WO2013076947A1 - A method for positively or negatively regulating the assembly of newly synthesized cenp-a to exogenous alphoid dna containing cenp-b boxes in mammalian cell lines - Google Patents

Abstract

We have discovered that de novo CENP-A assembly and kinetochore formation on human centromeric alphoid DNA arrays is regulated by a histone H3K9 acetyl/methyl switch. 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 similarly with the tethering of CENP-A deposition factors hMis18alpha or HJURP. HAT tethering bypasses the need for hMis18alpha, but requires HJURP for de novo kinetochore assembly. In contrast, tethering of H3K9 tri-methylase (Suv39h1) to the array causes methylation of H3K9, preventing de novo CENP-A assembly and kinetochore formation. CENP-A arrays assembled de novo by this mechanism can form kinetochores of human artificial chromosomes (HACs) that are propagated indefinitely in human cells.

Description

A METHOD FOR POSITIVELY OR NEGATIVELY REGULATING THE ASSEMBLY OF NEWLY SYNTHESIZED CENP-A TO EXOGENOUS ALPHOID DNA CONTAINING CENP-B BOXES IN MAMMALIAN CELL LINES

This invention relates to a method for positively or negatively regulating the assembly of newly synthesized CENP-A to exogenous alphoid DNA containing CENP-B boxes in mammalian cell lines., and to a method for forming a mitotically stable human artificial chromosome (HAC) that is indefinitely propagated or inherited independently from host chromosomes in human cell line.

The kinetochore is responsible for accurate chromosome segregation. During mitosis, kinetochores assemble on specialized centromere chromatin^1,2 composed of specific nucleosomes containing the essential histone H3 variant CENP-A³. Recent studies have identified several factors, including the Mis18 complex and HJURP^4-10, involved in the deposition of newly synthesized CENP-A at pre-existing CENP-A chromatin regions^10-14. 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^15-20. 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^21,22, 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^23-25. Canonical H3 nucleosomes co-purify with CENP-A in oligonucleosomes²⁶, and some classes of CENPs (e.g. CENP-T, -W) are suggested to bind only to H3 nucleosomes²⁷. 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.

US 6,297,029 B1 US 6,569,643 B2 US 2009/0136924 A1

Barnhart M. C. et al. HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore. J Cell Biol. 194, 229-243 (2011). Gascoigne, K. E. et al. Induced Ectopic Kinetochore Assembly Bypasses the Requirement for CENP-A Nucleosomes. Cell 145, 410-422 (2011).

The fundamental question addressed by this invention 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^28-30.

We generated de novo human artificial chromosomes (HACs) with a functional kinetochore assembly from naked alphoid DNAs introduced into human cultured cells, HT1080. These HAC centromeres accurately mimic the structure and mitotic behavior of endogenous centromeres, thus HAC system provides a powerful tool to study mammalian kinetochore assembly. However, to date de novo HAC formation with functional kinetochore assembly on input alphoid DNAs has been reported efficient only in HT1080 cells.

Failure to detect HAC formation in other commonly used human cell lines, such as HeLa, complicates the study of molecular mechanisms. Endogenous centromeric alphoid DNA repeats are enriched by euchromatic modifications (H3K4me2 and H3K9ac) in addition to CENP-A in HT1080 cells, whereas the repeats are inclined into heterochromatic (H3K9me3) in HeLa cells.

We have first demonstrated that tethering of the histone trimethylase Suv39h1 negatively regulates de novo CENP-A chromatin assembly and HAC formation on input alphoid DNAs carrying multiple tet-O sequences in HT1080, whereas tethering of histone acetyl-transferases p300 or PCAF that promote acetylation of H3K9 results in assembly of newly synthesized CENP-A on exogenous alphoid DNA arrays and positively regulates HAC formation and kinetochore establishment in HeLa cells.

Remarkably, HAT inducing de novo CENP-A chromatin assembly requires HJURP but bypasses the need for hMis18alpha, 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 can be 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 switch that acts upstream of HJURP.

These results indicate that epigenetic chromatin assembly balance on alphoid DNA in an individual cell line is crucial for the de novo kinetochore assembly.
Thus, the present invention relates to the following aspects.
Aspect 1:
A method for positively or negatively regulating the assembly of newly synthesized CENP-A to exogenous (or transfected) alphoid DNA containing CENP-B boxes in a host cell, comprising acetylating or methylating H3K9 in said alphoid DNA (alpha-satellite DNA), respectively, in the host cell line.
Aspect 2:
The method of Aspect 1, wherein the exogenous alphoid DNA is comprised in an artificial DNA construct or integrated in a chromosome of the host cell.
Aspect 3:
The method of Aspect 2 wherein the artificial DNA construct is a mammalian artificial chromosome and the host cell is a mammalian cell.
Aspect 4:
The method of Aspect 3 wherein the mammalian artificial chromosome is a human artificial chromosome (HAC) and the mammalian cell is human a cell.
Aspect 5:
The method of Aspect 4 wherein the HAC contains at least one exogenous gene to be expressed in the human cell.
Aspect 6:
The method of any one of Aspects 1-5 wherein the acetylating or methylating H3K9 is carried out by tethering exogenous histone acetyltransferase (HAT) or histone methyltransferase or their enzymatically active domain, or their functionally equivalent analogue to the alphoid DNA.
Aspect 7:
The method of Aspect 6 wherein the exogenous histone acetyltransferase or histone methyltransferase or their enzymatically-active domain, or their functionally equivalent analogue is fused to tet repressor (tetR) (tetR-fusion protein), and the exogenous alphoid DNA carries tet operator (tetO) sequence that allows the tethering of said tetR-fusion protein to the alphoid DNA.
Aspect 8:
The method of Aspect 7 wherein the exogenous alphoid DNA has 30 - 60 Kb and comprises repeats of alpha 21-I alphoid dimmer (alpha 21-I alphoid^tetO repeats) where the CENP-B box is contained in a monomer of the dimmer and the tetO sequence is contained in the other monomer instead of the CENP-B box.
Aspect 9:
The method of any one of Aspects 6-8 wherein the histone acetyltransferase is selected from the group consisting of p300, PCAF, KAT7(HB01), KAT6A(MOZ) and KAT8(MOF).
Aspect 10:
The method of any one of Aspects 6-9 wherein the histone methyltransferase is Suv39h1.
Aspect 11:
The method of any one of Aspects 6-10 wherein the tetR-fusion protein is expressed in the host cell.
Aspect 12:
The method of any one of Aspects 5-11 wherein the human cell has a relatively high H3K9me3 level.
Aspect 13:
The method of any one of Aspects 1-12 wherein the assembly of newly synthesized CENP-A to the exogenous alphoid DNA depends on the presence of a histone chaperone (CENP-A deposition factor).
Aspect 14:
The method of Aspect 13 wherein the histone chaperone is HJURP.
Aspect 15:
A method for forming a mitotically stable artificial DNA construct that is indefinitely propagated or inherited independently from host chromosomes in a host cell, comprising positively regulating the assembly of newly synthesized CENP-A by any one of the method of Aspects 1-14 so as to recruit inner and/or outer kinetochore proteins to CENP-A so that a functional kinetochore will be formed de novo on the artificial DNA construct.
Aspect 16:
The method of Aspect 15 wherein the artificial DNA construct is an artificial chromosome.
Aspect 17:
A mitotically stable human artificial chromosome (HAC) that is formed by the method of Aspect 16 and will be propagated or inherited for many generations without any further tethering of the exogenous histone acetyltransferase or their enzymatically-active domain, or their functionally equivalent analogue.
Aspect 18:
The human artificial chromosome of Aspect 17, which is stable for more than 60 days in culture of the host cell.
Aspect 19:
A human cell line or tissue comprising the mitotically stable human artificial chromosome of Aspect 17 or 18.

Tethering of histone acetyltransferases (HATs) induces de novo assembly of CENP-A and functional kinetochore on ectopic alphoid^tetO DNA, and can culminate in de novo formation of stable human artificial chromosomes (HACs). HAT-induced de novo CENP-A assembly appears to mimic the natural process. It requires the activity of specific CENP-A deposition factor HJURP. The HAT normally responsible for de novo CENP-A assembly and its key substrates in addition to H3K9 remain to be identified. Nonetheless, this observation that tethered HAT activity in canonical H3 chromatin can induce de novo CENP-A and outer kinetochore assembly by adjusting the modification status of H3K9 represents a major step towards understanding the epigenetic regulation of kinetochore assembly.

Regulation of the balance between H3K9ac (promoting CENP-A assembly) and H3K9me3 (inhibiting it) may be critical not only for de novo kinetochore assembly in our artificial system, but also for genome stability. These extremely large kinetochore formation at the ectopic site bundled an excess amount of mitotic spindles and thus arrested the cell cycle as a normal kinetochore function. Kinetochore size has to be regulated appropriately on the huge array of alphoid DNA. Adjusting the balance between H3K9 acetylation and methylation might provide a mechanism to minimize inappropriate CENP-A assembly and the formation of ectopic centromeres on native chromosomes.

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 alpha21-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, standard deviation (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 Fig. 10a and Table 2. Columns indicate non-alphoid repetitive DNA controls (black), type I alphoid DNA (white) and type II (gray), respectively. Error bars, standard deviation (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 micro m. 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, standard deviation (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 micro m. (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, standard deviation (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, standard deviation (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, standard deviation (n=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 alpha21-I alphoid^tetO DNA containing plasmid (pWTO2R; see Supplementary Fig. 12). (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 alpha21-I alphoid^tetO 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, standard deviation (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 micro m. (e) Summary of HAC formation. Bars indicate a frequency of HAC formation in the cells expressing protein fusions. Error bars, standard deviation (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 0 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 x(1-R)⁶⁰ (ref. 18). All HAC cell lines showed high stability (HAC loss rate >0.001). 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 micro m. (d) Schematic timetable for gene depletion and new CENP-A assembly assay. HeLa-HAC-R5 cells were firstly transfected with siRNA. After 24 hours 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) hMis18alpha or HJURP depletion using siRNA. siRNAs for hMis18alpha (sihMis18alpha) and for HJURP (siHJURP) as well as for a negative control (siNegative) were used for transfection. Total RNA was purified two days after transfection and quantified by real-time PCR. hMis18alpha or HJURP mRNA levels were normalized by HPRT transcripts. Horizontal axis indicates relative hMis18alpha or HJURP mRNA level against a negative control (siNegative). Error bar, standard deviation (n=3). (f) HA-CENP-A assembly frequency on endogenous centromere was counted in each sample (n>100). Error bar, standard deviation (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, standard deviation (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 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 alphoid^tetO 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 alphoid^tetO DNA. Cells were stained with DAPI, anti-GFP (green) and anti-HA (red). Arrowheads indicate alphoid^tetO DNA integration sites. Scale bar, 5 micro m. (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, standard deviation (n=3). (e) Frequency of de novo HA-CENP-A assembly on ectopic alphoid^tetO 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, standard deviation (n=3). (f and g) hMis18alpha tethering assay under HJURP depletion. The frequencies shown in panel d and e were counted (n>100). Error bar, standard deviation (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 micro m. (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, standard deviation (n=3). 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 Fig. 5a. After 48 hours incubation, cells were arrested in metaphase and spread on cover glass for immuno-staining. (b) Examples of high order centromere proteins assembly at ectopic alphoid^tetO 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 alphoid^tetO DNA integration site (red). The indicated images were obtained with tetR-EYFP-HJURP tethering. The results obtained with other fusions are shown in Supplementary Fig. 19. Scale bars, 3 micro m. (c) A frequency of ectopic HA-CENP-A assembly per total spread cells was counted (n>100). Error bar, standard deviation (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, standard deviation (n=3). 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 micro m. (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, standard deviation (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, standard deviation (n=3). P-values obtained with t-test are indicated. Figure 8 Cell type specific chromatin modifications and de novo CENP-A assembly activity on alphoid DNA. (a) Cell lines obtained from HAC formation assay with HT1080 and HeLa cells. pWTR11.32 plasmid (see Fig. 1b) was transfected into HT1080 or HeLa cells. HT1080 transformants, W0203 (HAC) and W0210R-8 (Integration) were obtained in previous work (ref. 21). HeLa transformants, HLW-Int-09 (Integration) and HLW-Int-22 (Integration), were obtained in this work. (b) No CENP-A was assembled on the ectopic alphoid DNA integration sites in HeLa cells. ChIP analysis was carried out with normal IgG and indicated antibodies. PCR primers used for quantitative PCR are shown at the top. Error bars, standard deviation (n=3). Figure 9 HeLa cell has de novo CENP-A assembly activity on transfected alphoid DNA. (a) A schematic diagram of competitive PCR detection using the synthetic alphoid DNAs. The alpha21-I 11mer wild type high order repeating (HOR) unit and the CENP-B box (CENP-B binding site) mutant 11mer HOR unit can be amplified with the same primer set keeping initial ratio of these DNA copies. PCR product from CENP-B box mutant 11mer contains two nucleotide substitutions, which produced a recognition site of restriction enzyme, EcoRV. PCR products derived from each alphoid DNA can be distinguished by EcoRV digestion and agarose gel electrophoresis. (b) Example of competitive PCR. The alpha21-I 11mer (WT) and CENP-B box mutant (MT) DNA were mixed at several ratios, and amplified by competitive PCR. PCR products were digested with EcoRV and applied on agarose gel electrophoresis. (c) Transient transfection and ChIP assay with a reporter plasmid. The pW/M11.64 contains both 60 kb of alpha21-I 11mer and 60 kb of CENP-B box mutant 11mer repeat (ref. 20). The pW/M11.64 was transfected to HT1080 or HeLa cell, and harvested 2, 4, 6 and 8 days after transfection for following ChIP assay. A competitive PCR detection was carried out with immuno-precipitated and input DNA. Capital letters indicates; ChIP sample with normal IgG (IgG), anti-CENP-A (A) and anti-CENP-B (B). Upper or lower band indicates PCR product derived from wild type or mutant 11mer repeat DNA, respectively. Figure 10 H3K9me3 and CENP-A assembly in human primary fibroblasts. (a) Schematic diagram of primers for human alphoid DNAs or other repetitive DNA sequences used in this invention. Although human alphoid DNA is basically repetitive sequence of 171bp monomers, each chromosome's alphoid DNA has sequence variations. Type I alphoid is homogeneous repeat of chromosome specific HOR unit. Type II alphoid is diverged monomeric repeat (refs 47,48). These indicated HOR DNA sequences are known as D17Z1 (17 alphoid), D21Z1 (21 alphoid), DXZ1 (X alphoid) and DYZ3 (Y alphoid). All the HOR except DYZ3 contain CENP-B box. Sat2, D4Z4 and DYZ1 were analyzed as controls for heterochromatic repeat. 5S ribosomal DNA sequence was used as a control for transcribed repetitive DNAs. One additional set of primers was designed for Alu elements that are dispersed traces of retrotransposon. The primer DNA sequences are shown in Table 2. (b) ChIP profiling of centromere chromatin with TIG-7 and hTERT-BJ1 cells. ChIP analysis was carried out with normal IgG and indicated antibodies. PCR primers used for quantitative PCR are shown at bottom. Vertical axis indicates enrichment against normal IgG control. Columns indicate non-alphoid repetitive DNA controls (black), type I alphoid DNA (white) and type II (gray), respectively. Error bars, standard deviation (n>3). (c) Human primary fibroblasts exhibit de novo CENP-A assembly activity. pW/M11.64 was transfected to TIG-7 or hTERT-BJ1 cells. Transfected cells were cultured for 8 days under presence of selective drug (G418), and then harvested for ChIP analysis. ChIP was carried out with normal IgG, anti-CENP-A and anti-CENP-B antibodies. Immno-precipitated DNAs were quantified by competitive PCR detection. Capital letters indicate; input DNA (I), precipitates with normal IgG (G), precipitates with anti-CENP-A antibody (A) and precipitates with anti-CENP-B antibody (B), respectively. Figure 11 p300 and PCAF localize at kinetochore. (a) Mitotic HeLa cells were spread on cover glass, and stained with anti-CENP-A (red), anti-p300 (green) and anti-PCAF (blue) antibodies. PCAF signals were detected on all kinetochores. p300 signals were also detected on many kinetochores. Similar result was obtained with HT1080 cells. Scale bar, 10 micro m. (b) Magnified images of the panel a. Selected two boxed areas shown in the panel a (top left, I and II) were magnified. (Most left panel) DNA, p300 and CENP-A signals are shown in blue, green or red, respectively. (Other panels) p300, CENP-A and PCAF signals are shown in green, red and blue, respectively. Scale bars, 3 micro m. Figure 12 Construction of alpha21-I alphoid^tetO repeats. (a) DNA sequence of original alpha21-I-EcoRI-2mer and tetO are shown. The tetO site (red box) was embedded at the position of the pseudo CENP-B box sequence. Recognition sites for restriction enzyme NheI or SpeI were added at the ends of alpha21-I alphoid^tetO 2mer sequence (orange boxes). (b) Schematic diagram for alpha21-I alphoid^tetO 2mer repeat construction. The alpha21-I alphoid^tetO 2mer repeating unit was cut out from a cloning vector with NheI and SpeI. Then this fragment was circularized and used as a template for rolling circle amplification (RCA) reaction. RCA products were assembled into tandem repeat arrays by transformation-associated recombination (TAR) cloning method (refs 43,44). For this invention, we used a TAR BAC isolate containing a 50 kb repeat of alpha21-I alphoid^tetO 2mer sequence (pWTO2R). Figure 13 De novo tetO-HAC formed in HeLa cells. HAC containing HeLa cells, p300-HAC-13, were arrested at mitotic phase and harvested. Then the cells were spread and stained with BAC DNA probe specific to introduced pWTO2R plasmid (red) and DAPI (blue). (top) HAC was stained with Pan-alphoid DNA probe with excess amount of unlabeled alpha21-I alphoid^tetO 2mer DNA. Although pan-alphoid DNA probe (green) can hybridize all alphoid DNA families, unlabeled DNA competed out pan-alphoid DNA signal on tetO-HAC (detailed method was described in ref. 18), indicating tetO-HAC was formed only with alpha21-I alphoid^tetO 2mer DNA. (middle) Intra- and inter-Alu PCR probe (green) stains almost all chromosomal arm regions but did not on tetO-HAC, indicating no detectable recruitment of host DNA fragment into de novo tetO-HAC. (bottom) Examples of metaphase tetO-HAC staining. Green signals obtained with indicated antibodies. Scale bars, 3 micro m. Figure 14 Suv39h1 tethering prevents de novo CENP-A assembly in HT1080 cell. (a) Schematic diagram for panel G. pWTO2R plasmid was transfected to HT1080 cells expressing tetR-EYFP or tetR-EYFP-Suv39h1 fusion protein. These tetR-EYFP and tetR-EYFP-Suv39h1 expressing cells were created by infection of retroviral expression vector. (b) Suv39h1 tethering inhibited de novo CENP-A assembly in HT1080 cells. pWTO2R transfected HT1080 cells were cultured under presence of selective drug (G418), and harvested for ChIP at two weeks after transfection. ChIP was carried out with normal IgG and indicated antibodies. Indicated primers were used for quantitative PCR (top). Error bars, standard deviation (n=3). Figure 15 HAC kinetochore is stably maintained without further HAT tethering. (a) p300-HAC-13 cell was cultured under presence of doxycycline (no tetR binding condition) for more than 60 generations and single colonies were isolated. HeLa-HAC-R5 cell was one of these isolated cell lines, which lost detectable tetR-EYFP-fusion expression during culturing. This HeLa-HAC-R5 cell was transfected with a plasmid construct expressing tetR-EYFP. Cells were blocked in colcemid and co-stained for the indicated centromere and kinetochore components after brief hypotonic treatment. CENP-A, CENP-C and CENP-T are the inner kinetochore components (first from top, second from top and bottom two panels, respectively). Furthermore, outer kinetochore components were assembled as determined by staining for the KMN components Hec1/Ndc80, hDsn1, hMis12 and hKNL1 (top to bottom panels). (b) In unperturbed mitotic cells, the HAC sister kinetochores (arrowheads) achieved alignment on the metaphase plate, with HAC kinetochores being under tension (top panel), and HAC sister chromatids segregated properly during subsequent anaphases (bottom panel) in all cells analyzed. Scale bar, 5 micro m. (c) ChIP analysis of alpha21-I alphoid^tetO (pWTO2R) transformants. Indicated cell lines were obtained from HeLa cells expressing tetR-EYFP (tetR-Int-06), tetR-EYFP-Suv39h1 (Suv39-Int-08), tetR-EYFP-p300 HAT domain (p300-HAC-13 and p300-Int-03) and tetR-PCAF HAT domain (PCAF-HAC-02), respectively. p300-HAC-13 and PCAF-HAC-02 are HAC cell lines. tetR-Int-06, Suv39-Int-08 and p300-Int-03 carry ectopic alpha21-I alphoid^tetO DNA integration sites. ChIP was carried out with normal IgG and indicated antibodies. Primer set for synthetic alpha21-I alphoid^tetO 2mer (tetO-2mer) was used for quantitative PCR. Error bars, standard deviation (n=3). Figure 16 HAT tethering induces new-CENP-A assembly and Suv39h1 tethering destabilizes tetO-HAC. (a) Frequency of HA-CENP-A assembly on endogenous centromere was counted in each sample shown in Figure 4C (n>200). (b) Frequency of HAT tethering-induced expanded HA-CENP-A assembly was counted in each sample shown in Figure 4C (n>200). (c) Destabilized tetO-HAC had lost CENP-A assembly. HeLa HAC-R5 cells expressing tetR-EYFP-Suv39h1 were stained with DAPI, anti-GFP (recognize EYFP, green) and anti-CENP-A (red). Arrowheads indicate a destabilized tetO-HAC outside of nuclei. Scale bar, 5 micro m. (d) Examples of destabilized tetO-HAC detected by FISH. HeLa HAC-R5 cells expressing tetR-EYFP-Suv39h1 were stained with DAPI, anti-GFP (recognize EYFP, green) and BAC DNA specific probe (red). Arrowheads indicate a destabilized tetO-HAC. (e) Suv39h1 tethering cumulatively destabilizes tetO-HAC. tetR-EYFP or tetR-EYFP-Suv39h1 was expressed in HeLa-HAC-R5 cells. Tethering of these proteins were induced by doxycycline removal. Cells were fixed at each time point and stained as shown in panel D. Destabilized tetO-HAC was counted (n>200) and HAC loss rate was calculated. (f) HAC retention rates of Suv39h1 tethered cells were also counted with the same method shown on panels d and e. (g) HAC loss rates for 14 days with tetR-EYFP fusions shown in Figure 4A. HAC loss rate was calculated with HAC retention rates at day 0 (N₀) or at day 14 (N₁₄) using the following formula: N₁₄=N₀ x (1-R)¹⁴ (ref. 18). Figure 17 Integrated alpha21-I alphoid^tetO DNA array has no CENP-A assembly. (a) There was no CENP-A assembly at ectopic alpha21-I alphoid^tetO integration site. Mitotic HeLa-Int-03 cells transiently expressing tetR-EYFP were spread and stained with DAPI (blue), anti-GFP (recognize EYFP, green) and anti-CENP-A (red). Green signal indicates tetR-EYFP that binds to ectopic integration site of alpha21-I alphoid^tetO DNA array. (b) ChIP analysis of HeLa-Int-03 cells. ChIP was carried out with normal IgG and indicated antibodies. A set of primers for synthetic alpha21-I alphoid^tetO repeat (tetO-2mer) was used for quantitative PCR. Error bars, standard deviation (n=3). Figure 18 CENP-A-HA assembly on the ectopic alphoid^tetO integration site. (a) CENP-A-HA was expressed with same procedure to Fig. 5b. Cells were stained with DAPI, anti-GFP (green) and anti-HA (red). Scale bar, 5 micro m. (b) Distribution of CENP-A-HA with tetR-EYFP-fusions tethering. CENP-A-HA localization patterns were divided as whole nuclei (green column), dots signals on only endogenous centromere (yellow column) and dots signals on centromere and tetO site. Cells containing tetR-EYFP spots were counted in each sample (n>100). Error bar, standard deviation (n=3). There was no large difference between HA-CENP-A and CENP-A-HA. Figure 19 Kinetochore protein assembly on CENP-A deposited alpha21-I alphoid^tetO site. Examples of CENP-E assembly at ectopic alpha21-I alphoid^tetO DNA integration site. HeLa-Int-03 cells were co-transfected with HA-CENP-A and each tetR-EYFP fusion protein expressing vector as shown in Fig. 6a. After 48 hours incubation, mitotically arrested cells were spread and stained with DAPI (blue), anti-GFP and anti-CENP-E (red). Images were obtained by tethering of tetR-EYFP-p300HD (top), tetR-EYFP-PCAFHD (middle) or tetR-EYFP-hMis18alpha (bottom), respectively. Scale bars, 5 micro m. Figure 20 Induced ectopic kinetochores bundle microtubules. (a) HeLa-Int-03 cells were stained with DAPI (blue), anti-GFP (green) and anti-alpha-tubulin (red) antibodies. Tethering of tetR-EYFP-alone did not disturb a symmetric spindle formation (top). In contrast, tethering of tetR-EYFP-HJURP induced bundled microtubules to tetO site and abnormal spindle shape (bottom). Scale bars, 5 micro m. (b) HeLa-Int-03 cells were stained with DAPI, anti-CENP-A (green) and anti-alpha-tubulin (red) antibodies. Tethering of tetR-EYFP-fusions except tetR-EYFP-Alone induced huge CENP-A assembly signal and abnormal spindle shape. Scale bars, 5 micro m. All tetR-EYFP-fusions were dissociated tetO site by addition of doxycycline one hour before staining. (c) Cell cycle distribution of tetR-EYFP-fusions tethered cells. HeLa-Int-03 cells were stained as in panel b. Cells in meta-/prometa-, ana-/telo- and interphase were counted (n>100). Error bar, standard deviation (n=3). Figure 21 No CENP-I assembled on mislocalized CENP-A. HeLa-Int-03 cells were transfected with HA-CENP-A expressing vector. Mitotically arrested cells were spread and stained with DAPI (blue), anti-HA (green) and anti-CENP-I (red). Approximately 20% of spread showed HA-CENP-A mislocalized signal on the chromosomal arm region (top left in the panels). However, a kinetochore protein CENP-I was localized only at centromere but not on chromosomal arm regions. Scale bar, 10 micro m. Figure 22 ChIP controls for Figure 7e. tetR-EYFP-Suv39h1 expressing HeLa-HAC-R5 cells were cultured with in the presence or absence of doxycycline for three days as shown in Fig. 7e, and then the harvested same sample sets as described in Fig. 7a. ChIP assay was carried out with normal IgG and indicated antibodies. Indicated primer sets were used for quantitative PCR (top). Error bars, standard deviation (n=3).

According to the present invention, the assembly of newly synthesized CENP-A to exogenous (or transfected) alphoid DNA containing CENP-B boxes is positively or negatively regulating in a host cell by acetylating or methylating H3K9 in said alphoid DNA (alpha-satellite DNA), respectively, in the host cell line. The assembly of newly synthesized CENP-A to the exogenous alphoid DNA may depend on the presence of a histone chaperone (CENP-A deposition factor) such as HJURP.

The exogenous or transfected alphoid DNA containing CENP-B boxes may be comprised in an artificial DNA construct such as a cloning or expression vector, or integrated in a chromosome of a host cell. The artificial DNA construct includes a mammalian artificial chromosome such as, for example, human artificial chromosome (HAC).

Any kind of cell or cell line known in the art may be used as the host cell in the present methods, including mammalian cell or cell lines of rodents such as mouse, and human cell or cell lines. Preferably human cell lines with a significantly higher level of H3K9me3 (heterochromatin-associated modification) than that in cells such as HT1080 that has H3K9ac (euchromatin-associated modification). Accordingly, examples of the human cell lines according to the present invention include HeLa, TIG7 and hTERT-BJ1 cells.

The artificial DNA construct according to the present invention may contain at least one exogenous gene to be expressed in the host cell and any other sequence elements known in the art that may perform a certain function in the host cell.

Any gene known in the art may be contained as the exogenous gene in the exogenous alphoid DNA. Such exogenous gene may be expressed by any method known in the art in the host cell, cell line or tissue, or an organism such as animals and human that is transplanted with these cell, cell line or tissue comprising the host cell line for any purpose or application such as therapy or treatment of diseases or disorders of the organism.

Additionally, the artificial DNA construct may optionally contain any elements known in the art including promoter, enhancer, operator, selection marker cassette, replication origin, etc, depending on its purpose, structure, function, vectors used for its construction and the like.

According to the present invention, the acetylating or methylating H3K9 is preferably carried out by tethering exogenous histone acetyltransferase (HAT) or histone methyltransferase or their enzymatically-active domain such as HAT-domain, or their functionally equivalent analogue to the alphoid DNA.

The histone acetyltransferase preferably used in the present invention is selected from the group consisting of p300, PCAF, KAT7(HB01), KAT6A(MOZ) and KAT8(MOF), and any other known one that has substantially the same activity or function as any one of the above ones. The histone methyltransferase preferably used in the present invention is Suv39h1 or any other known one that has substantially the same activity or function as Suv39h1.

The enzymatically active domain of the above enzymes such as HAT-domain of the histone acetyltransferase or their functionally equivalent analogue may be also used in the present invention. The functionally equivalent analogue of the enzyme may be a recombinant or naturally-occurring polypeptide having an amino acid sequence with 90% or more, preferably 95% or more, more preferably 98% or more of identity with the amino acid sequence of one of the above enzymes, or a polypeptide having an amino acid sequence wherein one to several amino acid residues, for example, one to three, five or ten amino acid residues have been substituted (preferably by means of conservative substitution), deleted, or added in the amino acid sequence of one of the above enzymes as long as the polypeptide has the histone acetyltransferase or histone methyltransferase activity with respect to H3K9. A polynucleotide encoding such recombinant polypeptide may be easily designed and prepared by those skilled in the art using a conventional gene-engineering technique.

In order to determine the identity or homology between two amino acid sequences, they may be preliminarily treated into an optimum condition for comparison. For example, a gap may be inserted into one of the sequences to optimize the alignment with the other sequence, followed by the comparison of amino acid at each site. When the same amino acid exists at a corresponding site of the first and second sequences, these two sequences are considered to be identical with respect to said site. Identity between two sequences is shown by a percent ratio of the number of the identical sites over the total number of amino acids between the two sequences.

The term "identity" in this specification means a ratio of an amount (or a number) of the amino acids in an amino acid sequence, which are determined to be identical with each other in the relationship between two sequences, showing an extent of the correlation between the two polypeptide sequences. The identity may be easily calculated. The term "identity" or "homology" is well known in the art, and many methods for the calculation of such homology are known, among them. For example, Lesk, A. M. (Ed.), Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, D. W. (Ed.), Biocomputing: Informatics and Genome Projects, Academic Press, New York, (1993); Grifin, A. M. & Grifin, H. G. (Ed.), Computer Analysis of Sequence Data: Part I, Human Press, New Jersey, (1994); von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press,New York, (1987); Gribskov, M. & Devereux, J. (Ed.), Sequence Analysis Primer, M-Stockton Press, New York, (1991) . A general method for the determination of the homology between two sequences is disclosed, for example, in Martin, J. Bishop (Ed.), Guide to Huge Computers, Academic Press, San Diego, (1994); Carillo, H. & Lipman, D., SIAM J. Applied Math., 48: 1073 (1988) . A preferable method for the determination of the homology between two sequences is, for example, one designed to obtain a largely related part between said two sequences. Some of them are provided as a computer program. Preferable examples of the computer programs for the determination of the homology between two sequences include GCG program package (Devereux, J. et al., Nucleic Acids Research, 12(1): 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol., 215: 403 (1990).

More specifically, the exogenous histone acetyltransferase or histone methyltransferase or their enzymatically-active domain or their functionally equivalent analogue is fused to tet repressor (tetR) (tetR-fusion protein), and the exogenous alphoid DNA carries tet operator (tetO) sequence that allows the tethering of said tetR-fusion protein to the alphoid DNA. Those skilled in the art may easily prepare an appropriate expression vector comprising a polynucleotide encoding the tetR-fusion protein and transfect a host cell with the thus prepared expression vector in a known manner, so that the tetR-fusion protein will be expressed in the host cell.

As described in the present specification, the exogenous alphoid DNA preferably has 30 - 60 Kb and comprises repeats of alpha 21-I alphoid dimmer (alpha 21-I alphoid^tetO repeats) where the CENP-B box is contained in a monomer of the dimmer and the tetO sequence is contained in the other monomer instead of the CENP-B box. Such exogenous alphoid DNA may be constructed by a known method such as rolling circle amplification (RCA) using an appropriate plasmid vector, yeast artificial chromosome (YAC) and/or bacterial artificial chromosome (BAC), for example, by those skilled in the art in known methods in the art such as those described in the present specification.

According to the present invention, a mitotically stable artificial DNA construct such as an artificial chromosome including HAC that is indefinitely propagated or inherited independently from host chromosomes in a host cell is formed by positively regulating the assembly of newly synthesized CENP-A by the above method so as to recruit inner and/or outer kinetochore proteins to CENP-A, so that a functional kinetochore will be formed de novo on the artificial DNA construct. The inner and/or outer kinetochore proteins include CENP-C,-E,-I-T, hKNL1, Hec1, hDsn1 and hMis12.

The present invention relates to a mitotically stable human artificial chromosome (HAC) that is formed by the above method and will be propagated or inherited for many generations without any further tethering or artificial binding of the exogenous histone acetyltransferase or their enzymatically-active domain, or their functionally equivalent analogue to the HAC.

The human artificial chromosome formed according to the above method is stable for a relatively long period of time, for example, more than several-ten days such as more than 60 days in culture of the host cell under normal conditions depending on the cell.

The present invention therefore relates also to any cell, cell line or tissue comprising said mitotically stable human artificial chromosome, or an organism such as animals and human that is transplanted with these cell, cell line or tissue.

The present invention may be easily carried out by those skilled in the art in accordance with the description of the present specification by means of standard techniques well known to those skilled known methods in the art, especially by using the methods described in Examples below. Unless specifically described in the specification, experimental procedures and treating conditions may be referred to the literatures well known in the art, such as J. Sambrook, E. F. Fritsch & T. Maniatis, "Molecular Cloning", 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N. Y. (1989) and D. M. Glover et al. ed., "DNA Cloning", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995) (DNA cloning), and with H. A. Erlich ed., PCR Technology, Stockton Press, 1989 ; D. M. Glover et al. ed.," DNA Cloning", 2nd ed., Vol. 1, (The Practical Approach Series), IRL Press, Oxford University Press (1995) and M. A. Innis et al. ed., "PCR Protocols", Academic Press, New York (1990) (PCR) . A commercially available agent and kit were used in accordance with protocols attached thereto. Especially, US 6,297,029 B1 and US 6,569,643 B2 may be referred to with respect to the basic structure, preparation and any other information about some aspects of a human artificial chromosome (HAC) containing CENP-B boxes, the entire disclosure of which is incorporated herein by reference.

METHODS:
Cell culture and transfection
HT1080 (tetraploid) and HeLa cells were grown in Glutamax I (Invitrogen) supplemented with 10% FBS at 37 degrees Celsius (C) in 5% CO₂ atmosphere. For transfections, Lipofectamin 2000 (Invitrogen), Lipofectamine (Invitrogen) or Fugene HD (Roche) was used for siRNA, BAC plasmid DNAs (pWTR11.32, pMTR11.32, pW/M11.64 and pWTO2R) or usual plasmid vectors, respectively. Retrovirus infection method used for tetR-fusions expression was previously described (ref. 29). For depletion experiments, siRNAs for Suv39h1 were obtained from Darmacon as a pool (D-009604-01, D-009604-02, D-009604-04 and D-009604-06), and siRNA sequence for hMis18alpha or HJURP depletion was referred Fujita et al. (ref. 10) or Dunleavy et al. (ref. 11), respectively.

ChIP
Cells were trypsinized and harvested in a centrifuge tube. Cells were washed with PBS and then fixed with 0.61% (for tetR-EYFP or EFYP fusions) or 0.3% (for histones) formaldehyde at 25 degrees C for 10 min. ChIP procedure was previously described (ref. 21). Antibodies used for ChIP are shown in Table 1. Immuno-precipitated DNAs were de-fixed at 65 degrees C for more than 4 hr and purified by phenol/chloroform extraction following proteinase K treatment. Purified DNA was quantified by the competitive PCR (Supplementary Fig. 9) or real-time PCR (BIORAD). For real-time PCR detection, SYBR Green I containing reagent was used (BIORAD). PCR primer sequences used for ChIP assay are shown in Table 2.

Construction of synthetic alphoid DNA arrays and expression vectors
The alpha21-I-EcoRI-2mer sequence was amplified with two primer sets; 2mer-F1 and 2mer-R2 for the first half, and 2mer-F2 and 2mer-R1 for the last half of alpha 21-I EcoRI 2mer. These two PCR products were independently cloned into a pUC vector and confirmed by DNA sequencing. Then, two fragments were combined and SpeI and NheI sites were added at the ends by a general cloning method. This synthetic dimer was named as alpha21-I alphoid^tetO 2mer (Supplementary Fig. 12). This alpha21-I alphoid^tetO 2mer was cut out from the vector with NheI and SpeI, purified from agarose gel, self ligated and used for rolling circle amplification (RCA). RCA reaction was carried out with phi29 polymerase (NEB) and short primer set for alphoid DNA sequences (refs 43,44). RCA products were co-transfected to yeast cells along with a cloning vector, termed pHook, and assembled arrays were recovered by transformation-associated recombination (TAR) cloning. pHook is BAC108L-based plasmid created in this invention, which contains Kanamycin/neomycin selection marker cassette, yeast ARS and CEN sequences and specific hook sequences for alpha21-I alphoid^tetO 2mer. Details of pHook vector sequence are available upon request. YACs carrying alphoid DNA arrays were purified and transfected to E. coli cells, and then single colonies were picked up for insert length screening by PFGE gel electrophoresis. Using this procedure, we obtained ~50-kb arrays of alpha21-I alphoid^tetO-2mer repeats in the pHook vector. The final construct was termed pWTO2R, and used for tetR-fusions tethering.

The tetR-EFYP gene was amplified from the pFB-tetR-EYFP-neo plasmid (ref. 29) with primers tetR-F and EYFP-R. PCR product was cut with Eco52I and EcoRI and cloned into NotI and EcoRI site of pQCXIP (Clontech). The constructed plasmid was named as pQC-TRYF-IP. The genes used for tetR-EYFP fusion proteins were obtained by RT-PCR. PCR product of Suv39h1, p300 HAT domain (amino acid 1283-1673) or PCAF HAT domain (amino acid 451-832) was cut with XhoI and NotI (for Suv39h1 and p300) or SalI and NotI (for PCAF), and cloned into XhoI and NotI site of pQC-TRYF-IP. Primer DNA sequences used in this invention are shown in Table 3. These plasmids were transfected to amphopack 293 cells (Clontech) with pVSV-G (Clontech) plasmid, and produced retrovirus were harvested and infected to HT1080 or HeLa cells according to a previously described method (ref. 29). For plasmid-based tetR-EYFP-fusion proteins expression, the genes were cloned into the pJETY3 vector. pJETY3 was constructed from pJTI Fast DEST vector (Invitrogen). Briefly, EF1 promoter, tetR-EYFP gene and IRES-Hyg gene sequences were inserted between PstI (1801) and EcoRI (4375) sites of pJTI Fast DEST vector by multistep DNA subcloning. Details of pJETY3 vector sequence are available upon request. PCR products of Suv39h1, p300HD, PCAFHD, hMis18alpha and HJURP genes were cloned into pJETY3.

Preparation of mitotic cells and chromosome spreads
For mitotic arrest, cells were treated with 350 nM of the highly reversible microtubule destabilizing drug, TN-16 (WAKO; refs 45,46), for 2 to 6 hours in the growth medium. Mitotic cells were harvested by pipetting, washed with PBS, incubated in a hypotonic buffer (20 mM Tris pH7.4, 1 mM EGTA and 40 mM KCl) and then spread on cover glass (Matsunami) by Cytospin3 (Shandon). Following immuno-staining and/or FISH were carried out according to the previously described method (refs 18,21). For ChIP with mitotic or post mitotic cells (Fig. 7), mitotically arrested cells were harvested with the method described above, firstly. Then a portion of the mitotic cells was fixed for ChIP, and the remaining were washed with PBS two times and plated in petri dish. After 1 hour incubation, unattached mitotic cells were washed out with PBS by pipetting. Attached cells were harvested for ChIP analysis at each time point. ChIP procedure is described in above section.

Cell staining (in Fig. 15)
HeLa-HAC-R5 cells were transiently transfected with a plasmid expressing tetR-EYFP. Transfection with 2 micro g plasmid DNA was carried out using Nucleofector Kit R (Amaxa) at programme setting I-013, essentially according to the manufacturer's instructions. Cells were subsequently seeded onto no. 1.5 glass coverslips in pre-warmed DMEM + GlutaMAX-I (Invitrogen) supplemented with 10% FBS and Penicillin / Streptomycin. For analysis of unperturbed mitoses two days after transfection, cells were fixed in 4% PFA / PBS for 10 minutes at room temperature prior to permeabilization in 0.2% PBS / Triton X-100 for 5 minutes and subsequent immuno-fluorescence staining as described below. For analysis of HAC kinetochore structure, transfected cells were incubated in the presence of 0.1micro g/ml Karyo MAX Colcemid (Invitrogen) for 5 hours at 37 degrees C followed by brief hypotonic swelling in 75 mM KCl prior to fixation as above.

Immuno-fluorescence staining was essentially carried out as described previously (ref. 30). In brief, PFA fixed cells were pre-blocked in 3% BSA in PBS / 0.2% Tween (PBS-T) at 37 degrees C following PFA fixation and Triton permeabilization. Incubation with primary antibodies diluted in 1% BSA in PBS-T was performed in a humid chamber at 37 degrees C for one hour, followed by washing in PBS-T and incubation with fluorescently labeled (Texas Red or Cy5) secondary antibody (Jackson Research), diluted in 1% BSA / PBS-T, for 30 minutes at 37 degrees C. Coverslips were subsequently mounted in VectaShield with DAPI (Vector Labs).

Z-stacks with a spacing of 0.2 micro m were acquired on a DeltaVision microscope setup based on an Olympus IX-71 inverted microscope stand coupled to a Photometrics Cool Snap HQ camera. Stage, filters, shutter and camera were controlled by SoftWorx (Applied Precision), and an Olympus UPlanSApo 100x oil immersion objective (numerical aperture of 1.4) was used during image acquisition. All images were acquired at 1x1 binning and deconvolved with SoftWorx. Maximum intensity projections of the relevant HAC-containing sections were generated for display purposes.

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 (Fig. 1a and Supplementary Fig. 8).
Surprisingly, HeLa cells, TIG7 human fetal primary and hTERT-BJ1 immortalized fibroblasts, could efficiently assemble CENP-A chromatin de novo, but declined rapidly (Fig. 1b, c and Supplementary Fig. 9 and 10). 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 (Fig. 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 (Fig. 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 (Fig. 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 and hTERT-BJ1 cells (Fig. 1d and Supplementary Fig. 10). The ChIP data were confirmed by a stronger H3K9me3 staining intensity at mitotic centromeres in HeLa cells (Fig. 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 (Fig. 2a). Suv39h1 over-expression increased both levels of the enzyme itself and H3K9me3 on centromeric alphoid DNAs in HT1080 cells (Fig. 2b). These results fit with the observations that mouse cells doubly null for Suv39h1 and Suv39h2 (Suv39h^dn)³¹.

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; Fig. 2c-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 the ectopic alphoid DNA array²⁰. 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^22,32. Furthermore, the acetyltransferases p300 and PCAF (p300/CBP associated factors³³) both localize at functional, but not at inactive, centromeres (Supplementary Fig. 11)^34,35.

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 (Fig. 3a). Into the tetR-EYFP expressing cells, we then introduced a 50 kb synthetic DNA array based on the alpha21-I alphoid dimer sequence with a tetO site where the CENP-B box would be on one monomer (pWTO2R; Fig. 3a and Supplementary Fig. 12). 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.

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 (Fig. 3b,c). 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 alpha21-I alphoid^tetO repeat were detected in HeLa cell lines expressing tetR-EYFP-p300 or tetR-EYFP-PCAF (in 8% or 14% of cell lines, respectively as a predominant effect; Fig. 3b,d,e and Supplementary Fig. 13). Importantly, HAC formation was never detected when the synthetic alpha21-I alphoid^tetO repeat was introduced into cells expressing tetR-EYFP or tetR-EYFP-Suv39h1 (Fig. 3e). Similarly, alphoid^tetO-based HAC formation was never observed in HT1080 cells expressing tetR-EYFP-Suv39h1 (Fig. 3e and Supplementary Fig. 14).

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 (Fig. 3f). Microscopic and ChIP analyses showed that the HACs remained mitotically stable by recruiting inner and outer kinetochore proteins CENP-A, -C, -T, hKNL1, Hec1, hDsn1 and hMis12 (Supplementary Fig. 15a,b) in the absence of bound exogenous HAT fusion proteins (Fig. 3f; Loss of tetR fusion binding to the HAC was confirmed by ChIP - Supplementary Fig. 15c).

Thus, HAT domain recruitment to the synthetic alpha21-I alphoid^tetO 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 G1⁷. To test whether the same chromatin modifiers that potentiate de novo kinetochore assembly also affect CENP-A maintenance 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; Fig. 4a,b). We then asked if HA-CENP-A (a mark for newly assembled CENP-A) assembled on the HAC and endogenous centromeres at 24 hrs (i.e. one complete cell cycle in HeLa cells) after transfection.

Tethering of tetR-EYFP alone did not affect the assembly of newly synthesized HA-CENP-A onto either the HAC or endogenous centromeres (Fig. 4c and Supplementary Fig. 16a). In contrast, tethered tetR-EYFP-Suv39h1 specifically reduced HA-CENP-A assembly on the HAC centromere (Fig. 4c and Supplementary Fig. 16b). This was coupled with destabilization of the HAC, detected as lagging chromosomes and micronuclei (Supplementary Fig. 16c-g).

Unexpectedly, tethering of p300HD or PCAFHD induced HA-CENP-A hyper-assembly not only at the HAC centromere, but covering the entire HAC sequence in a significant proportion of cells (34% and 40% in Fig. 4c and Supplementary Fig. 16b).

We next tested whether the known canonical CENP-A deposition factors hMis18alpha and HJURP are involved in HAT-induced CENP-A assembly. We first depleted hMis18alpha or HJURP by siRNA knockdown and then tethered tetR-fused HAT proteins to the synthetic alphoid array (Fig. 4d,e). hMis18alpha depletion reduced HA-CENP-A assembly at both endogenous centromeres and the HAC centromere (Fig. 4f and red bars in Fig. 4g). However, HA-CENP-A assembly on alphoid^tetO DNA was restored by tethered HAT fusions, (Fig. 4g, orange bars). In these cells, the restored HA-CENP-A assembly was increased indeed in cells showing no HA-CENP-A assembling signals on the endogenous centromeres in which the hMis18alpha depletion is effective (Fig. 4g, orange bars. P<0.05).

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 alphoid^tetO array (Fig. 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 (Fig. 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 alphoid^tetO 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 Fig. 17).

Tethering of tetR-EYFP-p300HD or tetR-EYFP-PCAFHD induced HA-CENP-A hyper-assembly on the ectopic array in 33% and 66% of cells (Fig. 5a-e). A similar effect was observed after tethering the CENP-A assembly factors, tetR-EYFP-hMis18alpha or tetR-EYFP-HJURP (HA-CENP-A hyper-assembly in 32% and 100% of cells, respectively - Fig. 5c-e). CENP-A assembly at the ectopic site induced by tetR-EYFP-hMis18alpha tethering was diminished by HJURP depletion (Fig. 5f,g), consistent with Barnhart et al. (ref. 36). In controls, tethering of tetR-EYFP alone or tetR-EYFP-Suv39h1 did not induce HA-CENP-A assembly at the ectopic site (Fig. 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 alphoid^tetO array by the HAT tetherings in addition to the usual assembly patterns of those histone H3 (Fig. 5h,i and Supplementary Fig. 18). Thus, HAT activity is sufficient to trigger the specific assembly of newly synthesized CENP-A on alphoid DNA without requiring the prior binding of other essential kinetochore proteins.

HAT tethering induces de novo functional kinetochore assembly at the ectopic site
We next investigated whether ectopic CENP-A assembly driven by chromatin acetylation or tethered hMis18alpha or HJURP can induce assembly of the outer kinetochore in HeLa cells (Fig. 6a).

CENP-A assembled on ectopic alphoid^tetO arrays was maintained in metaphase cells, where the ectopic HA-CENP-A was always detected as an extended region weakly stained with DAPI (Fig. 6b,c). HA-CENP-A-coated arrays were observed in 19% or 82% of metaphase cells expressing tetR-EYFP-hMis18alpha or tetR-EYFP-HJURP, respectively (compared to 32% and 100% in interphase cells). HAT-induced CENP-A assembly was less stable until metaphase cells (8% of cells, compared with 33%~66% of interphase cells - Fig. 5e and 6c). Continuous tethering of HAT activity at the ectopic site through the interphase cycles may destabilize the CENP-A chromatin.

Remarkably, the essential inner or outer kinetochore markers CENP-T, -I and -E^27,37 assembled on the ectopic array following CENP-A assembly (red arrowheads, Fig. 6b,d and Supplementary Fig. 19). These proteins accumulated at greater levels than at the centromeres of host chromosomes (Fig. 6b, green arrowheads). Such an induced hyper-kinetochore assembly at the ectopic sites can bundles an excess amount of microtubules and resulted in aberrant spindle formation. And, therefore, the cells could not exit from metaphase (Supplementary Fig. 20c). In contrast, kinetochore assembly was not observed on nonspecifically assembled CENP-A at whole chromosomal arm regions (Supplementary Fig. 21), consistent with Gascoigne et al. (ref. 38).

These results demonstrate that tethering of HAT activity or Mis18alpha can induce HJURP-dependent de novo CENP-A chromatin assembly and subsequent assembly of the functional outer kinetochore on an ectopic alphoid DNA array.
Centromere H3K9 acetylation normally occurs in a short time window following metaphase

Although forced HAT tethering can induce CENP-A and kinetochore protein assembly on alphoid^tetO DNA, the level of H3K9 acetylation on endogenous alphoid DNA is normally very low - almost undetectable in unsynchronized HeLa cells (Fig. 1d). This raises the question of whether CENP-A assembly induced by acetylation of H3K9 is biologically relevant. If centromere acetylation does normally occur, it may be during only a brief cell cycle window - possibly coinciding with the localization of hMis18alpha and HJURP to centromeres. HJURP centromere localization is high at two hours after release from a metaphase arrest, and rapidly decreases thereafter⁴.

Indeed, ChIP analysis revealed that H3K9 acetylation levels increased temporarily on endogenous and HAC centromere alphoid DNAs at one hour after release from a metaphase arrest, but fell again by three hours after the release (Fig. 7a-c). The temporary increase in H3K9ac can be blocked by tetR-EGFP-Suv39h1 tethering under the condition controlled with/without doxycycline and CENP-A level fell (dox-; Fig. 7e and Supplementary Fig. 22).

These results confirm the presence of intrinsic acetylation activity on centromere chromatin, and show that it is restricted to a short time window from anaphase through early G1. Taken together these results indicate that H3K9 acts as an acetyl/methyl switch to regulate CENP-A assembly. If it is acetylated, the chromatin can bind chaperones and assemble CENP-A chromatin. If it is trimethylated, CENP-A assembly is inhibited.

Discussion:
Centromeric chromatin acetylation induces de novo heritable kinetochore assembly
Tethering of histone acetyltransferases (HATs) induces de novo assembly of CENP-A and functional kinetochore on ectopic alphoid^tetO DNA, and can culminate in de novo formation of stable human artificial chromosomes (HACs). HAT-induced de novo CENP-A assembly appears to mimic the natural process. It requires the activity of specific CENP-A deposition factor HJURP. The HAT normally responsible for de novo CENP-A assembly and its key substrates in addition to H3K9 remain to be identified. Nonetheless, this observation that tethered HAT activity in canonical H3 chromatin can induce de novo CENP-A and outer kinetochore assembly by adjusting the modification status of H3K9 represents a major step towards understanding the epigenetic regulation of kinetochore assembly.

Recent exciting studies demonstrated that tethering of CENP-C and CENP-T³⁸ or HJURP^36, to an ectopic LacO array induced the assembly of a functional outer kinetochore. However those kinetochore-like structures were not tested if they were stably inherited. In our invention, importantly, de novo formed kinetochores through CENP-A assembly mechanism direct accurate segregation of the resulting HACs for many generations without any requirement for continued tethering of the exogenous HAT. Thus, our data suggest that proper assembly of CENP-A chromatin is critical for long-term epigenetic maintenance of centromere activity.

H3K9 ac/me3 are positive and negative regulators of CENP-A assembly, respectively
The notion that CENP-A assembly may normally be linked to chromatin acetylation^10,22,32 is strongly supported by our detection of a pulse of histone H3 acetylated on lys 9 (H3K9ac) during a brief window following release from a mitotic arrest. This timing corresponds remarkably well with the observed localization of hMis18alpha and HJURP at kinetochores^10-12 and is the cell cycle window in which CENP-A assembly normally occurs¹³.
Although Suv39h1 over-expression increased levels of H3K9me3 on centromeric alphoid DNA, the functions of endogenous centromeres on host chromosomes were not impaired. However, tethering of Suv39h1 to the alphoid^tetO kinetochore blocked the pulse of centromeric H3K9 acetylation normally seen during mitotic exit and interfered with the assembly of newly synthesized CENP-A on the established HAC centromere. Thus, although kinetochores do contain limited H3K9me3-containing chromatin regions²⁴, the CENP-A chromatin core in the active kinetochore must be protected from Suv39h1-induced H3K9 tri-methylation during mitotic exit.

Regulation of the balance between H3K9ac (promoting CENP-A assembly) and H3K9me3 (inhibiting it) may be critical not only for de novo kinetochore assembly in our artificial system, but also for genome stability. These extremely large kinetochore formation at the ectopic site bundled an excess amount of mitotic spindles and thus arrested the cell cycle as a normal kinetochore function. Kinetochore size has to be regulated appropriately on the huge array of alphoid DNA. Adjusting the balance between H3K9 acetylation and methylation might provide a mechanism to minimize inappropriate CENP-A assembly and the formation of ectopic centromeres on native chromosomes.

The role of centromeric heterochromatin may vary in different organisms. In fission yeast, heterochromatin is important not only for sister chromatid cohesion, but also for de novo CENP-A assembly^4,39-41. Understanding this contrast between fission yeast and human CENP-A assembly clearly requires additional study.

Breaking the HAC barrier
Since the first HAC formation assay, it has been unclear why de novo kinetochore formation could occur in HT1080 cells but not in other popular cell lines, such as HeLa. Indeed, in some quarters, this was taken to suggest that HAC formation in HT1080 might in some way be an aberrant process. Here, we could show a very simple H3K9 acetyl/methyl switch model to explain this host cell specificity for HAC formation. Assembly of a core of CENP-A sufficient to establish an epigenetically stable active centromere appears to require H3K9ac.

We have shown that tethering of HAT activity to the input alphoid DNA array breaks the kinetic barrier provided by the very brief window of acetylation that occurs during mitotic exit. This appears to allow sufficient time for CENP-A to assemble into "core" regions of sufficient size to be stably maintained^22,42. Thus the synthetic tetO-alphoid/tetR-fusion tethering system now allows us to induce de novo kinetochore assembly on both newly introduced synthetic alphoid DNA arrays as well as pre-existing arrays integrated into chromosome arms. This offers a powerful approach to analyzing epigenetic centromere/kinetochore formation and maintenance.

The present invention fulfills several utilities for human health. HACs generated from synthetic DNA arrays with an inducing system of CENP-A chromatin can be used for inventioning the expression of full-size genes or groups of genes including human disease genes with no upper limit for the size of DNA region to be cloned. They can also be used in gene therapy studies as a novel efficient system for gene delivery.

The following documents are incorporated herein by reference in their entirety.
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The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to those skilled in the art upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The following claims are intended to cover the components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.

Claims (19)

1. A method for positively or negatively regulating the assembly of newly synthesized CENP-A to exogenous (or transfected) alphoid DNA containing CENP-B boxes in a host cell, comprising acetylating or methylating H3K9 in said alphoid DNA (alpha-satellite DNA), respectively, in the host cell line.
2. The method of Claim 1, wherein the exogenous alphoid DNA is comprised in an artificial DNA construct or integrated in a chromosome of the host cell.
3. The method of Claim 2 wherein the artificial DNA construct is a mammalian artificial chromosome and the host cell is a mammalian cell.
4. The method of Claim 3 wherein the mammalian artificial chromosome is a human artificial chromosome (HAC) and the mammalian cell is human a cell.
5. The method of Claim 4 wherein the HAC contains at least one exogenous gene to be expressed in the human cell.
6. The method of any one of Claims 1-5 wherein the acetylating or methylating H3K9 is carried out by tethering exogenous histone acetyltransferase (HAT) or histone methyltransferase or their enzymatically active domain, or their functionally equivalent analogue to the alphoid DNA.
7. The method of Claim 6 wherein the exogenous histone acetyltransferase or histone methyltransferase or their enzymatically-active domain, or their functionally equivalent analogue is fused to tet repressor (tetR) (tetR-fusion protein), and the exogenous alphoid DNA carries tet operator (tetO) sequence that allows the tethering of said tetR-fusion protein to the alphoid DNA.
8. The method of Claim 7 wherein the exogenous alphoid DNA has 30 - 60 Kb and comprises repeats of alpha 21-I alphoid dimmer (alpha 21-I alphoid^tetO repeats) where the CENP-B box is contained in a monomer of the dimmer and the tetO sequence is contained in the other monomer instead of the CENP-B box.
9. The method of any one of Claims 6-8 wherein the histone acetyltransferase is selected from the group consisting of p300, PCAF, KAT7(HB01), KAT6A(MOZ) and KAT8(MOF).
10. The method of any one of Claims 6-9 wherein the histone methyltransferase is Suv39h1.
11. The method of any one of Claims 6-10 wherein the tetR-fusion protein is expressed in the host cell.
12. The method of any one of Claims 5-11 wherein the human cell has a relatively high H3K9me3 level.
13. The method of any one of Claims 1-12 wherein the assembly of newly synthesized CENP-A to the exogenous alphoid DNA depends on the presence of a histone chaperone (CENP-A deposition factor).
14. The method of Claim 13 wherein the histone chaperone is HJURP.
15. A method for forming a mitotically stable artificial DNA construct that is indefinitely propagated or inherited independently from host chromosomes in a host cell, comprising positively regulating the assembly of newly synthesized CENP-A by any one of the method of Claims 1-14 so as to recruit inner and/or outer kinetochore proteins to CENP-A so that a functional kinetochore will be formed de novo on the artificial DNA construct.
16. The method of Claim 15 wherein the artificial DNA construct is an artificial chromosome.
17. A mitotically stable human artificial chromosome (HAC) that is formed by the method of Claim 16 and will be propagated or inherited for many generations without any further tethering of the exogenous histone acetyltransferase or their enzymatically-active domain, or their functionally equivalent analogue.
18. The human artificial chromosome of Claim 17, which is stable for more than 60 days in culture of the host cell.
19. A human cell line or tissue comprising the mitotically stable human artificial chromosome of Claim 17 or 18.
    

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