein was not ubiquitylated in vitro ( Figure 2A, lanes 6-8; Figure 3E, lane 5; see Figure S3A for verification of purified recombinant proteins), and this absence of ubiquitylation strongly suggested that dimerization is required for CENP-A K124 ubiquitylation.

Heterodimerization with Ubiquitylated CENP-A Is Required to Ubiquitylate New CENP-A
To evaluate the importance of heterodimerization (i.e., a dimerization of old CENP-A -new CENP-A) for CENP-A localization at the centromere, we hypothesized that the loss of CENP-A dimerization and centromere localization by the H115A/L128A mutation could be rescued by the addition of a dimerization domain that has previously been used to force the dimerization of specific proteins (Wang et al., 2002). To test this hypothesis, we fused 28 or 24 amino acids derived from the dimerization domain of the Saccharomyces cerevisiae PUT3 protein (ScPUT3 dimerization domain; Figure S4A, "PD") or the Drosophila melanogaster Ncd (DmNcd dimerization domain; Figure Figure S6). In summary, the status of centromere localization of each Flag-tagged CENP-A protein matches that of ubiquitylation ( Figure S4A, right two columns). These results indicate that CENP-A heterodimerization with pre-existing ubiquitylated CENP-A is required for ubiquitylation and centromere localization of new CENP-A.

Overexpression of Monoubiquitin-Fused CENP-A K124R Induces Formation of Neocentromeres in Human Cells
In Drosophila cell lines, CENP-A overexpression causes mislocalization of CENP-A into noncentromeric regions (Heun et al., 2006). These ectopic centromeres are able to attract downstream kinetochore proteins and cause chromosome segregation defects, presumably as a result of dicentric activity (Heun et al., 2006). In humans, overexpression of CENP-A induces misloading of CENP-A at noncentromeric regions and assembly of a subset of kinetochore components, including CENP-C, hSMC1, and HZwint-1 (Van Hooser et al., 2001). However, the microtubule-associated proteins CENP-E and HZW10 were not recruited, and neocentromeric activity was not detected (Van Hooser et al., 2001). Recently, Lacoste et al. reported that ectopic mislocalization of CENP-A in human cells depends on the H3.3 chaperone DAXX rather than the specific centromeric CENP-A chaperone HJURP . We previously reported that addition of monoubiquitin to the CENP-A K124R mutant restores centromere targeting and interaction with HJURP; this finding demonstrated that monoubiquitylation is an essential signaling modification required for efficient interaction with HJURP and subsequent recruitment of CENP-A to centromeres (Niikura et al., 2015). Therefore, we hypothesized that if CENP-A K124 ubiquitylation is required to determine the location of the centromere, overexpression of constitutively ubiquitylated CENP-A K124R-Ub (K48R) could induce the formation of neocentromeres at noncentromeric locations.
We induced transient overexpression of Flag-CENP-A K124R-Ub (K48R) or Flag-CENP-A WT in HeLa Tet-Off cells for 48 h and performed 4-color immunostaining of chromosome spreads (staining for DAPI; Flag; endogenous CENP-B; and endogenous central-outer kinetochore proteins CENP-I, HEC1, or SKA1). Endogenous CENP-B was stained as preassembled native centromeres, and endogenous central-outer kinetochore proteins (CENP-I, HEC1, or SKA1) or chaperone proteins (HJURP or DAXX) were stained as both native and "putative" ectopic (neo-) centromeres. Structures with Flag and central-outer kinetochore protein-positive but CENP-B-negative signals were counted as "putative" neocentromeres at ectopic sites in HeLa Tet-Off cells (the average number of neocentromeres per cell of Flag-K124R-Ub [K48R] was 0.75 as determined by CENP-I immunostaining, 0.59 as determined by HEC1 immunostaining, and 0.46 as determined by SKA1 immunostaining) (Figures 5A-5E, 5G, and 5H).
Because SKA1 requires microtubules to localize to centromeres (Hanisch et al., 2006), we interpreted the result regarding SKA1 immunostaining as indicating that overexpression of constitutively monoubiquitylated CENP-A induces functional neocentromeres. This finding suggests that CENP-A ubiquitylation plays a role to determine the location of the centromere. The number of "paired" putative neocentromeres with SKA1-positive sister chromatids significantly increased from 48 h to 96 h after the induction of Flag-CENP-

CENP-A Ubiquitylation Contributes to Kinetochore Assembly in Addition to CENP-A Deposition at the Centromere
New centromeres can be generated artificially at ectopic sites through the assembly of CENP-A nucleosomes at Lac operator (LacO)-containing arrays (Barnhart et al., 2011;Logsdon et al., 2015;Mendiburo et al., 2011). We applied this LacO/LacI ectopic centromeric chromatin assembly system to address the ability of CENP-A (WT or mutants) fused to the Lac repressor (LacI) to recruit CENP-A chaperons and/or kinetochore components at arrays of Lac operator (LacO) sequences on chromosome 1 of U2OS cells (Janicki et al., 2004;Logsdon et al., 2015).
The K124R mutation significantly reduced the recruitment of CENP-A chaperons (HJURP and DAXX) and outer kinetochore proteins (HEC1 and SKA1) at LacO arrays, after ectopic loci forcibly were determined through LacO-LacI interaction ( Figures S7C-S7F). Addition of monoubiquitin to the CENP-A K124R mutant, when compared with CENP-A WT, significantly restored and enhanced the recruitment of CENP-A chaperons and outer kinetochore proteins at LacO arrays ( Figures S7C-S7F).
Taken together, these results suggest that CENP-A ubiquitylation contributes not only to determining the position of centromeric loci, but also to the assembly of kinetochores after ectopic loci were forcibly determined (see Discussion).

DISCUSSION
Our results of in vivo and in vitro ubiquitylation assays using the constitutively ubiquitylated CENP-A mutant clearly showed that ubiquitylated CENP-A is required for ubiquitylation of nonubiquitylated CENP-A. Therefore, the heterodimer (i.e., a dimer of old CENP-A and new CENP-A) is presumably recognized by the CUL4A complex, and the new CENP-A is ubiquitylated and maintained at the centromeres (Niikura et al., 2015). Our previous studies showed that CENP-A K124 is ubiquitylated in the M and G1 phases (i.e., at the time of CENP-A deposition at centromeres) (Jansen et al., 2007;Niikura et al., 2015). Based on these results, we provide two models of epigenetic inheritance of CENP-A ubiquitylation for the control of CENP-A deposition and maintenance at centromeres (Figure 7 and see further discussion below).

Models of the Inheritance of the Centromere Location
Bui et al. reported that native CENP-A nucleosomes are tetrameric during the early G1 phase, are converted to octamers at the transition from the G1 phase to the S phase, and revert to tetramers after DNA replication (Bui et al., 2012). CENP-A binds to HJURP during the G1 and G2 phases but not during the S phase (Bui et al., 2012). However, the current model of interconversion between tetrameric and octameric CENP-A nucleosomes in the cell cycle remains controversial (Fukagawa and Earnshaw, 2014;Hasson et al., 2013;Miell et al., 2013;Padeganeh et al., 2013), although the structures of the homotypic and heterotypic CENP-A particles have been solved (Arimura et al., 2014;Tachiwana et al., 2011). Therefore, we provide two models of epigenetic inheritance of CENP-A ubiquitylation: a tetramer model ( Figure 7A) and an octamer model ( Figure 7B).  (Bui et al., 2012), because of the dimerization of the CENP-A tetrameric nucleosome ( Figure 7A, larger dots circle in top). CENP-A nucleosomes, where each nucleosome has one single CENP-A molecule, are divided/diluted between the two daughter centromere-DNA sequences, and either is replaced with an H3 nucleosome or leaves a nucleosome-free gap during replication/S phase ( Figure 7A). In the octamer model, two CENP-A dimers in one nucleosome are split/diluted between the two daughter centromere-DNA sequences, and one CENP-A molecule either is replaced with one H3 molecule or leaves a molecule-free gap during replication/S phase ( Figure 7B).

Dunleavy et al. reported that histone H3.3 is deposited at centromeres in the S phase as a placeholder for CENP-A that is newly assembled in the
The following lines of evidence were collected from our studies and others. Zasadzinska et al. demonstrated that HJURP itself dimerizes through a C-terminal repeat region, which is essential for centromeric assembly of nascent CENP-A (Zasadzinska et al., 2013). Dunleavy et al. showed that HJURP localizes at centromeres during late telophase, which is when newly synthesized CENP-A is incorporated at centromeres in humans (Dunleavy et al., 2009). Phosphorylation and DNA binding of HJURP were suggested to determine its centromeric requirement and function in CENP-A loading (Muller et al., 2014). Our previous UbFC analysis suggested that K124-ubiquitylated CENP-A exists at centromeres and the nuclear region (Niikura et al., 2015). In addition, we previously showed that K124ubiquitylated CENP-A is found in the insoluble chromatin fraction (Niikura et al., 2015). Our previous study indicated that CENP-A K124 ubiquitylation is required for efficient interaction with HJURP (Niikura et al., 2015), and in the present study, our in vitro and in vivo ubiquitylation assays revealed that HJURP itself contributes to CENP-A K124 ubiquitylation ( Figures S3B, S3D, and S3E). Addition of purified HJURP itself did not induce ubiquitylation of CENP-A in vitro (in the absence of ubiquitylated CENP-A) but enhanced CENP-A ubiquitylation about 2-fold in the presence of CENP-A K124R-Ub (K48R) ( Figure S3B, compare lanes 4-6; Figure S3D, compare lanes 3-5). Heterodimerization of new CENP-A with pre-existing ubiquitylated CENP-A is required for ubiquitylation of new CENP-A and localization of new CENP-A to the centromere (Figures  4 and S4-S6). K124R-Ub (K48R) mutant increased signals of "punctuated" centromere localization of HJURP, whereas K124R mutation abrogated the centromere localization of HJURP ( Figure S7E, 'HJURP' and 'Merge'). The evidence summarized in this paragraph supports our proposed models ( Figures 7A and 7B) in which HJURP preferentially binds to ubiquitylated, preassembled "old" CENP-A, which resides predominantly in nucleosomes, especially at the initial step of the ubiquitylation of nascent ("new") CENP-A (Figure 7, anaphase/telophase). During this process, newly synthesized, free CENP-A targets ubiquitylated centromeric CENP-A through its attraction to HJURP, which is preassembled with "old" ubiquitylated, centromeric CENP-A (Figure 7, telophase/early G1). Subsequently, new CENP-A is ubiquitylated in the proximity of the nucleosome and/or inside the nucleosomes in a heterodimerization-dependent manner (old CENP-A -new CENP-A) during the M and G1 phases (Figure 7, telophase/early G1), and HJURP partly contributes to ubiquitylation ( Figures S3B, S3D, and S3E). In the tetramer model, heterodimerization could be internucleosomal ( Figure 7A, telophase/early G1). Thus, in these models, ubiquitylation and the location of the centromere are inherited epigenetically.

Neocentromere Formation
To date, "functional" neocentromeres have not resulted from the experimental mistargeting of overexpressed CENP-A in humans (Van Hooser et al., 2001). In our study, overexpression of the monoubiquitin fusion protein Flag-CENP-A K124R-Ub (K48R) led to sufficient recruitment of HJURP and central-outer kinetochore components (i.e., CENP-I, HEC1, and SKA1) to noncentromeric chromatin regions. In particular, SKA1 recruitment on the ectopic centromere verified that the neocentromeres are functional, because SKA1 centromere localization requires the formation of kinetochore-microtubule interactions (Hanisch et al., 2006;Schmidt et al., 2012). In our experiment, SKA1-positive, putative neocentromeres replicated and were inherited epigenetically between cell divisions.

Functions of CENP-A Chaperons, HJURP, and DAXX
Our assay using the LacO/LacI ectopic centromeric chromatin assembly system clearly revealed that CENP-A ubiquitylation contributes to the recruitment of CENP-A chaperons (HJURP and DAXX) and outer kinetochore components (HEC1 and SKA1) at LacO arrays ( Figure S7). It is possible that ubiquitylation of CENP-A contributes to maintain and stabilize ectopic neocentromeres in humans; in Drosophila melanogaster the E3 Ligase CUL3/RDX controls centromere maintenance by ubiquitylating and stabilizing CENP-A in a CAL1-dependent manner (Bade et al., 2014).
Results of our immunoprecipitation assays using cell lysates showed that the affinity of Flag-CENP-A-K124R-Ub (K48R) with endogenous DAXX is lower than that of Flag-CENP-A WT (Figures 6C and 6D); this difference in affinity supports this model ( Figure  6E). However, we found no significant difference between CENP-A WT and K124R-Ub (K48R) mutant regarding their affinity to endogenous HJURP ( Figures 6C and 6D). We speculate that the CENP-A binding to HJURP may have been saturated in the immunoprecipitation experiments using the cell lysates. Otherwise, this might be because the binding of CENP-A with HJURP is not stable through the cell cycle (Bui et al., 2012;Foltz et al., 2009) We also found that the fusion of monoubiquitin to the CENP-A K124R mutant significantly restored and enhanced the recruitment of CENP-A chaperons (HJURP and DAXX) and outer kinetochore proteins (HEC1 and SKA1) at LacO arrays ( Figures S7C-S7F); this result seemed to be inconsistent with the results regarding neocentromeres in Figures 6A and 6B. However, the results of the LacO/LacI ectopic centromeric chromatin assembly assay should represent the status "after" CENP-A assembly into chromatin, because in this system HA-LacI-CENP-A WT and mutants are forcibly incorporated into chromatin containing LacO arrays. Therefore, we speculate that HJURP and DAXX function in two different ways before and after CENP-A deposition at the centromere. Indeed, a dual chaperone function of HJURP in coordinating CENP-A and CENP-C recruitment was recently proposed (Tachiwana et al., 2015). DAXX also may have a dual role, because heat shock increases the accumulation of DAXX at CEN/periCEN (Morozov et al., 2012) and DAXX interacts with CENP-C (Pluta et al., 1998).

The Epigenetic Mark of Centromere Identity
CENP-A has been proposed to be the epigenetic mark of the centromere identity (Karpen and Allshire, 1997) on the basis of the following findings: CENP-A is localized only at active centromeres on dicentric chromosomes (Earnshaw et al., 1989;Sullivan and Schwartz, 1995;Sullivan and Willard, 1998;Warburton et al., 1997), CENP-A can be experimentally mistargeted to noncentromeric regions of chromatin, and this mistargeting leads to the formation of ectopic centromeres in model organisms (Fukagawa and Earnshaw, 2014). However, in humans, we have shown that overexpression of CENP-A itself is not sufficient for the creation of a neocentromere at a noncentromeric region. Ubiquitylation of CENP-A is necessary for the formation of neocentromeres and for the epigenetic inheritance of the centromere location. Considering that histone posttranslational modifications are defined as "epigenetic marks" traditionally, we propose that CENP-A ubiquitylation is a candidate for the epigenetic mark of centromere location, i.e., the centromere identity.

CENP-A Overexpression and Cancer
Ectopic incorporation of overexpressed CENP-A can lead to genomic instability (Amato et al., 2009;Au et al., 2008;Heun et al., 2006;Van Hooser et al., 2001), which occurs in particularly aggressive cancer cells and tissues (Biermann et al., 2007;McGovern et al., 2012;Tomonaga et al., 2003;Vardabasso et al., 2014;Wu et al., 2012). In humans, CENP-A overexpression can lead to its ectopic localization to chromosome regions with active histone turnover, as seen in cancer cell lines Tomonaga et al., 2003). At these ectopic loci, CENP-A forms heterotypic nucleosomes (CENP-A/H3.3) occluding CTCF binding, and their presence may increase DNA damage tolerance in cancer cells . Arimura et al. revealed a "hybrid" structure of the heterotypic CENP-A/H3.3 and suggested that the stable existence of the CENP-A/H3.3 nucleosome may cause ectopic kinetochore assembly (Arimura et al., 2014), which could lead to neocentromere formation and chromosome instability in cancer cells (Biermann et al., 2007;Lacoste et al., 2014;McGovern et al., 2012;Tomonaga et al., 2003;Wu et al., 2012). Our models of epigenetic inheritance of CENP-A ubiquitylation suggest that errors in CENP-A targeting, heterodimerization, and/or ubiquitylation induce abnormal accumulation of heterotypic nucleosomes ( Figure 7B, bottom). Hence, our findings may provide a basis for potential insights into understanding the mechanisms of cancer development.

Antibodies and Reagents
Please see Table S1 for antibodies, Table S2 for siRNA sequences, Table S3 for plasmid vectors, and Table S4 for baculovirus extracts used in this study.
CENP-A −/F hTERT RPE1 cells were generously given Dr. Don W. Cleveland, Ludwig Institute for Cancer Research, Department of Cell and Molecular Medicine, University of California at San Diego, and cultured as described previously (Fachinetti et al., 2013). Retro-Cre was added to CENP-A −/F RPE1 cells infected with retrovirus produced by transient cotransfection of 293T cells with pBabe-puro-Cre, psPAX2, and pMD2.G (Table S3). Four days after retro-Cre infection ( Figure S2A), cells were further infected with retrovirus produced by stable Plat-GP cells (Cell Biolabs, Inc) with pQCXIP-Flag-CENP-A or transient cotransfection of Plat-GP cells with the indicated pQCXIP constructs and pCMV-VSG-G (Table S3). For in vivo ubiquitylation assay of CENP-A −/F RPE1 cells, pCGN-HA-Ubiquitin was also transfected by using Fugene HD (Promega) 4 days after retro-Cre infection ( Figure S2A; see also CENP-A In Vivo Ubquitylation Assay). Cells were collected or fixed 6 days after retro-Cre infection and used in each analysis.

CENP-A In Vivo Ubiquitylation Assay
The in vivo CENP-A ubiquitylation assay was performed as described previously with the following minor modifications (Niikura et al., 2015). HeLa Tet-Off cells were transfected with the indicated expression vectors (see Cell Culture and Transfection) and incubated with 5 µM MG132 (CALBIOCHEM) for 24 h. Cells were then collected and lysed in denaturing buffer A1 (20 mM Tris-HCl pH 7.4, 50 mM NaCl, 0.5% Nonidet P-40, 0.5% deoxycholate, 0.5% SDS, 1 mM EDTA, 50 µM MG132, and complete EDTA-free protease inhibitor cocktail [Roche]) by a sonication and freeze-thaw process. Proteins were immunoprecipitated, and the immunoprecipitates underwent Western blot analysis with the indicated antibodies.
For CENP-A −/F RPE1 cells, the experiment was performed according to the time-course scheme in Figure S2A. Cells were infected with retroviruses harboring the indicated vector constructs, cultured (see Cell Culture and Transfection in Experimental Procedures), collected 6 days after retro-Cre infection, lysed, and analyzed as described for the HeLa Tet-Off cells.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Figure 1. CENP-A Monoubiquitylation or Diubiquitylation Is Epigenetically Inherited between Cell Divisions
(A) Schematic of the experimental protocol for Figures 1 and S1. When endogenous CENP-A (blue) was reduced to less than 10% of the normal level, exogenously expressed Flagtagged CENP-A (green) was not ubiquitylated and did not localize to the centromere (panel [2]). However, coexpression of the monoubiquitin-fused CENP-A K124R mutant (white) restored monoubiquitylation and diubiquitylation of Flag-tagged CENP-A and centromeric localization of Flag-tagged CENP- A (panel [3]). The C-terminal Flag tag on CENP-A (white "F" in black rectangle) is indicated.      (Table S2; see also Chromosome Spreading in Supplemental Experimental Procedures). Forty-six hours after transfection, cells were arrested at prometaphase by treatment with nocodazole for 2 h, and their chromosomes were examined