Assembly and characterization of heterochromatin and euchromatin on human artificial chromosomes
© Grimes et al; licensee BioMed Central Ltd. 2004
Received: 2 June 2004
Accepted: 22 September 2004
Published: 27 October 2004
Human centromere regions are characterized by the presence of alpha-satellite DNA, replication late in S phase and a heterochromatic appearance. Recent models propose that the centromere is organized into conserved chromatin domains in which chromatin containing CenH3 (centromere-specific H3 variant) at the functional centromere (kinetochore) forms within regions of heterochromatin. To address these models, we assayed formation of heterochromatin and euchromatin on de novo human artificial chromosomes containing alpha-satellite DNA. We also examined the relationship between chromatin composition and replication timing of artificial chromosomes.
Heterochromatin factors (histone H3 lysine 9 methylation and HP1α) were enriched on artificial chromosomes estimated to be larger than 3 Mb in size but depleted on those smaller than 3 Mb. All artificial chromosomes assembled markers of euchromatin (histone H3 lysine 4 methylation), which may partly reflect marker-gene expression. Replication timing studies revealed that the replication timing of artificial chromosomes was heterogeneous. Heterochromatin-depleted artificial chromosomes replicated in early S phase whereas heterochromatin-enriched artificial chromosomes replicated in mid to late S phase.
Centromere regions on human artificial chromosomes and host chromosomes have similar amounts of CenH3 but exhibit highly varying degrees of heterochromatin, suggesting that only a small amount of heterochromatin may be required for centromere function. The formation of euchromatin on all artificial chromosomes demonstrates that they can provide a chromosome context suitable for gene expression. The earlier replication of the heterochromatin-depleted artificial chromosomes suggests that replication late in S phase is not a requirement for centromere function.
In the post-sequencing phase of genome characterization, it is important to understand the contribution of non-coding sequences to higher-order genome structure and stability. Maintenance of genome integrity and the faithful transmission of genetic information in mitosis and meiosis are essential to organism survival and are critically dependent on two repetitive chromosomal elements. Telomeres protect against chromosomal truncation or fusion events , while centromeres ensure faithful chromosome segregation through cell division [2–4]. Failure in the function of these elements can lead to genomic instability, with often catastrophic consequences in humans such as miscarriage, congenital birth defects or cancer. In contrast to the telomere, whose properties have been well explored at the genomic and molecular levels , the human centromere remains relatively poorly characterized, and experimental systems for the genomic study of centromere formation and behavior are only just being developed and optimized [6–14].
Defining the minimal DNA sequences required for centromere function on a normal human chromosome has proved challenging, owing to the complex nature of inter- and intra-chromosomal homology and variability in genomic DNA content near the primary constriction. Common to all normal human centromeres are large amounts of alpha-satellite DNA, which is comprised of a family of diverged 'monomers' of around 171 base-pairs (bp) that have been amplified in multimeric groups (higher-order repeats) on different chromosomes to form chromosome-specific arrays typically megabases in length [15–17]. In addition, the core of higher-order repeat alpha-satellite is, where examined in detail, surrounded by other alpha-satellite sequences that fail to form a recognizable higher-order structure (so-called 'monomeric' alpha satellite) [10, 18–20]. Together, the two types of centromeric repeat span up to several megabases of genomic DNA at each centromere region and account for much of the largest remaining gaps in the human genome sequence assembly [21, 22]. Support for a critical role for alpha-satellite DNA in centromere function comes from recent studies on the human X chromosome, where the most abundant alpha-satellite sequence at this centromere, DXZ1, has been shown to be sufficient for centromere function [10, 23] and, more generally, from studies demonstrating the formation of de novo centromeres on human artificial chromosomes following transfection of some types of alpha-satellite sequences into human cells [6–14].
Paradoxically, despite conservation of the functional role of the centromere in every eukaryotic cell, DNA sequences at eukaryotic centromeres are quite divergent in sequence even between closely related species [24, 25]. Although primary genomic sequence has not been conserved at eukaryotic centromeres, they do, nonetheless, share features in common such as a structure based on tandem repeats, overall AT-rich composition, and packaging into specialized centromeric chromatin marked by the presence of centromere-specific histone H3 (CenH3) variants (reviewed in [4, 26, 27]). The ability of different genomic sequences to fulfill centromeric requirements in different species is in accord with data showing that the DNA normally associated with the genetically mapped centromere on normal human chromosomes is not always sufficient or necessary for centromere function. Rare chromosomal rearrangements can result in either dicentric chromosome formation, where one centromere is typically inactivated [28, 29], or in the formation of neocentromeres, where a centromere assembles on DNA that is not associated with the normal centromere genomic locus (reviewed in ). Together, these observations suggest that epigenetic factors are critical for centromere function  and point to the as-yet incompletely understood interplay of underlying genomic DNA sequences located in the centromeric region and their ability to package into specialized centromeric chromatin [2, 4, 27].
Recent evidence suggests that a complex system of epigenetic modifications based on histone variants and histone tail modifications is important for centromere activity (reviewed in [4, 31]), in much the same way as a histone code is involved in determining the transcriptional competence of DNA . Although the epigenetic basis of centromere function is not yet fully defined, a strong candidate for specifying the site of the functional centromere (kinetochore-forming region) is the family of CenH3 variants, which are conserved from yeast to humans and are essential to viability of the organism (reviewed in ). In humans and flies, CenH3 is restricted to the centromere where CenH3- and typical H3-containing nucleosomes exist in an alternating arrangement, generating a unique chromatin structure that may be important for centromere function [33, 34].
The most completely studied complex eukaryotic centromere at the molecular level is that of the fission yeast Schizosaccharomyces pombe. Detailed analyses of a 40-kilobase (kb) S. pombe centromere revealed that it encompasses both the kinetochore, as defined by the exclusive association of Cnp1, the fission yeast CenH3, with the central core element  and adjacent repeats enriched for heterochromatin-associated factors  that are important for centromeric cohesion [37–40]. Within the heterochromatic domains, histone H3 is methylated at lysine 9 (H3MeK9), resulting in the recruitment of the heterochromatin protein HP1-homolog Swi6 .
There is substantial evidence that HP1 is involved in setting up and/or maintaining a repressed chromatin state in several epigenetic systems (reviewed in ). HP1 proteins are conserved and localize to centromere regions in human and mouse cells [43–45]. Human cells express three HP1 isoforms, HP1α, HP1β and HP1γ. HP1α and HP1β localize primarily to pericentromeric regions, while HP1γ is dispersed at sites along chromosome arms . Furthermore, modified H3MeK9 nucleosomes, which create a binding site for HP1 (reviewed in ), have also been localized cytologically to centromere regions in flies and mice [44, 47–53]. These observations suggest a model in which local modifications of chromatin composition represent a crucial and highly conserved element necessary for the specification and/or maintenance of complex eukaryotic centromeres . Consistent with these models, chromatin immunoprecipitation assays with highly specific antibodies have shown that both mouse minor and major satellite DNA sequences exhibit trimethylation of histone H3 at lysine 9 [51, 53]. However, while the association of histone modifications typical of repressive heterochromatin has been clearly demonstrated for sequences that flank the functional centromere, it is less certain what modifications, if any, may characterize the CenH3-containing chromatin of the functional centromere itself. Indeed, many of the characteristics historically assigned to pericentromeric DNA (that is, repressive heterochromatin and late-replication in S phase [54, 55]) may be features of the surrounding heterochromatin, more so than of the functional centromere per se.
One way to address the interacting and complementary role(s) of DNA sequence and trans-acting chromatin factors in human centromere function is through the construction of detailed genomic maps of human centromeric regions and evaluation of their associated proteins [10, 19, 56, 57]. An alternative empirical approach is to construct minimal human artificial chromosomes from defined alpha-satellite DNA sequences [6–14] as tools for evaluating the essential genomic requirements of centromere specification. Indeed, previous studies have shown that the human CenH3 - centromere protein A (CENP-A) - is deposited at the centromere on artificial chromosomes constructed from alpha-satellite DNA [12, 13, 58]. However, it is not known whether heterochromatin formation is required for centromere establishment and propagation and/or whether de novo centromeres on human artificial chromosomes without large amounts of adjacent heterochromatin demonstrate the same chromatin characteristics as either normal human centromeres or human artificial chromosomes with large amounts of heterochromatin.
In the present study, we have characterized the nature of heterochromatin and euchromatin formed on a series of human artificial chromosomes derived from higher-order repeat alpha satellite from chromosomes X or 17 [12, 14]. While large artificial chromosomes contain substantial amounts of heterochromatin (characterized by the presence of modified H3MeK9 nucleosomes and HP1α) and replicate later in S phase, small artificial chromosomes show features more consistent with the euchromatin of the chromosome arms, including the presence of histone variants typical of expressed euchromatin and replication earlier in S phase. These data suggest that the chromatin environment required for de novo centromere formation and function is likely to be generally conducive to gene expression, as will probably be required for either gene-transfer experiments and/or functional genomic applications of the artificial chromosome technology. Further, the data raise the possibility that functional centromeres may adopt a novel chromatin state that is, contrary to what has been long assumed, quite distinctive from that of conventional heterochromatin.
To examine the chromatin composition of human artificial chromosomes, we used a panel of artificial chromosomes formed after transfection with vectors containing either synthetic chromosome 17 (D17Z1) or cloned X chromosome (DXZ1) alpha-satellite sequences [12, 14]. Each of the artificial chromosomes tested contains a functional de novo centromere assembled from the transfected DNA, as well as at least one copy of a functioning gene used as a selectable marker. Together, this panel of artificial chromosomes provides an opportunity to examine the nature of heterochromatin and euchromatin assembled on the transfected DNA sequences. The high mitotic stability and de novo composition of artificial chromosomes generated from D17Z1 (17-E29, 17-D34 and 17-B12) or DXZ1 (X-4 and X-5) have been described [12, 14]. As a more direct measure of artificial chromosome segregation errors, we have used an assay that allows cells to undergo anaphase but cannot complete cytokinesis . Using fluorescence in situ hybridization (FISH), artificial and host chromosome segregation products can be measured and nondisjunction or anaphase lag defects recorded.
Artificial chromosome segregation errors
Number (%) of chromosome mis-segregation events*
Number of cells analyzed
Chromatin formation on artificial chromosomes
Variation in levels of heterochromatin-associated factors correlates with artificial chromosome size
Euchromatin forms on artificial chromosomes
For their potential use as gene-transfer vectors or as general vehicles suitable for interrogation of genome function, human artificial chromosomes must also be capable of forming euchromatin to support gene expression. Indeed, one would hypothesize that at least small amounts of transcriptionally active chromatin must form during artificial chromosome formation to permit expression of the selectable marker gene(s) contained on the transfected constructs [10, 12, 14]. It has previously been shown using immunocytochemical methods [62, 63] that methylation of histone H3 at lysine 4, an epigenetic modification associated with transcriptionally permissive chromatin [64–66], is generally enriched on autosomes and depleted at the repressed inactive X chromosome and human centromere regions.
Previous structural analyses of artificial chromosomes indicate that they consist of input DNA multimers arranged as blocks of alpha-satellite DNA interspersed with vector sequences [7, 11, 12]. This structural organization is consistent with the presence of multiple selectable marker genes and differs from the large uninterrupted blocks of alpha-satellite DNA found at all human centromeres that are typically under-represented for this active chromatin mark (Figure 3). Because mitotically stable artificial chromosomes can have permissive as well as repressive chromatin present, these data suggest that this chromatin configuration does not significantly disturb mitotic centromere function.
Two modes of artificial chromosome replication timing
While the genomic determinants of potential origins of DNA replication in the human genome, as well as of their timing of replication during S phase, are still not well understood, the generally accepted paradigm is that expressed sequences replicate in the first half of S phase, while non-expressed sequences replicate in the second half . Consistent with this pattern, alpha-satellite DNA, as well as constitutive heterochromatin (such as that found on the Yq arm), replicate in the mid to late S phase period [54, 55, 68, 69]. In the present study, we have asked whether D17Z1-based artificial chromosomes replicate at a similar time to endogenous chromosome 17 alpha-satellite DNA. To determine the time of replication, unsynchronized cells were pulsed with bromodeoxyuridine (BrdU) for 2 hours, followed by a thymidine chase for varying lengths of time before harvesting cells in metaphase (see Materials and methods). Detection of BrdU incorporation at sites of DNA replication was performed using indirect immunofluorescence with an anti-BrdU antibody on metaphase spreads.
Replication timing of artificial chromosomes
Early: 0-2 h S
Mid: 2-4 h S
Mid: 4-6 h S
Late: 6-8 h S
Human artificial chromosomes provide a novel system for analyzing cis- and trans-acting factors necessary for chromosome segregation and offer potential for both functional genomics and gene-transfer applications. The artificial chromosomes we used contain defined alpha-satellite DNA sequences [12, 14]. Studying how epigenetic components assemble with alpha satellite to form a de novo centromere on artificial chromosomes may reveal the critically important components and may help distinguish between those features that are characteristic of the functional centromere itself and those that are markers of the surrounding heterochromatin. Such a distinction is extremely difficult in normal human chromosomes but should be enhanced by the ability to generate a variety of different artificial chromosomes made with different input sequences.
Recent detailed molecular studies in the fission yeast have revealed that such epigenetic factors are critical for centromere function. The fission yeast CenH3, Cnp1, is deposited only at the central core domain, while heterochromatin (marked by methylation of histone H3 at lysine 9 and by binding of the HP1 homolog, Swi6) forms on the surrounding inverted repeats [35, 36, 41]. The yeast data, together with the observations that CenH3s are conserved and that H3K9-modified nucleosomes and HP1 proteins are often found close to the centromere in higher eukaryotes, have contributed to the development of models for centromere packaging in the larger chromosomes of multicellular eukaryotes, including mammals. In these models, a specific centromeric chromatin configuration, in which CenH3-containing chromatin is surrounded by pericentric heterochromatin, is conserved and may be an important determinant of centromere function [2–4].
While the data presented here are largely consistent with these models, they permit two important refinements. First, large amounts of heterochromatin (containing alpha satellite and marked by H3TrimK9/K27 staining, HP1α binding and late replication) are not required for effective chromosome segregation during mitosis; indeed, the small artificial chromosomes examined here do not contain detectable amounts of H3TrimK9/K27 (Table 2). Second, the cytological characteristics of heterochromatin (repressive chromatin and later replication in S phase), classically attributed to the centromere [54, 55], may instead reflect features of the surrounding heterochromatin and do not appear to define critical properties of the functional centromere. Our own data would argue that the functional centromere - at least as assembled on the smaller D17Z1-based human artificial chromosomes - is instead characterized by a distinctive chromatin containing CenH3 (CENP-A) that can form within regions epigenetically modified with markers of euchromatin (Tables 1 and 2). This conclusion is consistent with parallel work on the organization of centromeric chromatin of normal Drosophila and human chromosomes . The finding that CENP-A-containing chromatin can be deposited within euchromatin-rich artificial chromosomes that are highly mitotically stable (more than 99.9 % segregation efficiency per cell division) yet depleted for heterochromatin modifications, suggests that only a very small amount of heterochromatin may be required on an artificial chromosome (from observations in yeast [37–40] and chicken DT40 cells  this is presumably for assembling the cohesin complex), and that this could also be true for human centromeres.
This study also addresses the question of timing of replication of D17Z1-based artificial chromosomes. The smaller artificial chromosomes that completely overlap with CENP-A  and euchromatic modifications (Figure 3) replicate early in S phase whereas the larger artificial chromosomes that have assembled heterochromatin (H3TrimK9/K27 and HP1α) in addition to euchromatin replicate later in S phase (Table 3). The later onset of replication on the larger artificial chromosomes is similar to that of host chromosome 17 centromere regions that are also enriched for H3TrimK9/K27 and HP1α (Figures 1 and 2, Tables 2 and 3). With the caveats that higher-resolution methods will be required to determine the precise replication timing of the CENP-A domain on the artificial chromosomes, and that differences in vector DNA content may be influencing origin establishment and/or usage, our observations are consistent with local chromatin modification being an important factor influencing artificial chromosome replication.
Chromatin composition as a factor in determining replication timing has also been implicated in a study of a Drosophila minichromosome deletion series. In this study, replication timing was shifted to an earlier point in mid-S phase following deletion of large amounts of pericentromeric heterochromatin from the minichromosomes . Support for a direct role of chromatin composition in replication timing comes from studies in budding yeast, where regions associated with acetylated histones (an epigenetic mark of active chromatin) replicate earlier than those depleted for this histone modification . However, unexpected recent evidence from fission yeast has shown that centromeric heterochromatin replicates early in S phase, suggesting that chromatin composition is not a uniform determinant of replication timing in lower eukaryotes . As the euchromatin-rich and highly mitotically stable artificial chromosomes replicate in the first half of S phase (in 17-E29, the majority of artificial chromosomes (75%, n = 20) replicated in the first 2 hours of S phase (Table 3)) these findings challenge the current dogma that replication later in S phase is an obligatory function of the centromere. The present findings are also supportive of earlier studies suggesting that replication timing of CenH3-containing chromatin is not a determinant of the functional centromere [69, 71].
Cytological data indicate that the amount of CENP-A modified chromatin (in addition to several other kinetochore-associated CENPs) is similar on endogenous human chromosomes and on all artificial chromosomes regardless of the amount of total alpha satellite present. This suggests that the amount of CENP-A chromatin and/or the size of the kinetochore is regulated and/or limited in some manner [6–14, 58, 61]. In contrast, the results of the present study indicate that the heterochromatic fraction of centromeric DNA (on both endogenous chromosomes and artificial chromosomes) is highly variable. In line with current models, we did detect elevated levels of H3TrimK9/K27 modifications and HP1α, diagnostic of heterochromatin on large artificial chromosomes generated from chromosome 17 (D17Z1) or X (DXZ1) alpha-satellite DNA. However, no immunocytochemically detectable heterochromatin (H3TrimK9/K27) was associated with the smaller artificial chromosomes.
To evaluate their potential for characterization of genome sequences and, eventually, for gene transfer or gene therapy applications, we sought to determine the extent of transcriptionally competent chromatin formation in artificial chromosomes. Epigenetic modification of histone H3 by dimethylation at lysine 4 (H3DimK4), a marker of transcriptionally competent chromatin, was present on all artificial chromosomes tested. This contrasts with the staining pattern associated with the centromere regions on human metaphase spreads, where this modification is largely undetectable, probably reflecting the general absence of genes mapping to centromere regions (Figure 3). As selectable marker genes are expressed on artificial chromosomes, it may be presumed that at least a portion of the artificial chromosome chromatin structure is transcriptionally permissive, consistent with the positive staining for H3DimK4. In line with these observations, large human transgenes have been expressed from artificial chromosomes [74–76] and selectable marker genes on artificial chromosomes assemble acetylated histones, another marker of euchromatin . Furthermore, detection of transcription of genes within the CenH3 domain of a human neocentromere  and a rice centromere  suggests that CenH3-containing chromatin can be transcriptionally competent. The relationship between active and repressive chromatin and underlying genomic sequences on the larger artificial chromosomes is not known and will require more detailed follow-up analyses. As other detailed chromatin immunoprecipitation studies have shown that methylation of histone H3 at lysine 4 or lysine 9 seem to be mutually exclusive [64, 65], it will be interesting to find out how the two types of chromatin are assembled during artificial chromosome formation and to find out if there is a mechanism that prevents spreading of chromatin between the heterochromatic and euchromatic sub-domains. An advantage of the artificial chromosome system is the capacity to manipulate sequence content and to test directly the involvement of candidate sequences in gene expression, chromatin establishment or timing of DNA replication.
In this study we included one line, 17-C20, that contains de novo D17Z1-based artificial chromosomes that retain both inner and outer kinetochore components yet are highly mitotically unstable as a result of their rapid loss in the absence of selection and the very high segregation error rate (12.2%) detected in the anaphase assay (Table 1). The artificial chromosomes in this line have a global chromatin composition indistinguishable to that of similar-sized D17Z1-based mitotically stable artificial chromosomes, as both H3DimK4- and H3TrimK9/K27-modified nucleosomes and HP1α are assembled (Table 2). Our study has not revealed the cause of the segregation defect of artificial chromosomes in 17-C20, and so a more extensive examination of additional epigenetic markers or centromere-associated factors may be informative. Detailed anaphase segregation analyses of D17Z1- and DXZ1-based artificial chromosomes have revealed that there is a range of mitotic stability among artificial chromosomes ; future studies will aim to characterize the mechanistic basis of the segregation defects and the relative contribution of genomic and/or epigenetic factors to chromosome behavior.
In summary, we have shown that artificial chromosomes assemble transcriptionally permissive chromatin and that there is a link between artificial chromosome size and the assembly of heterochromatin. Our results with the artificial chromosome panel are largely consistent with current models proposing that the formation of heterochromatin within the vicinity of CENP-A chromatin is functionally important, although the amount of heterochromatin assembled is quite variable, suggesting either that it is required only in small amounts or that it perhaps could even be dispensable. Strikingly, the studies here on the chromatin composition of artificial chromosomes, in combination with studies on normal human centromeres , strongly suggest that the chromatin state of the functional centromere region (as defined by CenH3 association) is quite distinct from pericentric heterochromatin. The artificial chromosome system provides a new set of reagents for investigating the role of both defined alpha-satellite DNA sequences and trans-acting epigenetic factors that cooperate to form a functional human centromere. A fuller understanding of the structure-function relationships of the chromatin and DNA composition of artificial chromosomes is important not only to further our understanding of the role of centromeres in genome stability, but also for the potential development of artificial chromosomes for gene transfer applications.
Materials and methods
Characterization of cell lines containing mitotically stable human artificial chromosomes formed after transfection with either synthetic D17Z1 arrays (PAC17HT1.E29 (17-E29), PAC17HT1.D34 (17-D34), BAC17HT4.B12 (17-B12) or cloned DXZ1 sequences (X-4, X-5) have been described previously [12, 14]. The artificial chromosomes in 17-C20 were generated using VJ104-17α32 , hybridize with both D17Z1 and BAC vector probes, are de novo in composition and assemble CENP-A and CENP-E (data not shown). All artificial chromosomes were formed in human HT1080 cells. Cell lines were grown as described  and supplemented with either 100 μg/ml G418 (Gibco) (17-B12, 17-C20) or 2 μg/ml Blasticidin S HCl (ICN) (17-E29, 17-D34, X-5, X-6), as described .
Anaphase assays used to directly measure chromosome segregation defects in 17-B12 and 17-C20 (Table 1) were carried out as previously described . Assays were carried out at either 45 days (17-B12) or 12 days (17-C20) culture without selection. The spectrum orange-labeled D17Z1 probe (Vysis) hybridized with host 17 centromere regions and artificial chromosomes, whereas the spectrum green-labeled BAC vector probe VJ104  hybridized exclusively with the artificial chromosomes. Co-localization of vector and D17Z1 probes produced yellow fluorescence on the artificial chromosomes, which allowed them to be distinguished from the host D17Z1 sequences (data not shown).
Generation of clonal lines expressing Myc-tagged HP1α
The nucleotide sequence of human HP1α (NCBI Nucleotide database: S62077) was used in BLAST searches against entries in the human expressed sequence tag (EST) database using the NIH BLAST server . A representative HP1α cDNA clone (IMAGE 627533) was obtained from Research Genetics. DNA was prepared with the Wizard-plus mini-prep DNA purification system (Promega), and the cDNA was sequenced on an ABI 373 (Perkin-Elmer) with a fluorescence labeled dye-terminator cycle sequencing kit according to the manufacturer's instructions (PRISM Ready DyeDeoxy Terminator Premix from Applied Biosystems). The full coding sequence of IMAGE 627533 was PCR-amplified with primers incorporating an EcoRI restriction enzyme recognition site (HP1α forward primer, 5'-GGAATT CTGATGGGAAAGAAAACCAAGCG-3'; reverse primer, 5'-GGAATTCGCTCTTTGCTGTTT CTTTC-3') and subcloned using standard techniques  into pcDNA3.1-CT-Myc-His (Invitrogen). Subclones were sequenced to verify sequence integrity and orientation as above. The HP1α-Myc tagged construct (pHP1α-Myc) was transfected into 17-C20, 17-B12, 17-E29 or 17-D34 cell lines using lipofectamine (Invitrogen), resulting in the formation of clonal lines (17-C20-1.B22, 17-B12-1.B10, 17-E29-1.C23 and 17-D34-1.A2, respectively) that stably express Myc-tagged HP1α. G418 selection at 400 μg/ml was applied to select clonal lines 17-E29-1.C23 and 17-D34-1.A2. Since 17-C20 and 17-B12 cells are G418-resistant, pHP1α-Myc was co-transfected in the presence of a second construct, pPAC4  that carries a bsr marker gene. Clonal lines (17-C20-1.B22 and 17-B12-1.B10) resistant to 4 μg/ml Blasticidin S HCl (ICN) were selected and expanded. Confirmation of Myc-tagged HP1α expression was by immunofluorescence using a mouse monoclonal anti-Myc antibody (Invitrogen).
Immunofluorescence and fluorescence in situhybridization (FISH)
Metaphase spreads were prepared for immunofluorescence using previously described protocols . Primary antibodies to the dimethylated form of histone H3 at lysine 4 (anti-H3DimK4) were purchased from Upstate Biotechnology (anti-dimethyl-histone H3 (Lys4)). Modification of histone H3 by trimethylation at lysine 9 (H3TrimK9) was detected using an antibody to the tri-methylated form of histone H3 at lysine 9 purchased from Abcam (anti histone H3-tri methyl K9). This antibody cross-reacts with lysine 27 on histone H3 and is termed anti-H3TrimK9/K27 in the present study. The CENP-A antibody was a generous gift from Manuel Valdivia (Cadiz University, Spain) . Antibodies to H3DimK4, H3TrimK9/K27 and CENP-A were raised in rabbits. Primary and secondary antibody incubations were in 1× PBS supplemented with 1% BSA (Sigma). Secondary antibodies were purchased from Jackson ImmunoResearch. After immunofluorescence detection, 20-50 spreads were captured and their positions on the slide recorded. Slides were subsequently hybridized with an appropriate alpha-satellite probe to detect transfected and endogenous alpha-satellite sequences. FISH was carried out using standard protocols.
Replication timing assay
Cells were pulsed with 10 μM BrdU (Roche) in T25 cm2 flasks for 2 h intervals. Following three PBS washes, medium supplemented with 50 μM thymidine (Sigma) was added. Cells were left in thymidine-containing medium until chromosome harvest. At appropriate intervals, colcemid was added to block cells in mitosis. Cells were harvested and fixed in 3:1 methanol/acetic acid. Metaphase spreads on microscope slides were baked for 1 h at 60°C. Primary anti-BrdU antibody (Roche) was added for 1 h at room temperature. Rhodamine-donkey-anti-mouse secondary antibodies (Jackson ImmunoResearch) were used to visualize sites of BrdU incorporation. Typically, 25 metaphase spreads were captured following BrdU detection and their coordinates on the slide recorded. Subsequent analyses with a D17Z1 probe were used to confirm identity of artificial or endogenous chromosomes.
We thank Chris Yan for helpful discussions and Beth Sullivan for communicating results before publication. This work was supported by a Franklin Delano Roosevelt Award from the March of Dimes Birth Defects Foundation.
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