Title: Breaking the HAC Barrier: Histone H3K9 acetyl/methyl balance regulates CENP-A assembly
Abstract: Article3 April 2012Open Access Breaking the HAC Barrier: Histone H3K9 acetyl/methyl balance regulates CENP-A assembly Jun-ichirou Ohzeki Jun-ichirou Ohzeki Department of Human Genome Research, Laboratory of Cell Engineering, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USAPresent address: Laboratory of Cell Engineering, Department of Human Genome Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan Search for more papers by this author Jan H Bergmann Jan H Bergmann Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Scotland, UK Search for more papers by this author Natalay Kouprina Natalay Kouprina Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Vladimir N Noskov Vladimir N Noskov Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Megumi Nakano Megumi Nakano Department of Human Genome Research, Laboratory of Cell Engineering, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan Search for more papers by this author Hiroshi Kimura Hiroshi Kimura Graduate School of Frontier Biosciences, Osaka University, Suita, Japan Search for more papers by this author William C Earnshaw William C Earnshaw Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Scotland, UK Search for more papers by this author Vladimir Larionov Vladimir Larionov Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Hiroshi Masumoto Corresponding Author Hiroshi Masumoto Department of Human Genome Research, Laboratory of Cell Engineering, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan Search for more papers by this author Jun-ichirou Ohzeki Jun-ichirou Ohzeki Department of Human Genome Research, Laboratory of Cell Engineering, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USAPresent address: Laboratory of Cell Engineering, Department of Human Genome Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan Search for more papers by this author Jan H Bergmann Jan H Bergmann Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Scotland, UK Search for more papers by this author Natalay Kouprina Natalay Kouprina Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Vladimir N Noskov Vladimir N Noskov Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Megumi Nakano Megumi Nakano Department of Human Genome Research, Laboratory of Cell Engineering, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan Search for more papers by this author Hiroshi Kimura Hiroshi Kimura Graduate School of Frontier Biosciences, Osaka University, Suita, Japan Search for more papers by this author William C Earnshaw William C Earnshaw Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Scotland, UK Search for more papers by this author Vladimir Larionov Vladimir Larionov Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Hiroshi Masumoto Corresponding Author Hiroshi Masumoto Department of Human Genome Research, Laboratory of Cell Engineering, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan Search for more papers by this author Author Information Jun-ichirou Ohzeki1,2, Jan H Bergmann3, Natalay Kouprina2, Vladimir N Noskov2, Megumi Nakano1, Hiroshi Kimura4, William C Earnshaw3, Vladimir Larionov2 and Hiroshi Masumoto 1 1Department of Human Genome Research, Laboratory of Cell Engineering, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan 2Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 3Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Scotland, UK 4Graduate School of Frontier Biosciences, Osaka University, Suita, Japan *Corresponding author. Department of Human Genome Research, Kazusa DNA Research Institute, Laboratory of Cell Engineering, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba292-0818, Japan. Tel.:+81 438 52 3952; Fax:+81 438 52 3946; E-mail: [email protected] The EMBO Journal (2012)31:2391-2402https://doi.org/10.1038/emboj.2012.82 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The kinetochore is responsible for accurate chromosome segregation. However, the mechanism by which kinetochores assemble and are maintained remains unclear. Here we report that de novo CENP-A assembly and kinetochore formation on human centromeric alphoid DNA arrays is regulated by a histone H3K9 acetyl/methyl balance. Tethering of histone acetyltransferases (HATs) to alphoid DNA arrays breaks a cell type-specific barrier for de novo stable CENP-A assembly and induces assembly of other kinetochore proteins at the ectopic alphoid site. Similar results are obtained following tethering of CENP-A deposition factors hMis18α or HJURP. HAT tethering bypasses the need for hMis18α, but HJURP is still required for de novo kinetochore assembly. In contrast, H3K9 methylation following tethering of H3K9 tri-methylase (Suv39h1) to the array prevents de novo CENP-A assembly and kinetochore formation. CENP-A arrays assembled de novo by this mechanism can form human artificial chromosomes (HACs) that are propagated indefinitely in human cells. Introduction The kinetochore is responsible for accurate chromosome segregation. During mitosis, kinetochores assemble on specialized centromere chromatin (Cleveland et al, 2003; Allshire and Karpen, 2008) composed of nucleosomes containing the essential histone H3 variant CENP-A (Earnshaw and Rothfield, 1985). Recent studies have identified several factors, including the Mis18 complex and HJURP (Hayashi et al, 2004; Camahort et al, 2007; Fujita et al, 2007; Mizuguchi et al, 2007; Stoler et al, 2007; Pidoux et al, 2009; Williams et al, 2009), involved in the deposition of newly synthesized CENP-A at pre-existing CENP-A chromatin regions (Okada et al, 2006; Fujita et al, 2007; Jansen et al, 2007; Dunleavy et al, 2009; Foltz et al, 2009). However, the mechanism by which centromere chromatin assembles and is stabilized at specific genomic loci remains unclear. Centromeric DNA sequences are competent to form de novo functional kinetochores in yeasts, mouse and some human cell lines (Clarke and Carbon, 1980; Hahnenberger et al, 1989; Harrington et al, 1997; Ikeno et al, 1998; Moralli et al, 2006; Okada et al, 2007). Human centromeric alpha-satellite (alphoid) DNAs can induce high efficiency de novo CENP-A and functional kinetochore assembly and subsequent human artificial chromosome (HAC) formation when introduced into HT1080 human fibrosarcoma cells. HAC kinetochore formation is highly dependent on regular arrays of alphoid DNA sequences with CENP-B binding capacity (Ohzeki et al, 2002; Okamoto et al, 2007), although de novo kinetochore assembly is not a simple DNA-protein reaction. Chromatin modifications are thought to regulate functional kinetochore assembly and maintenance by an epigenetic mechanism. Recent studies of normal centromeres also suggest a possible involvement of canonical histone H3-containing nucleosomes in kinetochore function. In humans, CENP-A nucleosomes are localized to only a portion of the megabase-sized alphoid DNA arrays, where they are organized as multiple clusters interspersed with histone H3 nucleosomes (Blower et al, 2002; Sullivan and Karpen, 2004; Ribeiro et al, 2010). Canonical H3 nucleosomes co-purify with CENP-A in oligonucleosomes (Ando et al, 2002), and some classes of CENPs (e.g. CENP-T, -W) are suggested to bind only to H3 nucleosomes (Hori et al, 2008). Thus, epigenetic CENP-A-mediated kinetochore assembly could also be affected by the surrounding H3 chromatin state. Thus, functional kinetochore formation and maintenance may be influenced by additional factors that determine the modification status of centromeric chromatin. The fundamental question addressed by this study is how different chromatin fates are generated on alphoid DNA in human cells and what kind of chromatin directs functional centromere/kinetochore assembly. We found that competency for stable CENP-A assembly and de novo kinetochore assembly are correlated with the acetylation status of H3K9 on alphoid DNA in several different cell types. We therefore decided to manipulate H3K9 modifications during de novo kinetochore assembly using a synthetic alphoid DNA array carrying multiple tet operator (tetO) sequences that allow the tethering of chromatin modifiers into the array as tet repressor (tetR) fusions (Nakano et al, 2008; Cardinale et al, 2009; Bergmann et al, 2011). Tethering of tetR-EYFP-p300 or tetR-EYFP-PCAF, two histone acetyltransferase (HAT) domains that promote acetylation of H3K9, results in assembly of newly synthesized CENP-A on exogenous alphoid DNA arrays. Remarkably, HAT induction of de novo CENP-A chromatin assembly requires HJURP but bypasses the need for hMis18α, and spontaneously nucleates assembly of an outer kinetochore on the artificial DNA arrays. Indeed, in a technological breakthrough, these HAT-induced de novo CENP-A arrays can even lead to the formation of stable HACs that are maintained indefinitely in human cell lines that have previously proven refractory to HAC formation. Together, our data reveal that CENP-A assembly appears to be controlled by a histone H3K9ac/me3 balance that acts upstream of HJURP. Results Cell-type-dependent chromatin assembly on transfected human alphoid DNA De novo kinetochore assembly is efficient in HT1080 cells. However, neither stable de novo kinetochore formation nor CENP-A assembly on exogenous alphoid DNA occurs in many other commonly used human cell lines, including HeLa (Figure 1A and Supplementary Figure S1). Figure 1.Cell type specific chromatin modifications on transfected and endogenous alphoid DNA. (A) Summary of the HAC formation assay. The pWTR11.32 plasmid, which contains 60 kb of α21-I 11mer repeat (shown in panel B), was transfected to HT1080 or HeLa cells. Single transformants were isolated and analyzed for chromosomal events by FISH and microscopy. Examples of HAC and integration are shown as merged images. Signals in pictures indicate DNA (gray), BAC plasmid DNA (red) and CENP-A (green). (B and C) Time-course ChIP analysis. The pWTR11.32 or pMTR11.32 plasmid (panel B) was transfected to HT1080 or HeLa cell. Transfectants were cultured under presence of selective drug (G418), and harvested at 2, 3 and 4 weeks after transfection. ChIP assay was carried out with normal IgG and indicated antibodies (panel C). Primer set for synthetic 11mer repeats was used for quantitative PCR. Error bars, s.d. (n=2). (D) Chromatin modifications on human repetitive DNAs. ChIP assay was carried out with normal IgG and indicated antibodies. Primer sets used for quantitative PCR are specific to 5S ribosomal DNA (5S Ribo), satellite 2 (Sat2), D4Z4 repetitive DNA (D4Z4), DYZ1 repetitive DNA (DYZ1), Alu elements (Alu), 17 alphoid (17a), 21-I alphoid (21a, 21b), 21-II alphoid (21c), X alphoid (Xa, Xb) and Y alphoid DNA (Ya, Yb, Yc) sequences. More information for these primers is shown in Supplementary Figure S3A and Supplementary Table S2. Columns indicate non-alphoid repetitive DNA controls (black), type I alphoid DNA (white) and type II (gray), respectively. Error bars, s.d. (n?3). (E) Examples of metaphase chromosome staining. Mitotic cell spreads were stained with DAPI (gray), anti-H3K9me3 (green) and anti-CENP-A antibody (red). Scale bar, 3 μm. Download figure Download PowerPoint Surprisingly, HeLa cells, TIG7 human fetal primary, hTERT-BJ1 immortalized fibroblasts and U2OS osteosarcoma cells, all efficiently assemble CENP-A chromatin de novo, but CENP-A levels declined rapidly during subsequent cell culture (Figure 1B, C, Supplementary Figure S2 and S3C). The decrease in CENP-A levels on transfected alphoid DNA in HeLa cells was accompanied by a progressive increase in the heterochromatin-associated modification, H3K9me3 (Figure 1C). Detailed ChIP analysis of the chromatin modification status at several endogenous centromeres revealed that alphoid DNA appears more euchromatic in HT1080 cells than in HeLa (Figure 1D). Using CENP-A and CENP-B as controls, H3K9ac, a euchromatic modification, was readily detected on HT1080 alphoid DNA, but was much lower at HeLa centromeres (Figure 1D). In addition, HT1080 cells had substantially lower levels of H3K9me3 on alphoid DNA than on other repetitive DNA sequences, including satellite 2, D4Z4 and DYZ1. In contrast H3K9me3 levels on alphoid DNA were significantly higher in HeLa, TIG7, hTERT-BJ1 and U2OS cells (Figure 1D and SupplementaryFigure S3). The ChIP data were confirmed by a stronger H3K9me3 staining intensity at mitotic centromeres in HeLa cells (Figure 1E). Suv39h1 negatively regulates de novo CENP-A assembly on alphoid DNA at ectopic site The histone methyltransferase Suv39h1 may be one critical factor responsible for this difference between HT1080 and HeLa alphoid DNA chromatin. HT1080 cells express only 50% of the relative level of Suv39h1 mRNA found in HeLa cells (Figure 2A). Suv39h1 over-expression increased both levels of the enzyme itself and H3K9me3 on centromeric alphoid DNAs in HT1080 cells (Figure 2B). These results fit with the observations that mouse cells doubly null for Suv39h1 and Suv39h2 (Suv39hdn) have low levels of centromeric H3K9me3 (Peters et al, 2001). Figure 2.Suv39h1, histone H3K9 tri-methylase, negatively regulates ectopic CENP-A assembly. (A) Suv39h1 expression level. Total RNA was purified from each cell line, reversely transcribed and quantified by real-time PCR. Suv39h1 mRNA amounts were normalized by HPRT transcripts. Both Suv39h1 and HPRT genes are on X chromosome. (B) Exogenous Suv39h1 expression induced H3K9me3 modification on centromeric alphoid DNAs in HT1080 cells. EYFP-tagged Suv39h1 gene was transfected and cells were harvested more than four weeks after transfection. ChIP was carried out with normal IgG and a set of indicated antibodies. Primer sets shown at the top were used for quantitative PCR. Error bars, s.d. (n=2). (C) Examples of no ectopic CENP-A assembly in HeLa integration cell (HLW-Int-09). Mitotic cells were spread on cover glass, and stained with DAPI (blue), anti-CENP-A antibody (green), and BAC DNA probe (red). Scale bar, 5 μm.(D) Depletion of Suv39h1 with siRNA. siRNAs for the GFP gene (siGFP; control) or Suv39h1 (siSuv39h1) was transfected to HLW-Int-09 cell. Total RNA was purified and quantified by real-time PCR. Suv39h1 mRNA levels were normalized by HPRT RNA. Vertical axis indicates relative Suv39h1 mRNA level against a negative control (siGFP). Error bar, s.d. (n=3). (E, F) ChIP assay was carried out with HLW-Int-09 cells treated by siGFP or siSuv39h1. Normal IgG and a set of different antibodies were used for ChIP. Indicated primer sets were used for quantitative PCR (top). Error bars in panel E, s.d. (n=2). (F) HLW-Int-09 cells were harvested at three time points, 0, 4 and 7 days after siSuv39h1 transfection and used for ChIP analysis. Error bars, s.d. (n=3). Download figure Download PowerPoint Suv39h1 depletion by RNAi revealed a remarkable inverse correlation between CENP-A and H3K9me3 levels on an alphoid DNA array integrated ectopically on a chromosomal arm in HeLa cells (HLW-Int-09; Figure 2C and F). These results suggest that Suv39h1 suppresses ectopic CENP-A incorporation, presumably by maintaining H3K9me3 levels on alphoid DNA. However, Suv39h1 depletion alone and the accompanying transient increase in CENP-A were not sufficient for functional kinetochore formation on ectopic alphoid DNA arrays (Okada et al, 2007). Additional regulatory factors must be required for functional kinetochore formation de novo on alphoid DNA. HAT recruitment breaks the barrier for de novo kinetochore assembly Several observations suggest that histone acetyltransferases may be required for functional CENP-A assembly and subsequent kinetochore formation de novo (Nakano et al, 2003; Okamoto et al, 2007). Furthermore, the acetyltransferases p300 and PCAF [p300/CBP associated factors (Yang et al, 1996)] both localize at functional, but not at inactive, centromeres (Supplementary Figure S4) (Craig et al, 2003; Choi et al, 2009). To test the hypothesis that histone acetylation might antagonize H3K9me3 and promote functional CENP-A assembly, we expressed tetR-EYFP fused to the histone acetyl-transferase (HAT) domains of p300 or PCAF in HeLa cells (Figure 3A). Into the tetR-EYFP expressing cells, we then introduced a 50 kb synthetic DNA array based on the α21-I alphoid dimer sequence with a tetO site where the CENP-B box would be on one monomer (pWTO2R; Figure 3A and Supplementary Figure S5) (Ebersole et al, 2005; Kim et al, 2009). In this system, tetR fusion proteins bound to tetO sites within the synthetic alphoid DNA arrays can directly modify the chromatin environment at a single centromere or locus in human cells. Figure 3.Recruiting of histone acetyl-transferases induced de novo kinetochore formation in HeLa cell. (A) The expression constructs and BAC plasmid used in this Figure. TetR-EYFP gene was fused with Suv39h1, p300 HAT domain (p300HD) or PCAF HAT domain (PCAFHD). HeLa cell lines expressing these tetR-EYFP fusions were generated by retrovirus infection, and these cells were transfected with α21-I alphoidtetO DNA containing plasmid (pWTO2R; see Supplementary Figure S5). (B) Schematic timetable for ChIP and HAC assay. (C) Time-course ChIP analysis. Cells transfected by plasmid pWTO2R were harvested at 2, 3 and 4 weeks after transfection. Normal IgG and a set of specific antibodies were used for ChIP. A set of primers for α21-I alphoidtetO 2mer (tetO-2mer) was used for quantitative PCR. Columns indicate the results obtained with cells expressing tetR-EYFP (green), tetR-EYFP-Suv39h1 (blue), tetR-EYFP-p300HD (pink) or tetR-EYPF-PCAFHD (red) fusions, respectively. Error bars, s.d. (n=3). (D) Examples of a HAC (p300-HAC-13) formed in HeLa cell. Metaphase cells were spread and stained with DAPI (blue), anti-CENP-A antibody (green) and BAC DNA probe (red). BAC DNA probe visualizes a vector region of the introduced pWTO2R construct. Scale bar, 3 μm. (E) Summary of HAC formation. Bars indicate a frequency of HAC formation in the cells expressing protein fusions. Error bars, s.d. (n?2). Chi-square test of the predominant pattern for HAC formation frequency indicated significant differences. Asterisks * or ** indicate P values, (P<0.05) or (P<0.005), respectively. (F) HAC stability without HAT tethering. HAC containing cells were cultured for 60 days under presence of doxycycline (no tetR binding condition; left panel) and absence of selective drug (permissive condition for HAC loss). The number of HAC retention rate in 30–50 spread metaphase cells was scored by FISH using input BAC DNA specific probes (right panel). HAC loss rate was calculated with HAC retention rates at day 0 (N0) or at day 60 (N60) using the following formula:N60=N0×(1–R)60 (Ikeno et al, 1998). All HAC cell lines showed high stability (HAC loss rate >0.001). Download figure Download PowerPoint Tethering of either HAT domain fusion (tetR-EYFP-p300HD or tetR-EYFP-PCAFHD) to the synthetic alphoid DNA enhanced H3K9ac modification and CENP-A assembly, as demonstrated by time-course ChIP assays (Figure 3B, C and Supplementary Figure S6). In contrast, tethering of the tetR-EYFP-Suv39h1 fusion increased H3K9me3 levels and also decreased CENP-A assembly. This raised the question whether HAT domain recruitment could stimulate de novo kinetochore formation. Remarkably, stable HACs bearing the synthetic α21-I alphoidtetO repeat were detected in HeLa cell lines expressing tetR-EYFP-p300HD or tetR-EYFP-PCAFHD (in 8 or 14% of cell lines, respectively; Figure 3B, D and and Supplementary Figure S7). Importantly, HAC formation was never detected when the synthetic α21-I alphoidtetO repeat was introduced into cells expressing tetR-EYFP or tetR-EYFP-Suv39h1 (Figure 3E). Similarly, alphoidtetO-based HAC formation was never observed in HT1080 cells expressing tetR-EYFP-Suv39h1 (Figure 3E and Supplementary Figure S8). Although exogenous HAT activity is required for initial de novo kinetochore formation in HeLa cells, once established, the de novo kinetochores no longer require this exogenous activity to maintain their structure and function. We initially observed that HACs were stably maintained in cell clones that no longer expressed the tetR-EYFP-HAT fusion construct, presumably due to the silencing of retrovirus integration sites. We therefore, directly tested whether de novo kinetochores remained functional following forced dissociation of the HAT domain fusions by culturing cells for more than 60 days in the presence of doxycycline (Figure 3F). Microscopic and ChIP analyses showed that the HACs remained mitotically stable and capable of recruiting inner and outer kinetochore proteins CENP-A, -C, -T, hKNL1, Hec1, hDsn1 and hMis12 (Supplementary Figure S9A, B) in the absence of bound exogenous HAT fusion proteins (Figure 3F; Loss of tetR fusion binding to the HAC was confirmed by ChIP—Supplementary Figure S9C). Thus, HAT domain recruitment to the synthetic α21-I alphoidtetO array renders HeLa cells competent for de novo kinetochore formation. Centromere chromatin modifications regulate newly synthesized CENP-A assembly Kinetochore maintenance requires the targeting of newly synthesized CENP-A to centromeres during mitotic exit/early G1 (Jansen et al, 2007). To test whether the same chromatin modifiers that potentiate de novo kinetochore assembly also affect newly synthesized CENP-A assembly at an established HAC kinetochore, we transiently co-transfected constructs expressing HA-tagged CENP-A (HA-CENP-A) plus various tetR-EYFP-fusion proteins into tetO-HAC containing HeLa cells (HeLa-HAC-R5; Figure 4A and B). We then asked if HA-CENP-A (a mark for newly assembled CENP-A) assembled on the HAC and endogenous centromeres at 24 h (i.e. one complete cell cycle in HeLa cells) after transfection. Figure 4.HAT tethering on tetO-HAC induced expansion of newly synthesized CENP-A assembly through HJURP. (A) A HAC cell line (HeLa-HAC-R5) was transfected with a set of tetR-EYFP-fusion expressing vectors. (B) Timetable for the experiment. HA-tagged CENP-A expression vector (pCDNA5-HA-CENP-A) was co-transfected with tetR-EYFP-fusion expressing vector. (C) Representative images of newly synthesized CENP-A assembly. Cells were stained with DAPI, anti-GFP (recognize EYFP; green) and anti-HA (red). Arrowheads indicate tetO-HAC position. Scale bar, 5 μm. (D) Schematic timetable for gene depletion and new CENP-A assembly assay. HeLa-HAC-R5 cells were firstly transfected with siRNA. After 24 h incubation, HA-CENP-A and a set of tetR-EYFP-fusion expression vectors were co-transfected. Cells were stained with DAPI, anti-GFP and anti-HA. (E) hMis18α or HJURP depletion using siRNA. siRNAs for hMis18α (sihMis18α) and for HJURP (siHJURP) as well as for a negative control (siControl: siNegative, ambion) were used for transfection. Total RNA was purified two days after transfection and quantified by real-time PCR. hMis18α or HJURP mRNA levels were normalized by HPRT transcripts. Horizontal axis indicates relative hMis18α or HJURP mRNA level against a negative control (siControl). Error bar, s.d. (n=3). (F) HA-CENP-A assembly frequency on endogenous centromere was counted in each sample (n?100). Error bar, s.d. (n=3). (G) A frequency of expanded HA-CENP-A assembly induced by HAT tethering (example is shown in panel C bottom) was counted in each sample (n?100). Error bar, s.d. (n=3). Column colors indicate subpopulations of cells, which had CENP-A assembly at endogenous centromere (red) and had no assembly (orange). *P-values of t-test are 0.001 (red column) and 0.017 (orange column). **P-values of t-test are 0.006 (red column) and 0.033 (orange column). Download figure Download PowerPoint Tethering of tetR-EYFP alone did not affect the assembly of newly synthesized HA-CENP-A onto either the HAC or endogenous centromeres (Figure 4C and Supplementary Figure S10A). In contrast, tethered tetR-EYFP-Suv39h1 specifically reduced HA-CENP-A assembly on the HAC centromere (Figure 4C and Supplementary Figure S10B). This was coupled with destabilization of the HAC, detected as lagging chromosomes and micronuclei (Supplementary Figure S10C-G). Unexpectedly, tethering of p300HD or PCAFHD induced HA-CENP-A hyper-assembly not only at the HAC centromere, but covering the entire alphoidtetO signal on the HAC in a significant proportion of cells (34 and 40% in Figure 4C and Supplementary Figure S10B). We next tested whether the known canonical CENP-A deposition factors hMis18α and HJURP are involved in this HAT-induced CENP-A assembly. We first depleted hMis18α or HJURP by siRNA knockdown and then tethered tetR-fused HAT proteins to the synthetic alphoid array (Figure 4D, E). hMis18α depletion reduced HA-CENP-A assembly at both endogenous centromeres and the HAC centromere (Figure 4F). However, stable HA-CENP-A assembly continued on alphoidtetO DNA with tethered HAT fusions following hMis18α depletion, which blocks CENP-A assembly on endogenous centromeres (Figure 4G, orange bars). The cell population, which had newly assembled HA-CENP-A on alphoidtetO DNA but no HA-CENP-A signals on endogenous centromeres, was relatively increased after hMis18α depletion (Figure 4G, orange bars. P<0.05). These results indicate that tethering of HAT fusions can partially rescue HA-CENP-A assembly in the absence of hMis18α. Importantly, HJURP depletion dramatically reduced HA-CENP-A assembly both on endogenous host centromeres and on the HAC. Furthermore, neither was rescued by tethering of HAT-fusion proteins to the HAC alphoidtetO array (Figure 4F, G). Thus, HJURP is required for HAT-mediated CENP-A assembly. Given that HAT tethering can potentiate de novo kinetochore formation on a HAC and induce HA-CENP-A hyper-assembly covering non-centromeric regions of the HAC (Figures 3 and 4), we next tested whether HAT-tethering can induce de novo CENP-A assembly on a chromosomal arm. We did this using a stable cell line (HeLa-Int-03), which carries an ectopic alphoidtetO chromosomal integration on which we have failed to detect any essential kinetochore-specific proteins other than CENP-B (which binds to the CENP-B box) (Supplementary Figure S11). Tethering of tetR-EYFP-p300HD or tetR-EYFP-PCAFHD induced HA-CENP-A hyper-assembly on the ectopic array in 27 and 47% of cells, respectively (Figure 5A and E). A similar effect was observed after tethering the CENP-A assembly factors, tetR-EYFP-hMis18α or tetR-EYFP-HJURP (HA-CENP-A hyper-assembly in 32 and 100% of cells, respectively—Figure 5C and E). CENP-A assembly at the ectopic site induced by tetR-EYFP-hMis18α tethering was diminished by HJURP depletion (Figure 5F), consistent with Barnhart et al (2011). In controls, tethering of tetR-EYFP alone or tetR-EYFP-Suv39h1 did not induce HA-CENP-A assembly at the ectopic site (Figure 5C–E). Moreover, no specific enhancement of the assembly of newly expressed HA-tagged histone H3.1 nor H3.3 was observed on the ectopic alphoidtetO array by the HAT tetherings in addition to the usual assembly patterns of those histone H3 (Figure 5H and Supplementary Figure S12). Figure 5.HAT and CENP-A deposition related factor could induce de novo ectopic CENP-A assembly. (A) Schematic diagram. HeLa-Int-03 cell line had ectopic integration site of alphoidtetO DNA on host chromosome (left). This ectopic site had no CENP-A assembly. In addition to the previous four constructs, two new tetR-EYFP-fusions were used for the experiment (right). (B) Schematic timetable for new CENP-A assembly assay. HeLa-Int-03 cells were co-transfected with HA-CENP-A and a set of tetR-EYFP-fusion expression vectors. (C) Representative images of newly synthesized CENP-A assembly on ectopically integrated alphoidtetO DNA. Cells were stained with DAPI, anti-GFP (green) and anti-HA (red). Arrowheads indicate alphoidtetO DNA integration sites. Scale bar, 5 μm. (D) A frequency of HA-CENP-A assembly on endogenous centromere per total HA-CENP-A expressing cells was counted in each sample (n?100). Error bar, s.d. (n=3). (E) Frequency of de novo HA-CENP-A assembly on ectopic alphoidtetO DNA integration site. HA-CENP-A signals on tetR-EYFP spot per total tetR-EYFP spots were counted in each sample (n?100). Error bar, s.d. (n=3). (F and G) hMis18α tethering assay under HJURP depletion. The frequencies shown in panel D and E were counted (n?100). Error bar, s.d. (n=3). (H) Representative images of newly synthesized histone H3.1 and H3.3 localization. HA-tagged histone