Assembly of additional heterochromatin distinct from centromere-kinetochore chromatin is required for de novo formation of human artificial chromosome.

Alpha-satellite (alphoid) DNA is necessary for de novo formation of human artificial chromosomes (HACs) in human cultured cells. To investigate the relationship among centromeric, transcriptionally permissive and non-permissive chromatin assemblies on de novo HAC formation, we constructed bacterial artificial chromosome (BAC)-based linear HAC vectors whose left vector arms are occupied by βgeo coding genes with or without a functional promoter in addition to a common marker gene on the right arm. Although HACs were successfully generated from the vectors with promoter-less constructs on the left arm in HT1080 cells, we failed to generate a stable HAC from the vectors with a functional promoter on the left arm. Despite this failure in HAC formation, centromere components (CENP-A, CENP-B and CENP-C) assembled at the integration sites correlating with a transcriptionally active state of both marker genes on the vector arms. However, on the stable HAC, chromatin immunoprecipitation analysis showed that HP1α and trimethyl histone H3-K9 were enriched at the non-transcribing left vector arm. A transcriptionally active state on both vector arms is not compatible with heterochromatin formation on the introduced BAC DNA, suggesting that epigenetic assembly of heterochromatin is distinct from centromere chromatin assembly and is required for the establishment of a stable artificial chromosome.


Alpha-satellite (alphoid) DNA is necessary for de novo formation of human artificial chromosomes (HACs) in human cultured cells.
To investigate the relationship among centromeric, transcriptionally permissive and nonpermissive chromatin assemblies on de novo HAC formation, we constructed bacterial artificial chromosome (BAC)-based linear HAC vectors whose left vector arms are occupied by ␤ ␤geo coding genes with or without a functional promoter in addition to a common marker gene on the right arm. Although HACs were successfully generated from the vectors with promoter-less constructs on the left arm in HT1080 cells, we failed to generate a stable HAC from the vectors with a functional promoter on the left arm. Despite this failure in HAC formation, centromere components (CENP-A, CENP-B and CENP-C) assembled at the integration sites correlating with a transcriptionally active state of both marker genes on the vector arms. However, on the stable HAC, chromatin immunoprecipitation analysis showed that HP1␣ ␣ and trimethyl histone H3-K9 were enriched at the nontranscribing left vector arm. A transcriptionally active state on both vector arms is not compatible with heterochromatin formation on the introduced BAC DNA, suggesting that epigenetic assembly of heterochromatin is distinct from centromere chromatin assembly and is required for the establishment of a stable artificial chromosome.
However, several lines of evidence also support the importance of epigenetic mechanisms. On stable di-centric chromosomes, caused by chromosomal rearrangements, most functional centromere proteins do not assemble on the inactive centromere despite the presence of human centromere-specific alphoid DNA and CENP-B Cleveland et al., 2003). More strikingly, on rearranged chromosome fragments from patients with mostly congenital abnormalities, a rare phenomenon may occur whereby a functional centromere, the 'neocentromere', forms and is maintained in the complete absence of alphoid DNA (Saffery et al., 2003). However, introduced alphoid yeast artificial chromosome (YAC) and/or bacterial artificial chromosome (BAC) DNA generates stable HACs in some transfected cell lines, whereas in the remainder of the transfectants it is integrated into host chromosomes and inactivated. It has not been established to date how epigenetic mechanisms lead to the different fates of the input DNA, active centromere assembly or centromere inactivation. CENP-A chromatin and centromere components, however, can be reassembled specifically on the ectopic alphoid YAC integration sites that correlate with transcriptional activation on the marker gene on the YAC sites (Nakano et al., 2003). The result indicates that the suppressed state on ectopic alphoid loci can be reversed by the epigenetic change of the adjacent chromatin to a transcriptionally active state. A lack of a unique marker sequence in centromeres of native chromosomes makes the analyses of the relationship between transcription and the centromere more difficult in higher eukaryotes. However, the presence of transcribed genes on CENP-A-assembled centromeric domains is found in a human neocentromere, and in a native centromere on rice chromosome 8 with relatively few copies of satellite DNA (CenO) (Saffery et al., 2003;Nagaki et al., 2004). In human and Drosophila, CENP-A-containing centromerechromatin domains are found to be enriched with dimethyl Lys4 of histone H3, which represents a transcriptionally permissive chromatin state (Sullivan and Karpen, 2004). This evidence suggests that the centromere is organized as a flexible and permissive chromatin for transcription and, therefore, different from the silent state usually associated with heterochromatin.
Chromosomes also need to maintain sister chromatid cohesion until the completion of metaphase for proper chromosome segregation. Centromeric cohesion is associated with heterochromatin at the pericentromeric regions. In fission yeast, cohesin plays a physical role in the cohesion of replicated chromatids. Cohesin is associated with heterochromatin protein HP1 homolog (Swi6) and, thus, is recruited to centromeric heterochromatin sites (Bernard et al., 2001;Nonaka et al., 2002). In higher eukaryotes, the disruption of heterochromatin causes the loss of proper cohesion and missegregation of chromosomes Valdeolmillos et al., 2004;Guenatri et al., 2004). Nevertheless, the relationships among heterochromatin-cohesion function, centromere-chromatin assembly and the presence of transcriptional genes in higher eukaryotic chromosomes are still unclear.
To investigate how centromere chromatin assembly and heterochromatin acquisition are affected epigenetically by transcriptional activities from linked genes on transfected naked DNA and the consequence for de novo artificial chromosome forming efficiency, we analyzed the fate of transfected linear alphoid BAC constructs containing a transcriptional unit with or without a functional promoter proximate to the insert alphoid DNA in addition to a common marker gene on the right arm. De novo stable HAC formation was only observed in transformed cell lines with the alphoid BAC bearing the non-functional promoter. Lower and variegated assembly of centromere components at integration sites was observed with the alphoid BACs bearing the functional promoter. Furthermore, chromatin immunoprecipitation (ChIP) analysis showed that heterochromatin protein HP1␣ and trimethyl histone H3-K9 were enriched on the non-transcriptional BAC arm on the stable HAC. These results suggest that interference in stable HAC formation with alphoid BAC constructs arise from the interruption of heterochromatin acquisition on the vector arm by the presence of an additional transcriptional unit on the arm.

DNA construction
The 7C5-basic BAC was constructed as follows. EcoRI-PuvII fragment (SV40-bsr cassette) with SV40 early promoter (including the enhancer element) and bsr gene from pSV2bsr (Kaken, Japan) was inserted into the SalI site of pBluescript II (Stratagene) after endfilling reactions. An XhoI and SalI fragment containing the SV40-bsr cassette from the resultant plasmid was then inserted in the XhoI site of pBeloBAC11 (BAC-SVbsr). Two 1.1 kb human telomeric DNA fragments derived from pMega⌬  inverted and cloned into a plasmid (piTel1.1) with ampR gene in the center, was cut with SalI and SpeI, end-filled, and inserted into the HpaI site of BAC-SVbsr DNA (BACTSVbsr). A 70 kb NotI fragment of the alphoid DNA from ␣7C5hTEL YAC  was inserted into the NotI site of the BACTSVbsr vector to produce the 7C5-basic BAC.
The loxP insertion plasmid vectors were assembled as follows. A synthetic loxP sequence was cloned between the SacI and NotI sites of pBluescript II. Into the SmaI and EcoRV digestion site of the resulting plasmid, an AluI fragment of kanamycin resistance gene (KanR) from pET30a (Novagen) was inserted (ploxK). A blunt-ended HindIII and SalI fragment containing ␤geo gene excised from pcDNA3␤geo (for ␤geo insertion vector), a blunt-ended SalI fragment containing CMV promoter (including the enhancer element) and ␤geo gene excised from pcDNA3␤geo (for CMV-␤geo insertion vectors with different orientations), or a blunt-ended SalI fragment containing SV promoter and ␤geo gene excised from psDNA3␤geo (for SV-␤geo insertion vector), was inserted into the blunt-ended BamHI site of ploxK. The DNA fragment of 1.2 kb insulator element (cHS4) at 5Ј end of the chicken ␤-globin locus (GI:1763620), was amplified from chicken genomic DNA by PCR (primer set; HS4-F/HS4-R), inserted into the SmaI site of pBluescript II, and then sequenced. Two tandem copy units of cHS4 fragments were inserted into the SmaI site of ploxK. Into these EcoRV sites, a blunt-ended SalI fragment containing CMV promoter and ␤geo gene excised from pcDNA3␤geo was inserted (for INS insertion vector).
Each HAC vector (7C5-SV, 7C5-SV/CMV, 7C5-SV/CMV rev , 7C5-SV/SV and 7C5-INS BAC) was assembled as follows: A part of plasmid vector containing replication origin was removed from each insertion vector with XhoI and SalI digestion and self-ligation. This self-ligated DNA and 7C5-basic BAC DNA were inserted into the loxP sites by Cre recombinase (Clontech).
Reverse transcriptase (RT)-PCR Total RNA was isolated from culture cells using RNeasy Mini Kit (QIAGEN). First-strand cDNAs were synthesized by M-MLV reverse transcriptase (Takara) using 1 g of total RNA with random hexanucleotides (Roche). PCR was performed using 1:40 v/v (equivalent to 25 ng RNA) of cDNA reaction and ExTaq (Takara). Amplified DNA was analyzed by agarose-gel electrophoresis and/or real-time PCR. The primers are summarized in supplementary material, Table S1.

Chromatin immunoprecipitation (ChIP) assay
ChIP assays were carried out according to the method from the previous description (Nakano et al., 2003). Cells were cross-linked in 1% formaldehyde for 10 minutes. Immunoprecipitated DNA with anti-CENP-A (mAN1), anti-trimethyl histone H3-K9 (Upstate) and anti-GFP (monoclonal, Roche), and the soluble chromatin (as input) were quantitated by real-time PCR (Nakano et al., 2003). ChIP assays using anti-acetylated histone H3 (Upstate) were performed according to the manufacturer's instructions.

Construction of HAC vectors with varied transcription activities on the left vector arm
Stable human artificial chromosomes (HACs) are composed of a multimerized structure of an input alphoid YAC DNA if linear DNA molecules that end with telomere sequences are used . To investigate how transcriptional activity and resulting chromatin structures affect de novo centromere-chromatin assembly and HAC formation, we first constructed the basic alphoid BAC (7C5-basic BAC). It contains 70 kb of type-I alphoid (␣21-I) array, inverted human telomeric repeats placed at both ends of the molecule by I-SceI digestion, a bsr gene driven by the SV40 early promoter (including the enhancer element) on the right side of the alphoid array and a loxP sequence on the left side (Fig. 1A). Next, we obtained three derivative HAC vectors by the Cremediated loxP recombination system (Fig. 1B). Although all HAC vectors have the ␤geo coding gene, the ␤geo gene of the 7C5-SV BAC has no promoter. The 7C5-SV/CMV BAC has the ␤geo gene driven by a CMV promoter (including the enhancer element), and the 7C5-INS BAC has a CMV-␤geo gene flanked by 1.2 kb sequences of chicken ␤-globin 5ЈHS4 region (cHS4) which is known to function as an insulator even in human cells (Recillas-Targa et al., 2002). The transient expression levels tested on these similar ␤geo plasmid DNAs -that differ only in the promoter sequences -showed 10ϫ higher expression with the CMV promoter than those with the SV40 promoter in HT1080 cells as determined by ␤galactosidase activity (Fig. 1C).
We then confirmed whether the transcription from promoters worked appropriately on these three kinds of HAC vector constructs in HT1080 cells (Fig. 1D). The transcripts from the HAC vector DNA at 24 hours after transfection were analyzed by reverse transcription, followed by moderate (25 cycles) or a high (30 cycles) PCR amplification with specific primers for the BAC constructs. The level of transcription from the bsr gene on all of the BAC constructs was high. A few transcripts that extended into the insert alphoid DNA were detected with the higher number of PCR amplification cycles in all BAC transfected cells. The transcripts from the ␤geo gene on the left BAC vector arm were detected only in the cells transfected with the BAC constructs containing the CMV promoter. Insertion of the insulator decreased the transcriptional range downstream of ␤geo on 7C5-INS BAC drastically compared with that on 7C5-SV/CMV BAC. It is conceivable that transcriptional elongation has a tendency to pause with an insulator cHS4 sequence on the 7C5-INS BAC (Zhao and Dean, 2004). These HAC vectors contain different transcriptional activities on the left vector arm.
De novo HAC-forming efficiency was affected by the multiple insertion of active genes To examine efficiency of de novo HAC formation, the HAC vector DNAs (7C5-SV, 7C5-SV/CMV and 7C5-INS BAC; Fig.  1B) were introduced into HT1080 cells. Then, the obtained blasticidin S (BS)-or G418-resistant cell lines were analyzed by fluorescence in situ hybridization (FISH) with an ␣21-I 11mer alphoid DNA probe and a BAC DNA probe (Table 1). Five out of 30 analyzed cell lines (four of 21 analyzed cell lines in the second experiment) generated with the 7C5-SV BAC DNA transfection contained one copy of a minichromosome (HAC) signal with both probes in more than 50% of metaphase spreads ( Fig. 2A), similar to the HAC formation observed with the YAC-based ␣21-I alphoid constructs Masumoto et al., 1998). No host chromosomal DNA was 7C5-SV/CMV rev BAC is identical to 7C5-SV/CMV BAC but contains the CMV-␤geo cassette in reversed orientation. (C) ␤-Galactosidase activity of HT1080 cells was analyzed 24 hours after transfection of either SV-␤geo or CMV-␤geo plasmid DNA, or after trasfection of pBluescript II as a control. Luciferase activity of co-transfected luciferase expression vector (pRL-CMV, wako) was used for normalization. Data are the averages of three independent experiments. Error bar shows the s.e.m. (D) RT-PCR was performed 24 hours after transfection of each HAC vector DNA (7C5-SV, 7C5-SV/CMV or 7C5-INS BAC). PCR was carried out with 25 or 30 cycles against reverse transcribed cDNAs with specific primers (from top: ␤-actin, bsr, bsr-pA, right junction, left junction, kanR, ␤geo-pA, ␤geo1). Control reactions were performed against mock-transcribed cDNAs without reverse transcriptase (-RT).  (Fig. 1B). However, no de novo stable HAC formation was observed (Table 1). Therefore, when the HAC vector had been designed to permit transcripts for either directions on the left vector arm, we could not obtain de novo any stable HAC cell lines, whether we used identical ␣21-I alphoid DNA competent for centromere-kinetochore assembly or decreased the transcriptional activities by inserting the insulator.
Low level of assembly of centromere proteins at ectopic integrated sites To examine whether centromere proteins assemble on the input DNAs, we analyzed cell lines that contained a HAC or a host chromosome integration site by immunostaining and simultaneous FISH. In the S026 cell line, which contains a HAC composed of multimerized 7C5-SV BAC DNA (33 copies of the input BAC construct; Table 2), centromerekinetochore components CENP-A, CENP-B and CENP-C signals were detected on all HACs analyzed ( Fig. 2A, Table 2). The HAC was also very stable for an extended periods without the selective drug BS, with R=-0.0011 during 60 days of culture. We analyzed the cell lines in which introduced BAC DNAs had been integrated into a host chromosome (S013 cell line with 7C5-SV BAC DNA, K031 with 7C5-SV/CMV and IN010 and IN011 with 7C5-INS). Although CENP-B signals of weaker levels were detected in 60-100% of cells at the ectopic integration site of multimerized BAC DNAs, lower and variegated CENP-A and CENP-C signals were detected in about 6-26% of the cells (Fig. 2B, Table 2). We did not find any correlation among the levels of the variegated assembly at the ectopic integration sites, the total size of the alphoid DNA, which varied in different cell lines from 0.6 to 3.5 Mbp (multimers of 8-51 copies of the input DNA), and the differences in BAC constructs (Table 2). Although we could not obtain stable HAC de novo, the ability to assemble centromere-kinetochore components is not completely lost from these HAC formation-deficient alphoid BAC constructs. Reactivation as a centromere at the ectopic integration site of HAC formation-deficient BAC correlated with transcriptional activity Our previous study showed that CENP-A chromatin and centromere components can be reassembled specifically on the ectopic alphoid YAC integration sites that correlated with transcriptional activation on the marker gene on the YAC sites (Nakano et al., 2003).  2C, a loss rate R=0.0027 for further 60 days of culture without the selective drugs). These reformed minichromosomes were accompanied by various sizes of DAPI-stained host chromosome fragments acquired from the integration site following the breakage event (Fig. 2C). Therefore, functional centromere-kinetochore structure can still be assembled at the HAC-formation-deficient alphoid BAC integration site.
Journal of Cell Science 118 (24)  (Nakano et al., 2003). Total size of alphoid DNA composed of multimerized input DNA was calculated from copy number as being equivalent to 70 kb of inserted alphoid DNA.  We then analyzed the chromatin structure by chromatin immunoprecipitation (ChIP) with an anti-acetylated histone H3 antibody. The precipitated DNA samples were quantitated by real-time PCR with specific primers for alphoid BAC constructs. As shown in Fig. 3B, immunoprecipitates of acetylated histone H3 enriched transcriptionally active regions such as the endogenous promoter of the CENP-B gene (11.2-15.3%, controls with normal IgG were less than 0.04%), but did not enrich satellite 2 (sat2) control sequences (0.08-0.10%), which are located at pericentromeric heterochromatin regions of chromosome 1 and 16 (Espada et al., 2004). Acetylated histone H3 was abundant in the bsr gene (12.4-16.5%) on the right arm of the introduced alphoid BAC constructs in all cell lines. Under double selection conditions, acetylated histone H3 was enriched by the ␤geo gene (5.5%) in K031 cells, whereas less acetylated histone H3 was observed in the single drug selection (BS) of K031 cells and other cell lines (0.56-1.28%). In all cell lines, the levels of acetylated histone H3 correlated very well with the transcriptional activities of the BAC arms and were very low on the left and the right alphoid junctions, especially on ␣21-I alphoid DNA (0.09-0.17%), which is located in the centromere of the HAC or the integration sites of the BAC DNA, as well as at the centromere region of human chromosome 13 and 21.
Thus, consistent with our previous finding, an open chromatin structure that allows a transcriptionally active and hyper-acetylated histone H3 state proximate to the inserted alphoid DNA is coupled with the reassembly of CENPs, even on the HAC-formation-deficient alphoid DNA at the ectopically integrated site, and does not inhibit the formation of a centromere-kinetochore structure.
The transcription-capable structure itself conflicts with assembly required for the HAC To analyze further the antagonism between HAC formation and the addition of the CMV promoter on the left vector arm, we co-transfected 7C5-SV/CMV BAC DNA and 7C5-basic BAC DNA into HT1080 cells and selected with BS and G418. De novo HAC-forming-activity was recovered in up to 16% in analyzed cell lines (3 out of 19 analysed cells with 7C5 mix, see Table 1). The signals specific for the 7C5 SV/CMV BAC DNA were detected only on those HAC that overlapped with extrachromosomal signals detected by a BAC probe or alphoid probe (see supplementary material Fig. S2A). Thus, the conflict can be overcome by a further recruitment of CMV-promoter-less alphoid BACs without acquiring other chromosomal fragments. The existence of an additional transcription-capable structure on the left arm might conflict with an unknown structural assembly required for stable artificial chromosomes other than the centromerekinetochore.

Heterochromatin and centromere chromatin form distinct domains on a HAC
To explore the chromatin structure required for a stable HAC formation, ChIP and real-time PCR analysis was carried out to investigate the detailed distribution of CENP-A and lysine 9 trimethyl histone H3 (triMet H3-K9) as a heterochromatin marker (Peters et al., 2003) at the sequence level on the stable HAC (S026) and the BAC integration sites (K031) under the single (BS) or the double (BS/G418) selection.
Immunoprecipitation with the antibodies anti-CENP-A, anti-triMetH3-K9 or normal IgG allowed very low levels of recovery of endogenous CENP-B promoter region, on which neither CENP-A nor triMet H3-K9 assembly is expected (Fig.  4A), consistent with the abundance of acetylated histone H3 on this region (Fig. 3C). Immunoprecipitates against the control (normal mouse IgG) did not enrich any specific site of the 7C5-SV(or SV/CMV) BAC DNA on the HAC or the integration sites, or satellite2 DNA at the pericentromeric region (sat2 probe) (Fig. 4A). The distributions of CENP-A on the BAC DNA in all three cases were almost restricted to the ␣21-I alphoid DNA, including the left and right arm junctions (L1 and R1), whereas triMetK9-H3 was predominantly enriched at the transcriptionally silent left arm region of the HAC (S026) and the integration site under the single BS selection of K031 cells (Fig. 4A). Neither CENP-A nor triMet H3-K9 was enriched at the bsr gene on the right arm, which is a transcriptionally active and hyper-acetylated histone H3 state. The double selection of the same K031 cell line by additionally adding the drug G418 results in an increase of the levels of CENP-A assembly at alphoid-BAC vector junctions (L1 and R1), concurrent with a decrease of triMet H3-K9 levels on the left vector arm and the junctions (K031 BS+G418 in Fig. 4A). The results showed clear evidence that triMet H3-K9 assembled on the promoter-less left vector arm of the stable HAC (S026), and also assembled as an antagonism for the transcriptional activity on the left arm and for CENP-A assembly, at least on the alphoid-vector junctions at the BAC integration site (K031). Although we cannot distinguish ␣21-I alphoid inserts on the BAC constructs from the endogenous loci on chromosome 13 and 21centromeres with this alphoid probe, the higher enrichment of CENP-A was detected in the HAC and the integration cell line with the double selection than that in parental HT1080 cells (Fig. 4A).
Then, we focused our analysis on how heterochromatin protein (HP1) is distributed on HAC. In metaphase-arrested S026 cells, bright HP1␣ signal overlapped with BAC-probe signal on the HAC derived from 7C5-SV BAC DNA (Fig. 5B), and strong signals were detected at pericentromeric regions on native chromosomes (Guenatri et al., 2004) (Fig. 5A). To investigate HP1␣ assembly on the HAC by ChIP, we established HAC-containing S026 derivative cell lines that stably expressed YFP-tagged HP1␣ (Hayakawa et al., 2003) or YFP protein alone. The pericentromeric localization of YFPtagged HP1␣ proteins was confirmed prior to ChIP assay (supplementary material Fig. S3). The relative enrichment of each DNA fragment compared with the recovery of endogenous CENP-B promoter region by the immunoprecipitation analysis is indicated in Fig. 4B. The distributions of YFP-tagged HP1␣ on the HAC showed a similar pattern with triMet H3-K9 but not with CENP-A. YFP-tagged HP1␣ was enriched on the BAC left arm region (L2, L3 and L4 probes, 5.3-7.5-fold) similar to the positive control of satellite2 DNA (8.1-fold). Interestingly, ␣21-I alphoid DNA was also slightly enriched (2.7-fold), regardless of CENP-A abundance, indicating that some parts of the ␣21-I alphoid array were covered by heterochromatin. Therefore, the localization pattern of HP1␣ shown by ChIP analysis is consistent with the assembly of triMet H3-K9 on the promoter-less left vector arm of the stable HAC (Fig. 4A,B).

HP1␣ and aurora B kinase at the BAC integration sites
We next analyzed the distribution of HP1␣ at the BAC integration sites in metaphasearrested cells. Interestingly, an HP1␣ signal was not detected at the BAC-integrated site on the chromosome arm region in K031 cells, but was detectable at the canonical centromere region on the host chromosome (Fig. 5C). However, we detected the small HP1␣ signal on the reformed minichromosomes in K031 cells by double selection (Fig. 5D). The mitotic localization of HP1␣ is not consistent with the distribution of triMet H3-K9 at the BAC integrated sites on the chromosome arm or on the reformed minichromosome (Fig.  4A).
Therefore, we confirmed the localization of aurora B kinase in metaphase-arrested cells. Aurora B kinase behaves as a chromosomal passenger protein (CPP), forming a complex with INCENP, survivin and the recently identified Borealin (Gassmann et al., 2004). INCENP associates with pericentromeric heterochromatin and this pericentromeric localization depends on cohesin function early in mitosis (Ainsztein et al., 1998;Sonoda et al., 2001). As the results of immunostaining and simultaneous FISH analysis using anti-aurora B kinase antibody and BAC probe show, we could observe the localization of aurora B kinase on the de novo HAC and reformed minichromosome with an activated centromere from the alphoid BAC integration site ( Fig. 5D and data not shown). However, we could not detect aurora B kinase signal at the integrated loci on the chromosome arm region, even though the signal was detectable at the canonical centromere region on the host chromosome in K031 cells (Fig. 5C). Therefore, at a minimum, a pericentromeric heterochromatin-cohesion-related protein (HP1␣) and an inner centromere protein (aurora B kinase) were assembled on the HAC as well as the reformed minichromosome that correlates with the centromere function, but were not distinctively enriched on the alphoid BAC integration site of the metaphase chromosome arm, which does not have the centromere function.
Taken together, all these results strongly support the view  that heterochromatin assembly on the vector arm of the introduced alphoid BAC-YAC construct is incompatible with the insertion of a transcriptionally active CMV promoter (including the enhancer element), and crucial for de novo HAC formation after transfection and centromere-chromatin assembly on the ␣21-I alphoid array.

Discussion
Kinetochore formation does not conflict with a transcriptionally permissive chromatin proximate to alphoid DNA The centromere domains of humans and D. melanogaster are marked by an interspersed structure with dimethylated histone H3-K4 clusters and CENP-A clusters (Sullivan and Karpen, 2004). This recent observation supports the repeat-subunit model of centromere structure proposed by Zinkowski et al. (Zinkowski et al., 1991) and is consistent with a transcriptionally permissive state of centromere chromatin. Transcriptionally active genes within a functional centromere also have been found in a human neocentromere and the centromere of rice chromosome 8 (Saffery et al., 2003;Nagaki et al., 2004). These observations implicate a coexistence of transcriptional competence and kinetochore function within the same centromere domain, perhaps because the centromere is the repeat-subunit structure and these two events function during different phases of the cell cycle; transcription during interphase and kinetochore assembly during mitosis.
HACs are composed of a repeated chromatin structure on an input alphoid YAC-BAC multimer. However, we could not generate any stable de novo HAC when HAC vectors, containing the centromere-competent ␣21-I alphoid DNA, permitted transcription from the left vector arm in addition to the right arm. Our previous study had shown that transcriptional activity does not interfere with the assembly of centromere chromatin on the type-I alphoid YAC multimer (Nakano et al., 2003). Moreover, a structural change of chromatin -from a suppressed state to a transcriptionally active state -at the marker genes at the ectopic integration site of the alphoid YACs, using a short-term treatment with the The localization of HP1␣ (red), compared with kinetochore protein CENP-C (green) in metaphase HT1080 cell is indicated in A. The presence or absence of HP1␣ (green in c-f, h, i) or aurora B kinase (green in g-j) on the BAC (red) locus was analyzed in (B) de novo HACs (S026), (C) the integration site (K031) or (D) the reformed minichromosome (K031) in metaphase-arrested cells. Chromosomes were counterstained with DAPI (blue or gray). Arrows indicate the HACs or the integration sites of the alphoid BAC. Arrowheads indicate canonical centromeres on the host chromosomes. Bars, 10 m.
histone deacetylase inhibitor (TSA) or a long-term culture under drug selection, facilitates the reassembly of centromere components including CENP-A to the nearby inserted alphoid DNA. Present data also showed that, the reassembly of centromere components at the ectopically integrated site of even a HAC-formation-deficient ␣21-I alphoid BAC correlated with the transcriptional activation at the vector arm region (Fig.  2C, Fig. 3). Therefore, the active transcriptional state at the vector arms proximate to the insert alphoid DNA does not inhibit the assembly of centromere components directly (Fig.  6 model B).
All these observations do not conflict with the idea that a transcriptionally permissive chromatin state could also allow assembly of centromere chromatin composed of CENP-A nucleosomes. In addition, overexpression of CENP-A leads to mislocalization of the proteins across entire chromosome arms, especially euchromatic loci (Van Hooser et al., 2001). This indicates that CENP-A loading on chromatin might be Journal of Cell Science 118 (24) Fig. 6. Models of chromatin assembly and structural organization on input alphoid BACs. Chromatin assemblies and transcripts on one unit of multimerized alphoid BAC DNA (7C5-SV BAC in A, 7C5-SV/CMV BAC in B-D) are shown as hypothetical models. Chromatin states supported by RT-PCR and ChIP analyses are indicated by colored lines. Arrows below the vector maps show transcription level (width) and length. Even though the ChIP analysis represents the sum of chromatin structures formed on multimerized alphoid BAC units, our ChIP data show a tendency for distinct chromatin structures to correspond to the sequence structures of the alphoid BAC constructs, implying that the HAC is maintained as punctuated blocks of chromatin structures. (A) Open chromatin or euchromatin (green) at the transcriptional gene on the right arm enhances the assembly of centromere chromatin (red) on the inserted alphoid DNA. In addition, when heterochromatin assembly (blue) occurs at the left arm and at a part of alphoid repeats (Fig. 4), functional HAC is generated and stably maintained. (B) If the heterochromatin-formation domain on the left arm is replaced with euchromatin by inserting a transcriptional gene unit, centromerekinetochore components still can assemble on the alphoid array, but cohesion and inner centromere functions are absent. As a result, the unstable structure of the extra-chromosome is lost or integrated into a host chromosome. (C) Otherwise, at the integration site of a host chromosome, a silencing effect may be induced and/or heterochromatin spreads as indicated by triMet H3-K9 (Fig. 4A) into both non-selective marker gene and the insert alphoid DNA; consequently, centromere assembly on the alphoid DNA would be inactivated. (D) On the ectopic integration sites of multiple alphoid BAC DNAs, however, a chromatin opening also occurs stochastically in a part of the multiple array as indicated with the variegate assembly of the CENPs at the sites. If the open chromatin is selected again by the double selection as described in B, the chromatin opening and the functional assembly of kinetochore components on the ectopic alphoid array are accelerated and causes the reformation of minichromosomes accompanied by chromosome breakage events and with a part of the host chromosome fragment as a donor of heterochromatin-cohesion. regulated by the expression level of CENP-A and/or by the cell cycle (Shelby et al., 2000), and might occur on permissive chromatin but be displaced from histone H3 nucleosomes by some loading factors.
Our ChIP experiments, however, showed that CENP-A chromatin localizes almost exclusively at the inserted alphoid DNA on HACs but only rarely spreads across the BAC-YAC vector arm DNAs, including the transcribing marker genes (Fig. 4A) (Nakano et al., 2003). CENP-A chromatin preferentially assembled on AT-rich (~60% AT) synthetic alphoid repeats that contain CENP-B boxes, but not on GCrich (~40% AT) synthetic repeats based on a pBR322 vector fragment (Ohzeki et al., 2002). Therefore, these results suggest that the transcriptionally active chromatin state of marker genes increases the deposition activity of CENP-A nucleosomes on adjacent alphoid repeats but not on these GCrich transcribing genes and vector DNA themselves under normal cellular condition (Masumoto et al., 2004).
The role of heterochromatin and the inner side of the centromere for de novo HAC formation Heterochromatin protein HP1␣ assembly on HACs was recently observed on both BAC-based (Grimes et al., 2004) and YAC-based stable HACs (our unpublished results). Our data show that triMet H3-K9 and HP1␣ localize on the stable HAC and are enriched at the left arm of 7C5-SV BAC DNA (Fig. 4). This region includes the ␤geo coding gene without a functional promoter. By contrast, HAC-formation-deficient derivative alphoid BACs (7C5-SV/CMV and 7C5-INS) include a transcriptional ␤geo gene with the CMV promoter. We also failed to generate de novo stable HACs from the other derivatives on which the CMV promoter of the ␤geo gene was replaced by a SV40 promoter (data not shown) or the orientation of the CMV driven ␤geo gene was reversed (Table  1). However, the association of transcriptional activities is not inhibitory for the assembly of the functional centromere as discussed above. On the other hand, heterochromatin structures are incompatible with the transcriptional activation of the left arm by an additional promoter and even by the insulation of this region.
Why is a heterochromatin structure required for de novo HAC formation and maintenance? In many eukaryotes, the centromere is embedded in heterochromatin, which contributes to faithful chromosome segregation and genome stability. HP1␣ and triMet H3-K9 are enriched at the pericentromeric regions on mitotic chromosomes in human and mouse (Fig. 5) (Hayakawa et al., 2003;Peters et al., 2003;Guenatri et al., 2004). In fission yeast, Swi6 (a HP1 homologue) is physically associated with a cohesin protein Rad21, whose complex is essential for sister-chromatid cohesion and faithful chromosome segregation, and thus the association may explain the assembly of cohesin to the pericentromeric heterochromatin region (Bernard et al., 2001;Nonaka et al., 2002). In higher eukaryotes, pericentromeric heterochromatin domains and cohesion domains also overlap on mitotic chromosomes and the disturbance of the heterochromatic structure causes the loss of proper chromatid cohesion Valdeolmillos et al., 2004;Guenatri et al., 2004). Moreover, the cohesin complex does not only comprise sisterchromatid cohesion, but also plays a role in the accumulation of chromosome passenger proteins (CPP) to the inner centromeric region (Sonoda et al., 2001;Vagnarelli and Earnshaw, 2001;Vass et al., 2003). Aurora B kinase, one of the CPP components, dynamically and widely regulates mitotic events, kinetochore function and cytokinesis, and is also involved in cohesion releasing from chromosome arms and accumulating at inner centromere regions until just before onset of anaphase. Therefore, the accumulation of aurora B kinase is an important functional marker for the inner side of the centromere, which connects the kinetochore and heterochromatin-cohesion functions (Carmena and Earnshaw, 2003;Maiato et al., 2004). Real-time observation of HACs in living mitotic cells showed that HACs are accurately aligned at the spindle equator, and the sister chromatids of the HAC are resolved with the same timing as natural chromosome separation synchronized with mitotic cell-cycle progression (in preparation). Thus, de novo HACs also retain the normal mechanism controlling sister chromatid cohesion and its resolution. Indeed, our data show that HP1␣ and aurora B kinase localize to HACs in metaphase-arrested cells and on reformed minichromosomes that contain an activated centromere from the alphoid BAC integration site, but not on the ectopically integrated and inactivated site of the alphoid BAC on the chromosome arm region (Figs 4,5). HP1␣ and CPP might be more dynamically regulated on the mitotic chromosomes. Correspondingly, heterochromatin structure is also acquired close to the kinetochore domain at neocentromeres (Saffery et al., 2000;Aagaard et al., 2000). These results strongly suggest that, acquisition of heterochromatin structure is also necessary for introduced naked DNAs -coincident with the assembly of a kinetochore structure during an early stage of stable HAC formation -and, thus, the introduced DNA needs to supply appropriate sequences not only for the kinetochore-forming domain but also for the heterochromatin domain (Fig. 6 model A).
Potential influence of transcriptional activity upon epigenetic chromatin assembly and HAC formation Treatment of mouse cells with RNase causes release of HP1 and disturbance of the chromatin state of the pericentromere (Maison et al., 2002). In fission yeast, small interfering RNAs (siRNA) -generated by the cleaving double stranded RNA (dsRNA) by Dicer -is found to be associated with epigenetic chromatin modification and the establishment of heterochromatin Volpe et al., 2002). A conditional loss-of-function mutant of Dicer in a chicken cell line showed premature sister chromatid separation and mitotic defects without an abnormal assembly of CENP-A and C (Fukagawa et al., 2004). On the other hand, a Dicer-deficient mouse embryonic stem (ES) cell line showed defective differentiation and centromere silencing, but maintained ES morphology, cell viability and chromosome stability (Kanellopoulou et al., 2005), suggesting the existence of another mechanism for maintaining chromosomal stability in ES cells, in addition to Dicer-RNAi.
In our RT-PCR experiment, transcripts from both the left arm and the right arm sides continue into the alphoid array in the 7C5-SV/CMV BAC during transient transfection (Fig. 1D). Moreover, a suppression of transcriptional activity from the CMV promoter towards the alphoid array was observed in the cell line with the integration of the 7C5-SV/CMV BAC DNA. It is conceivable that the homologous alphoid repeats transcribed from both vector arms are potential targets of Dicer and thus contribute to the suppression of centromere chromatin assembly on the insert alphoid DNA (Fig. 6, model C). However, our results do not strongly support an explanation, only a potential dsRNA-mediated mechanism on alphoid DNA, because we failed to generate de novo HAC from 7C5-SV/CMV rev BAC (in which the orientation of the CMV driven ␤geo gene was reversed) precluding the presence of dsRNA on the alphoid DNA. It, therefore, appears that a transcribing structure on the left arm itself conflicts with HAC formation (Fig. 6 model B). In addition, we observed reformed minichromosomes accompanied by an activated centromere from integrated loci of the 7C5-SV/CMV BAC DNA (Fig. 2C) and HAC formation from the 7C5 mix (Table 1, supplementary material Fig. S2), despite being under BS and G418 double selection and requiring transcription on the left arm of the 7C5-SV/CMV BAC DNA. However, in these cases, a missing structure for heterochromatin-cohesion at the left vector arms on the reformed minichromosomes or HACs might be compensated for by the acquisition of a host chromosomal fragment (Fig. 6, model D) or the CMV promoter-less alphoid BAC. Once formed, these HACs and reformed minichromosomes are stable even under non-selective conditions. Therefore, the existence of transcription from both the left and the right arm towards alphoid DNA does not induce further suppression of the centromere chromatin assembly on the HAC.
From these results we conclude that the transcriptionally active chromatin on the left arm of input-DNA conflicts with de novo assembly of heterochromatin-cohesion on this site. Therefore, these chromosomes were not maintained stably only by forming a de novo centromere-chromatin structure, assembled on the inserted alphoid DNA (Fig. 6 model B). Thus, de novo HAC formation might arise from a delicate balance on the introduced naked alphoid BAC-YAC DNA multimer between transcriptionally permissive chromatin for centromere assembly and heterochromatinization (Fig. 6  model A).
At this moment, we do not know how heterochromatin structure is assembled preferentially at the left vector arm. The promoter-less structure of the left arm limits chromatin opening at the site and its GC-rich (GC content of 52-57% and also CpG-rich) structure might be a good candidate for targets of CpG methylation and methylated CpG-binding proteins (Rhee et al., 2002;Fujita et al., 2003;Espada et al., 2004;Jorgensen et al., 2004). CpG-rich vectors derived from bacterial DNA cause suppression of transgene expression in mammalian cells (Hodges et al., 2004;Chen et al., 2004). The interplay between such suppressive elements and factors may cooperatively effect the assembly of heterochromatin at the left arm. Recently, HP1␣ and HP1␥ were found to co-purify with the centromere component hMis12, suggesting that pericentromeric heterochromatin is possibly linked to centromere function (Obuse et al., 2004). We could not distinguish the difference between the alphoid DNA on the HAC and the alphoid DNA on the centromere of chromosome 21; we found that trimethyl histone H3-K9 and HP1␣ were slightly enriched also at the ␣21-I alphoid array (Fig. 4), suggesting that alphoid DNA, outside the centromere core region, is epigenetically inactivated and utilized as a part of pericentromeric heterochromatin. It has also been found that the maize centromere core repeat array (CentC) is transcribed and these transcripts are bound to centromere CENP-A chromatin (Topp et al., 2004). However, it remains unclear whether the transcripts play a role in centromere function and how the borders of the centromere and heterochromatin domains are defined epigenetically. Thus, further investigations are required to elucidate functional relationships between the primary DNA sequences, centromere chromatin, heterochromatin and the epigenetic modification correlating with transcriptional activity.