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. 2012 May 16;31(10):2391-402.
doi: 10.1038/emboj.2012.82. Epub 2012 Apr 3.

Breaking the HAC Barrier: histone H3K9 acetyl/methyl balance regulates CENP-A assembly

Jun-ichirou Ohzeki  1 , Jan H Bergmann, Natalay Kouprina, Vladimir N Noskov, Megumi Nakano, Hiroshi Kimura, William C Earnshaw, Vladimir Larionov, Hiroshi Masumoto
Affiliations

Breaking the HAC Barrier: histone H3K9 acetyl/methyl balance regulates CENP-A assembly

Jun-ichirou Ohzeki et al. EMBO J. .

Abstract

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.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
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.
Figure 2
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).
Figure 3
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).
Figure 4
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).
Figure 5
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 H3 was expressed with the same procedure to panel B. Cells were stained with DAPI, anti-GFP (green) and anti-HA (red). Scale bar, 5 μm. (I) Distribution of histone H3s. Histone H3.1 showed two localization pattern, whole nuclei (green column) and dots (yellow column). No specific enrichment was observed at tetO alphoid DNA other than the usual assembly pattern of these histone H3. Cells containing tetR-EYFP spots were counted in each sample (n?100). Error bar, s.d. (n=3).
Figure 6
Figure 6
Ectopic kinetochore proteins assembly induced by CENP-A recruiting factors. (A) Schematic diagram of metaphase cell preparation. HeLa-Int-03 was co-transfected with HA-CENP-A and a set of tetR-EYFP fusion protein expressing vectors. Six tetR-EYFP-fusions are shown in Figure 5A. After 48 h incubation, cells were arrested in metaphase and spread on cover glass for immuno-staining. (B) Examples of high order centromere proteins assembly at ectopic alphoidtetO DNA integration site. Spread mitotically arrested cells were stained with DAPI, anti-HA (green), anti-CENP-I (red) and anti-CENP-E (blue) (top). Another staining was also carried out with DAPI, anti-CENP-A (green), anti-CENP-T (red) and anti-CENP-B (blue) (bottom). Arrowheads indicate endogenous centromere (green) and ectopic alphoidtetO DNA integration site (red). The indicated images were obtained with tetR-EYFP-HJURP tethering. The results obtained with other fusions are shown in Supplementary Figure S14. Scale bars, 3 μm. (C) A frequency of ectopic HA-CENP-A assembly per total spread cells was counted (n?100). Error bar, s.d. (n=3). (D) A frequency of ectopic kinetochore proteins (CENP-T, CENP-I and CENP-E) assembly per total ectopic CENP-A signal positive cells (n=8∼100). Error bar, s.d. (n=3).
Figure 7
Figure 7
Centromere acetylation occurs within a short time window following metaphase. (A) Schematic diagram for cell sample preparation. Cells were arrested in metaphase for six hours, and then harvested and released to G1 phase. Mitotically arresting (pale blue), one hour post release (orange), three hours post release (green) and random culture cells (gray) were harvested. (B) Examples of phase contrast microscope images for cells at each time points. Scale bar, 20 μm. (C) Centromere acetylating activity in HeLa cell. ChIP assay was carried out with normal IgG and a set of specific antibodies using samples indicated in panel A. A set of primers was used for quantitative PCR (top). Error bars, s.d. (n=3). P-values obtained with t-test are indicated. (D) Schematic diagrams for Suv39h1 tethering. A HeLa-HAC-05 cell line expressing tetR-EYFP-Suv39h1 was established in the presence of doxycycline (dox). Three days before sample preparation, tetR-EYFP-Suv39h1 tethering to HAC was induced by dox washout. Then a set of four cell samples shown in panel A was harvested and used for ChIP. (E) Suv39h1 tethering represses the increase of centromeric H3K9ac level. HeLa-HAC-R5 cells expressing Suv39h1 were cultured with presence or absence of doxycycline for three days, and then harvested similar to that shown in panel A. ChIP assay was carried out with a set of specific antibodies. A set of primer was used for quantitative PCR (top). Error bars, s.d. (n=3). P-values obtained with t-test are indicated.
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