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Conceived and designed the experiments: JB. Performed the experiments: CK HSWW MM KB AS MGN. Analyzed the data: CK HSWW MM KB AS TL MGN JB. Wrote the paper: CK KB JB.
Centromeres are critically important for chromosome stability and integrity. Most eukaryotes have regional centromeres that include long tracts of repetitive DNA packaged into pericentric heterochromatin. Neocentromeres, new sites of functional kinetochore assembly, can form at ectopic loci because no DNA sequence is strictly required for assembly of a functional kinetochore. In humans, neocentromeres often arise in cells with gross chromosome rearrangements that rescue an acentric chromosome. Here, we studied the properties of centromeres in Candida albicans, the most prevalent fungal pathogen of humans, which has small regional centromeres that lack pericentric heterochromatin. We functionally delimited centromere DNA on Chromosome 5 (CEN5) and then replaced the entire region with the counter-selectable URA3 gene or other marker genes. All of the resulting cen5Δ::URA3 transformants stably retained both copies of Chr5, indicating that a functional neocentromere had assembled efficiently on the homolog lacking CEN5 DNA. Strains selected to maintain only the cen5Δ::URA3 homolog and no wild-type Chr5 homolog also grew well, indicating that neocentromere function is independent of the presence of any wild-type CEN5 DNA. Two classes of neocentromere (neoCEN) strains were distinguishable: “proximal neoCEN” and “distal neoCEN” strains. Neocentromeres in the distal neoCEN strains formed at loci about 200–450 kb from cen5Δ::URA3 on either chromosome arm, as detected by massively parallel sequencing of DNA isolated by CENP-ACse4p chromatin immunoprecipitation (ChIP). In the proximal neoCEN strains, the neocentromeres formed directly adjacent to cen5Δ::URA3 and moved onto the URA3 DNA, resulting in silencing of its expression. Functional neocentromeres form efficiently at several possible loci that share properties of low gene density and flanking repeated DNA sequences. Subsequently, neocentromeres can move locally, which can be detected by silencing of an adjacent URA3 gene, or can relocate to entirely different regions of the chromosome. The ability to select for neocentromere formation and movement in C. albicans permits mechanistic analysis of the assembly and maintenance of a regional centromere.
Centromere function is essential for proper chromosomal segregation. Most organisms, including humans, have regional centromeres in which centromere function is not strictly dependent on DNA sequence. Upon alteration of chromosomes, new functional centromeres (neocentromeres) can form at ectopic positions. The mechanisms of neocentromere formation are not understood, primarily because neocentromere formation is rarely detected. Here. we show that C. albicans, an important fungal pathogen of humans, has small regional centromeres and can form neocentromeres very efficiently when normal centromere DNA is deleted, and the resulting chromosomes are stably propagated. Neocentromeres can form either very close to the position of the deleted centromere or at other positions along the chromosome arms, including at the telomeres. Subsequently, neocentromeres can move to new chromosomal positions, and this movement can be detected by silencing of a counterselectable gene. The features common to sites of neocentromere formation are longer-than-average intergenic regions and the proximity of inverted or direct repeat sequences. The ability to select for neocentromere formation and movement in C. albicans permits mechanistic analysis of the assembly and maintenance of a regional centromere.
Centromeres, the DNA regions at which microtubules attach to and segregate daughter chromosomes, are essential for genome integrity. Point centromeres have been characterized extensively because of their small size and relative simplicity, especially in Saccharomyces cerevisiae. They are composed of one centromere-specific nucleosome that spans <200 bp of DNA, organized in a tripartite structure that includes a specific DNA binding site necessary for centromere function (reviewed in ,). In contrast, regional centromeres are found in most eukaryotes, including fungi other than a subgroup of the Saccharomycotina . They span very large DNA domains (10′s to 1000′s of kb) and are organized into core DNA regions associated with centromere-specific nucleosomes and flanked by highly repetitive DNA packaged into pericentric heterochromatin (reviewed in ). Regional centromere function is epigenetic in character –: rather than being dependent upon a specific DNA sequence, the presence of CENP-A, the centromere-specific histone H3 variant, defines the position of a functional centromere. It is thought that CENP-A is regulated by its stabilization at functional centromeres/kinetochores, because at non-centromeric loci CENP-A is removed and proteolyzed , (reviewed in ).
Neocentromeres, defined as functional kinetochores that assemble at ectopic positions, usually appear together with other chromosome rearrangement events (reviewed in ). Neocentromeres have the properties of active centromeres, and, by definition, they associate with CENP-A –. Over 90 examples of human neocentromeres have been documented, most involving the formation of supernumerary chromosomes and often associated with developmental disabilities or specific cancers . Many more neocentromeres likely escape detection because they are eliminated during development ,,. It is not known if neocentromere formation occurs first, followed by mutation of the natural centromere or, conversely, if mutation of the natural centromere leads to neocentromere formation.
The chromosomal position of neocentromeres is different in different organisms. In Drosophila, they have been found only adjacent to chromosome breaks that inactivated the original centromere ,. In S. pombe they appear only at telomeric loci . In contrast, human neocentromeres exhibit flexible adaptation to changes in chromosome structure, often appearing far from the site of the original centromere at either terminal or submetacentric loci (reviewed in ).
No specific DNA sequence properties necessary for functional regional centromere assembly have been identified. Regional centromeres in humans, flies, plants and fungi are composed of long tracts of repetitive DNA, yet repeat tracts are not absolutely required for centromere function or for the formation of neocentromeres (reviewed in , –). In S. pombe, pericentric heterochromatin formation is necessary for efficient de novo assembly of a functional kinetochore , as well as for formation of telocentric neocentromeres .
C. albicans, an opportunistic fungal pathogen that resides as a commensal in its human host, possess regional centromeres that are much smaller and simpler than other regional centromeres ,. Each of its 8 diploid chromosomes has a centromere that is regional based on its size (~3–4.5 kb that specifically associates with CENP-ACse4p ,), the lack of tripartite point-centromere DNA structure, the presence of several orthologs of proteins found only at regional centromeres, and the absence of orthologs of proteins found only at point centromeres .
Most notably, C. albicans CENs lack pericentric heterochromatin: CENP-A associated core sequences are not embedded in long tracts of repetitive DNA ,; there are no clear orthologs of either heterochromatin protein 1 (HP1) or of enzymes necessary for the methylation of histone H3 lysine 9; and there is no homolog of CENP-V, a protein that regulates the extent of pericentric chromatin in human cells . Consistent with a lack of pericentric heterochromatin, genes near the centromeres are transcribed at levels close to the average level of transcription across the genome (K. E. S. Tang and JB, data not shown). Furthermore periodic nucleosome spacing is seen at inactive centromere DNA and not at active CENs, suggesting that nucleosomes at active CENs do not associate tightly with a specific DNA sequence . Naked CEN7 DNA used to transform C. albicans did not permit de novo assembly of centromere function ,. Taken together, these observations suggest that, like other regional CENs, the assembly of a centromere and the inheritance of centromere function in C. albicans requires epigenetic properties conferred by the association of CENP-A and other kinetochore proteins, rather than by a specific DNA sequence.
The stoichiometry of microtubules and centromere-specific nucleosomes differs in different organisms. S. cerevisiae has only one CENP-A nucleosome and one microtubule per centromere, while S. pombe has ~2–3 CENP-A nucleosomes  and ~2–4 microtubules per centromere . This suggests that one microtubule is attached to kinetochore proteins assembled at each CENP-A nucleosome . In humans, the number of CENP-A nucleosomes is thought to be far larger than the number of microtubule attachments ,. In C. albicans there are ~8 CENP-ACse4p molecules per centromere, presumed to be organized into 4 centromere-specific nucleosomes, and only one microtubule per centromere . This suggests that only one of the four CENP-A nucleosomes at each centromere assembles a kinetochore structure that binds a microtubule. Thus, the prevailing model is that at C. albicans CENs, as at human centromeres, some CENP-A-containing nucleosomes bind microtubules while others do not.
Seven of the eight C. albicans centromeres are near short repeats; only the centromere of Chr7 (CEN7) does not have obvious repeat sequences nearby . Yet most analysis of C. albicans centromere function has been performed with CEN7 DNA, which is necessary for Chr7 stability . On Chrs 2, 3 and 6 there are direct repeats within ~3 kb of the centromere core sequence. On Chrs 1, 4, 5 and R the CENP-A bound centromere core DNA is flanked by a short inverted repeat (IR). The palindromic structure of the four centromeres with a flanking IR is most reminiscent of the structure of S. pombe and other regional centromeres.
Here, we studied the properties of C. albicans CEN5, a centromere with a palindromic structure, by replacing the CEN5 DNA and the flanking IR with a URA3 marker. We found that it behaved like a regional centromere: the resulting cen5Δ::URA3 Chr5 derivatives were stably maintained through mitosis by efficient formation of a neocentromere at one of several non-centromeric loci. Loss of the wild-type chromosome 5 homolog and homozygosis of the neoCEN5 homolog demonstrated that cells can survive in the absence of any CEN5 DNA. Some of the cen5Δ transformants formed a “proximal neoCEN” near cen5Δ::URA3, which subsequently moved onto, and silenced the URA3 gene. Other cen5Δ transformants formed “distal neoCENs” at several different loci far from the deleted CEN5 locus. Thus, neocentromere formation does not require a specific DNA sequence and can occur at several different chromosomal loci.
We previously identified isochromosome derivatives of Chr5, in which the derived chromosomes replaced either the right arm with left arm information (i(5L)), or replaced the left arm with right arm information (i(5R)) ,. The only DNA common to the i(5L) and i(5R) isochromosomes is the predicted Chr5 centromere (CEN5), including an inverted repeat (IR) flanking the central core (CC5) (Figure 1A). This CC5+IR structure resembles a simplified version of the central portion of typical regional centromeres (e.g., central core+innermost repeats in S. pombe centromeres). Telomere truncation constructs  replacing either the complete Chr5L arm  or the Chr5R arm (Table S1) yielded chromosome fragments (Figure 1A) similar in mitotic stability to that of the isochromosomes (<10−3 loss/division). Thus, the CC5+IR DNA is associated with centromere function in Chr5 and all its stable derivatives, implying that it provides centromere function.
To ask if CEN5 DNA is necessary for centromere function, we replaced CEN5 (CC5+IR) on one of the two Chr5 homologs with a PCR product that included a URA3 gene flanked by homology to regions just outside the IR (Figure 1B). In C. albicans laboratory strains, the two Chr5 homologs differ in at least two ways. First, the mating type-like locus (MTL) is heterozygous (MTLa or MTLalpha). Second, there are two LTR repeats present in the ‘long’ allele of CEN5 (one in the central core region and one to the left of the left side of the IR) (Figure 1B). In addition, the Major Repeat Sequence (MRS) on Chr5R can vary in size by >50 kb ,.
Initially, we found lower transformation frequencies for cen5Δ::URA3 constructs than for control myo1Δ::URA3, constructs that replaced a gene 100.2 kb from CEN5. For example, there were 3–5-fold fewer transformants per microgram of DNA for cen5Δ::URA3 than for the control when colonies were counted 3–4 days after transformation. The proportion of transformants that carried bona fide CEN5 deletions, detected by PCR amplification, was also 4–5 fold lower for the cen5Δ::URA3 than for the myo1::URA3 strains. However, for the cen5Δ transformation (and not for the control transformants), new colonies continued to appear for up to ~9 days after transformation. After 9 days, similar numbers of Ura+ transformants were obtained for the cen5Δ and the control transformations (Figure 1B). When the transformant colonies that appeared later were analyzed, the proportion of transformants that carried bona fide CEN5 deletions, as detected by PCR amplification and confirmed by Southern analysis (Figures 1C and S1A), was similar to, or slightly less than, that of a myo1Δ::URA3 control strain (Figure 1B). We also obtained transformants in which CEN5 was replaced with NAT1. Southern blots and hybridization to CEN5 were used to verify the correct insertion of these markers (Figure S1B–D).
The growth rates of the cen5Δ::URA3 strains were indistinguishable from the growth rates of wild-type strains and strains carrying URA3 at non-centromeric loci (Table 1, Figure 2). Similar levels of stability and growth rates were also seen in the cen5Δ::NAT1 transformants (Table 1). This result is different from what was seen with CEN7: deletion of the Chr7 CENP-ACse4p binding region was reported to yield only highly unstable chromosomes . Importantly, all of the correct cen5Δ::URA3 transformants exhibited relatively high levels of mitotic stability (Table 1), measured as the proportion of cells that retain the ability to grow in the absence of uridine.
Transformation of C. albicans sometimes causes unintended aneuploidies . To investigate this possibility, we analyzed the cen5Δ::URA3 transformants using comparative genome hybridization (CGH) and CHEF karyotype gels. CGH array analysis did not detect any copy number changes in any of the strains: like the parent, all were disomic for all 8 chromosomes (Figure 1D and data not shown). In general, CHEF karyotypes for all but strain YJB9907 were indistinguishable from the parent strain for Chr5 when stained with ethidium bromide (Figure 1E left panel). Only a Chr5-sized band was detected with a URA3 probe as well as probes for CEN5, Chr5L and Chr5R (Figure 1E and data not shown), indicating that no Chr5 rearrangements occurred. In one strain, (YJB9907) the two Chr5 homologs became separable and the two separable Chr7 homologs became inseparable (Figure 1E, left panel, lane 2), most likely because of recombination between the MRS repeats on these chromosomes . The CGH and CHEF gel analysis together indicate that no gross chromosomal rearrangements were evident in the cen5Δ strains. Thus, the cen5Δ strains retained two intact copies of Chr5 and genome integrity was maintained.
The relative stability of strains lacking CEN5 implies that a neocentromere, an ectopic assembly of a functional kinetochore onto non-centromeric DNA , formed on the Chr5 homologs carrying cen5Δ alleles. Importantly, these neocentromeres apparently formed in all recovered transformants in which CEN5 had been correctly replaced with a selectable marker.
Because C. albicans is diploid, all of the cen5Δ::URA3 strains retained one wild-type copy of CEN5. While centromeres generally function only in cis, phenomena that act in trans, such as transvection in Drosophila (reviewed in ), raised the possibility that the cen5Δ::URA3 homolog was able to segregate through some interaction with the wild-type CEN5 homolog of Chr5. To determine if cells could survive and divide in the absence of any CEN5 DNA, we utilized sorbose growth, which selects for cells that lose one copy of Chr5 . Strains were plated to sorbose medium lacking uridine to select for Ura+ cells carrying only one Chr5 homolog (Figure 3A, middle panel). The colonies that appeared on sorbose-uri medium were then streaked to rich medium (YPAD) and larger colonies (those that likely reduplicated the single copy of Chr5 , Figure 3A, lower panel) were streaked to SDC-uri to ensure that they retained the URA3 marker. PCR analysis confirmed retention of the cen5Δ::URA3 and failed to detect wild-type CEN5 or MTLa (Figure 3B) in several isolates, consistent with the idea that the wild-type Chr5/CEN5 homolog had been lost. CHEF analysis indicated that the karyotype of these strains was unaltered (Figure 3C). In the case of YJB9907, the parental Chr5 homologs had distinct mobilities, and the sorbose-selected derivatives (YJB9907-3s and YJB9907-6s) carried only the smaller homolog, which hybridized to URA3 and not to CEN5 (Figure 3C, lanes 5 and 8). CGH analysis indicated that the remaining Chr5 homolog was present in two copies (Figure 3D) following growth on SDC-uri. For YJB9929, the two derivatives (9929-1s and 9929-2s) showed similar results to YJB9907 and its derivatives. Both PCR and Southern analysis confirmed that the original CEN5 DNA was absent from both strains (Figure 3B&C).
The four strains lacking any CEN5 DNA exhibited growth rates that were slightly slower than the growth rates of wild-type and cen5Δ::URA3/CEN5 strains (Figure S1E, Table 1). This reduced fitness could be because the cells lacked CEN5 DNA completely or it could be due to the homozygosis of all markers on Chr5 that occurred as a consequence of sorbose selection and subsequent growth on rich medium (Figure 3A). To address this question, we performed growth rate experiments with strain YJB9726, which maintained and then reduplicated one CEN5 homolog, yet was homozygous for all genes on Chr5 because it had been propagated on sorbose (Table 1, Figure S1E). Importantly, the growth rates of strains with and without intact CEN5 were similar, indicating that CEN5 DNA is not necessary for chromosome propagation and stable strain maintenance. Rather, the slower growth is likely due to one or more genes that, when homozygous, cause reduced fitness. This supports the idea that neocentromeres formed in the cen5Δ::URA3 strains have no obvious deleterious effect on growth rate or fitness in standard laboratory growth conditions.
Two different classes of cen5Δ::URA3 transformants were distinguishable based on the rate at which cells acquire the ability to grow on 5-FOA (FOAR cells), a compound that is toxic to Ura+ cells . This assay is generally used as a proxy for mitotic loss rates and quantified using fluctuation analysis to identify median FOAR rates ,. The Class A transformants, termed ‘proximal neoCENs’ based on data described below, became resistant to 5-FOA at a rate of ~10−2–10−3/cell division and the phenotype was reversible. The Class B transformants (termed ‘distal neoCENs’ based on data described below) became irreversibly resistant to 5-FOA at a rate of ~10−5–10−6/cell division, similar to the rate of 5-FOA resistance of myo1Δ::URA3 strains (Figure 4A). Disruption of either Chr5 homolog (short or long, Figure 1B) yielded both Class A and Class B transformants, indicating that the class of transformant was not due to disruption of a specific CEN5 allele.
Southern analysis of the Class B FOAR strains indicated that, like myo1Δ::URA3 transformants, when these cells became FOAR, they had lost the URA3 marker, (Figure 4B). Thus, rare FOAR derivatives of Class B cen5Δ::URA3 transformants have either undergone a non-disjunction event to lose the entire chromosome carrying cen5Δ::URA3 or have lost the URA3 via recombination. To distinguish between these possibilities, we followed the segregation of markers on both arms of Chr5 (HIS1/his1Δ::NAT1 near the Chr5R telomere and MTL on Chr5L). At least 98% of the FOAR Class B derivatives had lost heterozygosity at HIS1 (on Chr5R) and, among those, 100% of them also lost heterozygosity at MTL (on Chr5L) (Figure 5). Thus, there is strong genetic support for the idea that whole chromosome loss was the cause of the FOAR phenotype in Class B transformants (Figure 5).
Several lines of evidence indicated that the higher rate of FOAR in Class A transformants was due to transcriptional silencing and not loss of URA3 DNA. First, the FOAR phenotype was reversible: transformants initially selected on SDC-uri gave rise to cells that subsequently grew on SD+5-FOA and these colonies could subsequently give rise to cells that grew on SDC-uri (Figure 4A). In each transfer (from SDC-uri (U) to SD+FOA (UF), then to SDC-uri (UFU), then to SD+FOA (UFUF) and so on), only a subpopulation of colonies appeared (~1/1000 cells). Importantly, the reversible FOAR phenotype was unique to the cen5Δ::URA3 strains; when URA3 was inserted adjacent to an intact CEN5 (e.g., in the 5L-URA3-TEL strain), it was not reversibly silenced (Figure 4A). Second, strains grown on SD+FOA retained URA3 DNA on Chr5, as detected by CHEF Southern (Figure 4B) and PCR (data not shown). Third, URA3 DNA sequence was unaltered in any of the FOAR Class A transformants (YJB9909, YJB9915, YJB9916 and YJB9926, data not shown), indicating URA3 did not acquire point mutations. Fourth, genetic analysis of Class A FOAR transformants carrying markers on Chr5L and Chr5R did not lose markers from either arm (Figure 5), reinforcing the idea that both Chr5 homologs were retained on 5-FOA. Finally, real time PCR detected ~16 fold lower levels of URA3 mRNA in cells grown on SD+FOA relative to those grown on SDC-uri. This indicates that URA3 expression was silenced at the transcriptional level in the Class A FOAR cells (Figure 4C). Taken together, these results indicate that URA3 DNA was silenced at the transcriptional level in most Class A FOAR transformants and therefore, that the cen5Δ::URA3 Chr5 homolog was stable to a similar degree in these cells and in the Class B cells. Thus, cen5Δ chromosomes are very stable, far in excess of the stability of minichromosomes in either S. cerevisiae or S. pombe that bear bona fide centromeres ,. This provides more evidence that the cen5Δ::URA3 homologs in both classes of transformants must carry a functional neocentromere.
Active regional centromeres and neocentromeres are, by definition, associated with CENP-ACse4p –,,. To identify the position of neocentromeres on cen5Δ::URA3 homologs, we performed chromatin immunoprecipitation (ChIP) experiments using antibody raised against an N-terminal peptide from C. albicans CENP-ACse4p (see Materials and Methods). We first analyzed Class A transformants to investigate the mechanisms linking neocentromere function and the growth phenotypes on 5-FOA or medium lacking uridine in these strains.
As expected, anti-CENP-ACse4p specifically recognized DNA in CEN4, CEN5 and CEN6 central core regions, but did not preferentially precipitate non-centromeric sequences such as the TAC1 gene on Chr5L (Figure 6A). In addition, in Class A transformants grown on SD+5-FOA, CENP-ACse4p preferentially associated with URA3, while when these strains were grown on SDC-uri, CENP-ACse4p associated with URA3 to a lesser degree (Figure 6B). This implies that the reversible silencing is due to reversible association of CENP-ACse4p with URA3. In contrast, in Class B Ura+ cells, CENP-ACse4p was not associated with URA3, and in FOAR cells the URA3 DNA had been lost from the strain and was not detectable in the lysate (Figure 6B).
To more precisely localize the active neocentromeres in Class A transformants, we analyzed DNA from anti-CENP-ACse4p ChIP experiments using PCR amplification of contiguous 400 bp DNA segments spanning a ~15 kb region including and surrounding CEN5. Consistent with previous studies ,, CENP-ACse4p was associated with the CC5 sequence and not with the IR or flanking Ch5L or Ch5R sequences (Figure 7A) in two different parental strains that each carried intact copies of both CEN5 homologs. Furthermore, in six independent cen5Δ::URA3 transformants, the wild-type CC5 DNA on the intact, unmodified homolog (which could be distinguished based on the presence or absence of the LTR insertion, Figure 1B) was specifically associated with CENP-ACse4p (Figure S2A and B). Thus, in all cen5Δ::URA3 strains, the centromere on the wild-type Chr5 homolog was not perturbed by the loss of CEN5 on the other homolog.
In addition, CENP-ACse4p was associated with DNA ~1 kb to the left of URA3 in all Class A transformants growing on SDC-uri. A similar pattern was seen in four independent transformants, including those carrying deletions of either CEN5 homolog (with and without the LTR) (Figures 7B and S2C). In contrast, when Class A transformants were grown on 5-FOA (URA3 gene silenced), CENP-ACse4p always associated with URA3 in all four independent Class A transformants, irrespective of the CEN5 homolog that had been disrupted (Figures 7B, S2C and data not shown). Importantly, when strains were cycled from SDC-uri to 5-FOA and then back to SDC-uri for two additional cycles (UFUFU) and then to SDC+FOA again (UFUFUF), the same patterns were retained: in cells grown on 5-FOA the CENP-ACse4p was associated mostly with URA3; in cells grown on SDC-Uri CENP-ACse4p enrichment was adjacent to the URA3 gene (Figure 7C). This suggests that the neocentromere occupied different positions when Class A cells were grown under different conditions and that its average position had shifted position by ~1–2 kb.
Analysis of Class A transformants in which the short vs long CEN5 homologs were disrupted revealed that the episemon LTR was only associated with CENP-ACse4p when the LTR was linked to the cen5Δ::URA3 homolog (Figure S2C). In addition, the LTR appeared to be a preferred site for CENP-ACse4p binding. When it was located on the same homolog as cen5Δ::URA3, the lateral spread of CENP-ACse4p from URA3 into the adjacent DNA was reduced. Thus, CENP-ACse4p associated with DNA flanking the left side of the IR only in cis and not in trans.
In the proximal neoCEN transformants, we detected the association of CENP-ACse4p with URA3 because of the FOAR counter-selectable phenotype of Ura− cells. Quantitative RT PCR indicated that the 16-fold silencing of URA3 occurred by affecting levels of mRNA (Figure 4C). To ask if association with CENP-ACse4p resulted in transcriptional silencing of genes other than URA3, we measured the expression levels of two genes located just left of cen5Δ::URA3 in Class A/proximal neoCEN strains. These two genes (orf19.3161 and orf19.3163) were expressed at higher levels in FOAR cells (when CENP-ACse4p was not associated with them) relative to their expression in Ura+ cells (when CENP-ACse4p was associated with them) (Figure 4C). This is consistent with the idea that association of a gene with CENP-ACse4p results in decreased transcription from that gene.
In the Class B strains, CENP-ACse4p associated with CC5 on the wild-type homolog and was not associated with other DNA fragments near cen5Δ::URA3 (Figure S2D). This suggested that the neocentromeres in Class B strains were located beyond the 15 kb region analyzed by ChIP, and thus are located too far from cen5Δ::URA3 to silence it. To locate the position(s) of neocentromeres in Class B transformants, we performed ChIP followed by massively parallel high-throughput sequencing (ChIP-SEQ) of one wild-type and two Class B transformants (YJB9907 and YJB9929). As expected, in each strain, the major region associated with CENP-ACse4p on each wild-type chromosome overlapped with the region previously described by Carbon and co-workers as the centromere ,, although it was smaller, ranging from 2–3.3 kb (Figure S3). The difference in sizes predicted is likely due to the improved resolution from the smaller average fragment size used in the ChIP pulldowns.
In all three strains, the degree to which each centromere, other than CEN5, associated with CENP-ACse4p was similar relative to the other centromeres (Figure 8A). In both of the cen5Δ::URA3/CEN5 strains, CENP-ACse4p associated with the remaining CEN5 copy at a level slightly higher than half the relative signal present on two copies of CEN5 in wild-type cells (Figure 8A).
Analysis of the ChIP-SEQ data also identified new CENP-ACse4p-associated DNA sequences on Chr5 in both Class B neocentromere strains (Figure 8B). In YJB9907 CENP-ACse4p was associated with a ~4 kb region near the left telomere. This peak (present in one copy, termed neoCEN-1) included 36% of the ChIP-SEQ reads relative to the average centromere (present in two copies). This is ~half as much CENP-ACse4p as was present in the CEN5 peak from the other Chr5 homolog. In strain YJB9929, two different regions of the genome exhibited small peaks of CENP-ACse4p association: ~1.2 kb around orf19.1978 on Chr5L (termed neoCEN-2) and a ~1 kb region overlapping orf19.6678 and it's 5′ upstream region on Chr5R (termed neoCEN-3) (6% and 8% of the signal seen in an average centromere, respectively). This suggests that the neoCENs associate with less CENP-ACse4p than does a wild-type centromere.
The positions of the predicted neocentromeres in the two Class B strains, as well as in wild-type strains, were then analyzed by standard ChIP analysis, using primers that amplified fragments across the regions identified by ChIP-SEQ (Figure S4). As predicted from the ChIP-SEQ analysis, in YJB9907 CENP-ACse4p was associated with unique, telomere-adjacent sequence within and 5′ to orf19.5698 (neoCEN-1), and in YJB9929 CENP-ACse4p was associated with neoCEN-2 and neoCEN-3 DNA. Thus, neocentromeres can form at positions far from the deleted cen5Δ::URA3 locus. For this reason, we term Class B transformants ‘distal neoCEN’ transformants as compared to the Class A ‘proximal neoCEN’ transformants.
Importantly, the neocentromere peaks were strain-specific. They never appeared in the wild-type strain or in the other neocentromere strain (Figure 8B). ChIP extracts of YJB9907 analyzed by PCR confirmed the presence of CENP-ACse4p near the left telomere and its absence in the neoCEN-2 or neoCEN-3 regions. Similarly, in YJB9929, CENP-ACse4p was associated with neoCEN-2 and neoCEN-3 and not with the neoCEN-1 region. Furthermore, on all other chromosomes, telomeric regions were not associated with CENP-ACse4p (data not shown). Thus, one unambiguous neocentromere is formed in YJB9907, while in YJB9929 at least two different DNA regions are associated with CENP-ACse4p and presumably function as neocentromeres.
Because dicentric chromosomes are highly unstable , and because YJB9929 exhibited a growth rate similar to that of YJB9907 and wild-type strains (Table 1, Figure 2), we hypothesized that the YJB9929 ChIP extract was prepared from a mixed population of cells carrying at least two different neocentromeres. To test this possibility, we analyzed 4 independent colonies from the YJB9929 culture by ChIP-SEQ. Despite the presence of a mixed population, we detect CENP-ACse4p peaks only at the neoCEN-3 position (as well as at the original CEN5 position, which is expected because this strain is heterozygous for CEN5/cen5Δ::URA3 (Figure 8B)). PCR of these ChIP extracts confirmed that CENP-ACse4p is located at the neoCEN-3 position in 3 of the 4 colonies (Figure S4). This is consistent with the stronger signal for neoCEN-3 relative to neoCEN-2 in the original YJB9929 isolate (Figure 8B). Thus, in different strains, neocentromeres formed at different loci and on both arms of Chr5.
We next determined the position of the neocentromeres in the sorbose-derived YJB9907s and YJB9929s strains lacking both wild-type CEN5 homologs. CENP-ACse4p ChIP-SEQ of strains YJB9907-3s and YJB9907-6s detected CENP-ACse4p associated with neoCEN-1, near the Chr5L telomere (Figure 8B), although the signal peak appeared to shift to the telomere-distal portion of the peak. PCR analysis of the two strains confirmed that CENP-ACse4p remained associated with this telomere-adjacent region. Furthermore, no major peak was detected at any other Chr5 locus tested (e.g, neoCEN-2 or neoCEN-3, Figure S4) and, consistent with the loss of CEN5 in these strains, no CEN5 DNA was associated with CENP-ACse4p. Thus, despite the fact that sorbose growth causes stresses that can result in chromosome loss, the neocentromere in strain YJB9907 was sufficiently stable to be maintained in the same general region during selection for Chr5 loss and reduplication/non-disjunction. Nonetheless, the shift in the peak position suggests that the neoCEN in YJB9907s strains may have undergone a local shift in its position.
In contrast, ChIP of YJB9929-1s and YJB9929-2s did not detect CENP-ACse4p associated with neoCEN-1, neoCEN-2 or neoCEN-3 (Figure S4). ChIP-SEQ of the two YJB9929-s strains identified a new, strong CENP-ACse4p signal associated with sequences ~170 kb from the Chr5L telomere (Figure 8B, neoCEN-4). PCR of ChIP samples from the two independent derivatives (Figure S4) confirmed that CENP-ACse4p was present at this locus. Thus, in strain YJB9929 exposed to the stress of growth in sorbose, the original neoCEN locations were lost and a new active neocentromere (neoCEN-4) arose at a distance of ~640 kb from the neoCEN-3.
It has been suggested that analysis of neocentromere DNA will identify features of DNA sequence necessary for centromere function . The only characteristic common to all C. albicans centromeres is that they are found in very long intergenic regions (average 7.5 kb, range 3.8–17.4 kb) (Figure 8C and ). In addition, seven of the eight C. albicans centromeres are flanked by repeats; four (including CEN5) are oriented as inverted repeats while three are oriented as direct repeats.
Like other regional centromeres, C. albicans centromeres have no common primary DNA sequence feature . Consistent with this, no sequence motifs common to centromeres and/or neocentromere regions (including flanking sequences upstream and downstream of the centromeres and neocentromeres) were identified using stringent search criteria and no obvious inverted repeats, palindromes or common sequence motifs were found at all centromeres and neocentromeres (using EMBOSS (http://pro.genomics.purdue.edu/emboss/) , JSTRING (http://bioinf.dms.med.uniroma1.it/JSTRING) and MEME ). Furthermore, neither the centromeres nor neocentromeres were enriched for any nucleotide or dinucleotide combination relative to total genomic DNA.
Importantly, the C. albicans neocentromeres identified here all shared two features seen at bona fide centromeres. First, for each of the neocentromeres, at least one intergenic region within the CENP-A binding peak was considerably larger than the average size of intergenic regions on Chr5 (902 bp) or across the entire genome (853 bp) (Figure 8C). Furthermore, all of the neocentromeres were found in close proximity to repeat sequences (Figure 8D). NeoCEN-1 is immediately next to the telomere-adjacent repeated TLO11 and the rho-5a LTR repeat and within 4 kb of the telomere repeats. NeoCEN-2 is centered on GIT2, with adjacent copies of GIT1 and GIT4, which encode predicted glycerophosphoinositol permeases that share 75% and 73% identity with GIT2 at the DNA level (determined by BLAST ). NeoCEN-3 is flanked by an inverted repeat that includes orf19.6676 and orf19.6678 on the left and orf19.6681 and orf19.1124.2 on the right. These gene pairs are 99% (orf19.6676 and orf19.1124.2) and 70% (orf19.6678 and orf196681) identical. In addition, the intergenic region adjacent to orf19.6678 includes an omicron-5a LTR element. Finally, neoCEN-4 is within a large (>4.4 kb) intergenic region that includes an LTR element (chi-5a) and is flanked by orf19.570 and orf19.575, which are >75% identical at the DNA sequence level and are organized in a direct repeat orientation. Thus, all four neoCENs include a large intergenic region as well as inverted or direct repeats. Furthermore, three of the four neoCENs also feature an LTR repeat in close proximity.
This study has revealed important new properties of C. albicans small regional centromeres, many of which are reminiscent of the properties of larger regional centromeres. First, the loss of centromere DNA results in formation of a stable neocentromere. CEN5 can be replaced with URA3 or NAT1 and, in bona fide transformants, the resulting Chr5 remains stable and cells are viable in the absence of any CEN5 DNA. Second, neocentromere formation is highly efficient, apparently occurring in all bona fide transformants. Third, while C. albicans lacks classic heterochromatin components such as HP1, histone H3K9 methyltransferases or DNA methyltransferases, an active centromere silences transcription of an associated gene. Fourth, neocentromeres can form at several different regions of Chr5, including within each of the chromosome arms and at a telomere-adjacent position. Fifth, loci that form neocentromeres have two common properties: a longer-than-average intergenic region and the presence of repeated DNA. Finally, under selective conditions, neocentromeres can move either locally (a few kb) or over long distances (100's of kb).
Taken together, our data suggest a model (Figure 9) in which all recovered transformants form a neocentromere, defined by the presence of CENP-ACse4p. In some transformants, the neocentromere assembles adjacent to cen5Δ::URA3 and subsequent selection for FOAR identifies those individuals in which URA3 is silenced by virtue of its association with CENP-ACse4p. Because this silencing and the association of CENP-ACse4p with URA3 are reversible, we propose that direct association with CENP-ACse4p nucleosomes is incompatible with active transcription and that the neocentromere moved onto the URA3 gene and silenced it in FOAR cells. In other transformants, the neocentromere assembles far from the deleted centromere and FOAR derivatives are usually due to chromosome loss. Furthermore, while exposure to sorbose promotes Chr5 loss in C. albicans , here we found that the telocentric neoCEN-1 remained relatively stable upon sorbose growth while, in a different strain, neoCEN-2 and neoCEN-3, which gave less robust CENP-ACse4p signals, were lost upon sorbose exposure and a new neocentromere, neoCEN-4, appeared at an entirely different locus.
Chromosomes carrying a neocentromere exhibited loss rates of ~10−4–10−5 per division. This is much more stable than minichromosomes in S. cerevisiae (reviewed in ) or in S. pombe  and resembles the high levels of neocentromere stability in humans (reviewed in ). Deletion of CEN5 did not affect cell growth rates unless all Chr5 markers were homozygous, which grew at least as well as CEN5-containing strains that were homozygous for all Chr5 markers (Table 1, Figure S1E). Thus, slow growth was due to homozygosis of one or more genes on Chr5 and not to the loss of CEN5. CEN5 deletion occurred with ~equal frequency on the two Chr5 homologs, suggesting that C. albicans requires two different alleles at one or more Chr5 loci to maintain optimal fitness.
The high level of stability in cen5Δ strains is consistent with the idea that C. albicans has regional centromeres. Yet deletion of CEN7 was reported previously to result in high levels of Chr7 instability (>50% loss per division) . CEN7 is the only C. albicans centromere lacking any flanking repetitive DNA . The role of repeat DNA in CEN5 structure, and whether or not it affects centromere function, remains to be determined. However, the presence of repeat DNA at each of the neocentromeres (Figure 8D) suggests that CEN7 may behave differently from all other C. albicans centromeres.
A notable property of C. albicans neocentromeres is that they form in all bona fide transformants. This result is remarkable for two reasons. First, it suggests that when a centromere is perturbed, it induces the formation of neocentromeres. While we cannot rule out that events can occur in the opposite order, our results clearly suggest that loss of a functional centromere stimulates the formation of a new one. Second, it indicates that, when induced to form, neocentromere formation is efficient, since replacement of CEN5 yields similar numbers of transformants as replacement of a non-essential gene on Chr5. This high level of neocentromere efficiency is unprecedented.
Despite its efficiency, the establishment of neocentromeres was not always rapid. Some transformants appeared early while others continued to appear over time, with high levels of correct transformants appearing as small colonies 7–9 days after transformation. In contrast, when a control gene on Chr5 was disrupted, most colonies appeared within a few days and no new small colonies appeared after several days. Importantly, C. albicans cen5Δ strains that initially appeared as small colonies subsequently grew with wild-type growth rate kinetics (Figure 2). This suggests that deletion of a wild-type centromere led to the epigenetic establishment of neocentromeres at new positions and that neocentromere assembly occurred over the first ~1 week after transformation. Since we isolated similar proportions of proximal and distal neocentromeres from cen5Δ::URA3 colonies that appeared early or late following transformation, the time at which transformants initially appeared was not a reflection of neocentromere position (e.g., proximal vs distal neoCEN). This slow appearance of transformants is reminiscent of the epigenetic nature of centromere establishment on minichromosomes in S. pombe. In this case, cells carrying minichromosomes with functional centromeres were selected because they grew more rapidly than cells carrying minichromosomes that had not yet formed functional centromeres .
During the course of this work, Ishii et al. (2008) demonstrated that excision of an S. pombe centromere results in several different cell fates. In a large proportion (~80%) of the cells, the chromosome became unstable, leading to chromosome missegregation and cell death. Cells are rescued at a frequency of <10−3  by two types of events: telomere-telomere fusion of the acentric chromosome with one of the other chromosomes or formation of a telocentric neocentromere. Such telomere-telomere fusions were not detected in any of the strains we studied here, although we have documented a telomere-telomere fusion event between Chr5 and an isochromosome (5L) in at least one drug-treated strain . Thus, while telomere-telomere fusions can occur in C. albicans, they do not appear to be a major mechanism of chromosome rescue following loss of wild-type centromere DNA.
In S. pombe, neocentromere formation was almost completely dependent upon the ability to form heterochromatin. The proportion of rescued cells carrying neocentromeres dropped from ~75% in wild-type cells to <10% in cells lacking Swi6 (an HP1 homolog), Clr4 (a histone H3 methyl transferase) or Dcr1 (the dicer homolog required for RNAi-dependent heterochromatin formation) . Clearly, C. albicans CEN5 exhibits epigenetic properties, such as neocentromere formation and movement, in the absence of classic pericentric heterochromatin. Accordingly, we were able to obtain cen5Δ::URA3 transformants in strains lacking the histone deacetylase ortholog of S. cerevisiae Sir2p. The efficiency of transformation and the proportion of Class A to Class B transformants were similar in sir2Δ/sir2Δ and isogenic wild-type (SIR2/SIR2) C. albicans strains (H.W., unpublished data). This result is consistent with the idea that canonical heterochromatin is not necessary for neocentromere formation. We propose that, rather than being required for kinetochore assembly per se, pericentric heterochromatin in larger regional centromeres may be required for the higher order centromere structure necessary to support the organization of multiple microtubule attachment sites per centromere. Because C. albicans has only one microtubule attachment per centromere/kinetochore, this type of centromere structure may be dispensable.
The neocentromeres formed in this study had two common features: large intergenic regions and repeated DNA (Figure 8C&D). Similarly, in organisms with larger regional centromeres, centromeres are positioned in ‘gene deserts’. Interestingly, 14 newly evolved centromeres in primate species formed in regions significantly devoid of genes in other primate genomes. . Furthermore, fine structure analysis (at the level of 10–20 kb regions within the ~2 Mb centromeric domians) of rice centromeres indicates that the very few genes found within a centromeric domain were located within subdomains associated with histone H3 rather than with CENP-A/CEN-H3 histones, suggesting that centromeres evolve from gene-poor regions ,. Here we found that association with CENP-ACse4p silences URA3 in C. albicans (Figures 4 & 7). Furthermore, active transcription through centromeric DNA inactivates kinetochore function in S. cerevisiae  as well as on a human minichromosome . In S. pombe, CENP-ACnp1 silences ura4+ in the centromeric central core in a concentration-dependent manner ,. We propose that the incompatibility of CENP-A nucleosomes and active transcription is a general feature of centromeres.
The second structural feature of neocentromeres is proximity to repeated DNA sequences. At seven of the eight native centromeres, inverted or direct repeats are found within a few kb of the CENP-A enriched core. Importantly, while a single LTR sequence is sometimes seen within the region, these repeats are different from pericentric repeats flanking larger regional centromeres in other organisms because they are often unique to the individual centromere. For example, the inverted repeat flanking CEN5 is composed of DNA found only on the two sides of the CEN5 central core. Similarly, the GIT1, GIT2 and GIT4 genes are found only in the neoCEN-2 region and orf.6676 and orf19.1124.2 are found only in the neoCEN-3 region (Figure 8D). We propose that a small amount of repeat DNA assists in the assembly of a functional kinetochore in a heterochromatin-independent manner.
The reversible nature of the change in URA3 expression status in Class A/proximal neoCEN transformants and the correlated change in CENP-ACse4p position relative to URA3 suggests that neocentromeres can move locally, at least in a subset of cells (1/1000) in the population. During selection for chromosome loss on sorbose medium, a poor carbon source, the general telocentric position of the neocentromere in YJB9907 was maintained, although it may have shifted a few kb distal to the telomere. Telocentric chromosomes are common in mice, and C. albicans CEN6 is telocentric, located ~50 kb from the Chr6R telomere, indicating that telomeres are one preferred site of centromere assembly, perhaps because they are generally devoid of essential genes and terminate in repetitive DNA.
In strain YJB9929 it appears that the neocentromere(s) were less stable. The presence of two different neocentromere positions YJB9929, which was originally isolated from a single colony, is also consistent with the idea that neocentromere positions are not fixed. Analysis of single colonies suggested that each cell most likely had only one neocentromere on Chr5 (Figure 8B). The smaller size of the neocentromeric DNA region associated with CENP-ACse4p may result in a smaller number of centromeric nucleosomes than at wild-type centromeres. Human neocentromeres are generally similar in size to normal centromere regions when the size of the constriction is measured by electron microscopy . However, levels of CENP-A at two human neocentromeres, measured with a GFP-CENP-A, are ~1/3 the level seen at most other centromeres , which was proposed to be due to less efficient loading of CENP-A at human neocentromeres . We propose that, in C. albicans, the neocentromeres remained functional despite the smaller amount of CENP-A because only one CENP-A nucleosome is required to attach to a single microtubule on each sister chromatid .
In contrast to the stable neocentromere in YJB9907s, in two independent YJB9929s strains obtained by sorbose selection for loss of one Chr5 homolog and reduplication of the remaining copy, the neocentromere moved to an entirely new position (neoCEN-4). No CENP-ACse4p was detected at the neoCEN-4 locus in any of the YJB9907 isolates or in any of the YJB9929 isolates prior to sorbose selection. Thus, upon sorbose selection in YJB9929 a neocentromere formed ~40 kb from neoCEN-2 on the same chromosome arm or ~640 kb from neoCEN-3 to the other chromosome arm.
We do not know if neocentromeres are repositioned by sliding along the DNA or by disassembling and reassembling at a new location. We propose that proximal neoCENs reposition by sliding locally (1–2 kb at URA3) because they tend to remain close by, resulting in reversible silencing when a neocentromere is within a few kb of URA3 and not when it is >200 kb away. Furthermore, similar local movements occur at distal neoCENs as evidenced by the slight shift of the neoCEN-1 peak. In contrast, because CENP-A is normally removed from non-centromeric DNA regions , it is tempting to speculate that an assembly/disassembly mechanism (Henikoff and Dalal 2005) contributes to distal neoCEN formation as well as to the movement of CENP-A from neoCEN-3 to neoCEN-4 during sorbose selection of strain YJB9929.
C. albicans centromeres are unique in that neocentromere formation at multiple positions is efficiently induced by deletion of a centromere. As in humans and Drosophila, chromosome breakage and healing (mediated by homologous recombination between URA3 and the CEN5 flanking DNA) accompanies neocentromere formation events in C. albicans cen5Δ transformants. This suggests that, while similar mechanisms operate in organisms that have pericentric heterochromatin, the process is simpler and much more efficient in C. albicans because the complexity of heterochromatin does not come into play.
Neocentromeres can form close to the site of the original centromere (as in Drosophila ), at distal loci with no sequence conservation (as in humans (reviewed in ) or near a telomere (as in S. pombe )). In humans, there are a small number of cases in which neocentromeres appear at ectopic positions and are not associated with any obvious chromosome rearrangement (reviewed in ,). While the formation of neocentromeres in C. albicans followed deletion of the wild-type centromere, we did not detect new aneuploidies or translocations in any of these strains. This is especially interesting in light of the high levels of chromosome rearrangements that can occur in C. albicans strains . Another unique aspect of C. albicans centromeres is that the movement of proximal neoCENs can be monitored by selection and counter-selection for URA3. This provides an extraordinarily powerful tool for direct studies of the mechanisms of local neocentromere movement and demonstrates that, while rare, short range neocentromere movement (several kb) is more frequent than movement over much longer distances.
There is much that we do not understand about regional centromeres. In part, this is because S. cerevisiae point centromeres cannot be used to study epigenetic phenomena, such as neocentromere formation and movement, that occur only at regional centromeres. The small size and simplicity of C. albicans regional centromeres greatly facilitates studies of the core functions of regional centromeres without the complications associated with long, complex repeat tracts and pericentric heterochromatin. In this initial study, we found that C. albicans centromeres form neocentromeres, despite having no pericentric heterochromatin. Furthermore, neocentromeres can form at multiple chromosomal loci including loci close to the deleted centromere, telomeric loci and other distal loci on both chromosome arms. Finally, neocentromeres can move over short or long distances to new loci and can silence a gene associated with it. Additional work will be needed to identify the environmental and genetic conditions that influence the rate of formation as well as the structural features that modulate the location and movement of neocentromeres.
Strains are listed in Table S1 and were constructed using PCR-mediated gene deletion with 70 nucleotides of homology to genomic DNA with gene disruption primers listed in Table S2 and a lithium acetate protocol . CEN5 was deleted by amplification of URA3 from pGEM-URA3 , which was inserted at CEN5 in both orientations. As an alternate approach, CEN5 was replaced with NAT1 from plasmid pMG2120 using primers 3161 and 3162. Strain YJB9779 (5L-URA3-Tel) was constructed by transforming strain YJB7617 with URA3-TEL amplified from pMG2192 using primers 2249 and 1489 to insert it 360 bp to the right of the IR (37 bp centromere proximal to the orf19.4219 coding sequence). pMG2192 was constructed by digesting pMM100  with Not1/Xho1 and 1000 bp CA7 telomere repeat was ligated to Xho1/Not1 cut pYPB1-ADH1 . Strain YJB9858 (TEL-NAT1-5R) was constructed by transforming YJB7617 with TEL-NAT1 amplified from pMG2194 using a forward primer (2313) that produces a fragment that can insert 1 kb to the left of the IR (660 bp centromere proximal to the orf19.3161 coding sequence) and a reverse primer (1489) with homology to telomere sequence in the plamid. pMG2194 was constructed by digesting pMM100  with ApaI and the 900 bp fragment containing the telomeric repeat was cloned into pMG2171 . C. albicans transformants were isolated after incubation on SDC-uri or YPAD+Nat at 30°C.
Transformation frequencies and the proportion of bona fide transformants was similar for cen5Δ and control strains. The frequency of insertion into the long and short CEN5 homologs was also similar when either earlier or later appearing transformants were analyzed. For example, for a cen5Δ::URA3 transformation, of the 61 transformants, 8 were correct insertions and 4 deleted CEN5 long and 4 deleted CEN5 short. Transformants were screened for those with bona fide insertions by PCR using primers (Table S2) flanking the insertion site together with primers within the inserted DNA as illustrated in Figure S1A. The CEN5 homolog that was disrupted with URA3 was identified by Southern analysis of conventional and CHEF karyotype gels using methods previously described . Probes were amplified with primers listed in Table S2.
C. albicans karyotypes were analyzed by CHEF gel electrophoresis as described previously  using conditions that optimize visualization of chromosomes 2–7. Comparative genome hybridization (CGH) was performed as described  and was plotted to the C. albicans genetic map using an updated version of the Chromosome_Map , based on Assembly 21 coordinates ,.
DNA was transferred from agarose gels to Magnacharge nylon membranes (GE Osmonics, Minnetonka, MN) . Membranes were probed overnight at 42°C and detected with anti-Digoxigenin-Alkaline Phosphatase and CDPstar essentially as described . Probes were prepared by PCR amplification using DIG-labelled nucleotides according to manufacturer's instructions (Roche, Indianapolis, IN).
Strains carrying cen5Δ::URA3 were grown on sorbose medium without added uridine for 7–9 days. Colonies that appeared were streaked to YPAD media and large colonies that appeared early were then streaked to SDC-uri medium to ensure that they had retained the URA3 marker. PCR analysis was performed to identify derivative strains in which the cen5Δ::URA3 fragment was retained, the wild-type CEN5 was no longer detectable, and that only one of the two MTL loci on Chr5 was retained, using primers listed in Table S2.
Growth rates were determined using overnight stationary phase cultures diluted 1105 in YPAD media covered with mineral oil in a 96-well plate (Corning, NY). Cultures were incubated in a microplate spectrophotometer (Sunrise, Tecan, San José, CA) at 30°C with constant linear shaking. OD readings were taken every 15 min. Doubling time was calculated based on the exponential constant of the fitted exponential curve of each well using software kindly provided by Sven Bergmann and Gil Hornung.
Mitotic stability was determined by comparing the proportion of cells containing the relevant marker (URA3 or NAT1) relative to the total number of cells in the population following growth under conditions selective for the marker . Fluctuation analysis of loss rates was performed as described  using the method of the median . Briefly, strains were streaked for single colonies and grown on SDC-uri for 2 days at 30°. Twenty independent colonies per strain were inoculated into 5 ml liquid non-selective medium (YPAD) and grown overnight at 30°C with shaking. Cultures were harvested by centrifugation and washed twice in 1 ml of sterile water. Appropriate dilutions were plated onto nonselective YPAD for total cell counts and selective media (SD+FOA) for selection of Ura- colonies. Plates were incubated at 30°C for 2–3 days, and colony counts were used to calculate the rate of FOAR/cell division . To distinguish loss of the URA3 marker from silencing, we analyzed FOA resistant colonies identified in loss rate fluctuation analyses done with strain YJB10064 (his1::NAT1/HIS1 ~38 kb from the Chr5R telomere) in which either CEN5 or MYO1 had been replaced with URA3. The resulting strains [YJB10169 (myo1Δ::URA3), YJB10233 (cen5Δ::URA3 Class A), and YJB10234(cen5Δ::URA3 Class B)] were replica-plated to SDC-his, SDC-uri, YPAD+Nat, and YPAD. FOAR colonies that re-grew when replica plated to SDC-uri were candidates for URA3 silencing. Images of the SDC-his, YPAD+Nat, and YPAD replica plates were overlaid in order to classify colonies as NatRHis+, NatSHis+, or NatRHis−, using the YPAD replica plate as a control for colony transfer. From each colony class obtained per strain, five colonies were analyzed by PCR amplification of the MTL loci using primers 1193, 1194, 1195 and 1197 (Table S2).
Rabbit polyclonal anti-CENP-ACse4p antibodies were raised against an N-terminal peptide (amino acids 1–18 (Ac-MARLSGQSSGRQTGQGTSC-amide)) of Ca CENP-ACse4p and affinity purified (Quality Controlled Biochemicals; Hopkinton, MA) following the protocol of Sanyal and co-workers .
ChIP was performed using extraction protocols modified from Weber et al.  and Carbon and Co-workers . Overnight cultures were diluted to an OD600 of ~0.3 and harvested at an OD600 of ~1.0. Cells were fixed in 1% formaldehyde for 15 min at 30°C and then treated with zymolyase (0.2 mg/ml+0.5 mM AEBSF) until 80–90% of cells were spheroplasts. Reactions were stopped with 1.2 M Sorbitol, 1 mM MgCl2, 20 mM PIPES pH 6.8, and sonicated to yield DNA fragments of 500–600 bp. Input lysate was collected after insoluble matter was precipitated twice by centrifugation. The lysate was incubated at 4°C for ~16 h with or without 4 µg/ml anti-CENP-ACse4p antibody. Antibody and associated DNA was incubated with Protein A-agarose beads at 4°C for ≥3 hrs, washed several times and then eluted twice at 65°C. Crosslinks were reversed overnight at 65°C followed by RNase A and proteinase K treatments. Proteins were removed by phenol-chloroform/isoamyl alcohol/chloroform extraction. Samples were ethanol precipitated, washed in 70% ethanol and resuspended in 80 ml TE.
Each Class A ChIP experiment was performed with the same isolate grown in SDC-uri and in SD+FOA. ChIP experiments were repeated with 4 different Class A strains (YJB9909, YJB9915, YJB9916 and YJB9926) analyzing 3 independent colonies from YJB9909 and YJB9915 and one colony each from strains YJB9916 and YJB9926. Two independent colonies from each of two different Class B strains (YJB9907 and YJB9861) also were analyzed. Contiguous fragments spanning ~15 kb across CEN5 were amplified using primer pairs listed in Table S2. Dilution PCRs were used to ensure that the amplification of each PCR product was in the linear range. PCR products were run on an 1.4% agarose gel and band intensities were quantified using ImageJ software (NIH) with a background correction macro. Intensities were calculated (+Ab/lysate) and normalized to the mean intensity of CEN5 central core PCR products in all strains except cen5Δ/cen5Δ strains, in which case it was normalized to the mean intensity of CEN4 central core products. “No-antibody” samples were run in parallel to ensure quality of the samples and reactions (e.g., Figure 6). Correlation coefficients for groups of samples were performed using the Excell ‘Correlation’ function .
Chromatin lysates and samples immunoprecipiated with CENP-ACse4p antibody (as described above) were processed for massively parallel high throughput sequencing using the Illumina Genome Analyzer Classic following manufacturer's instructions. Samples were nebulized to an average ~200 bp fragment size. The ends were repaired and an “A” base was added to the 3′ ends. Illumina adapter oligos were ligated to the fragments and these were purified on a 2.0% agarose gel by excising a region of the gel corresponding to 200 base pairs. After purification using a Qiagen Gel Extraction kit,(Qiagen, Valencia, CA), the adapter modified DNA fragments were enriched by PCR. The enriched fragments were purified using a Minelute PCR purification kit from Qiagen and quantified using the Quant-iT dsDNA HS Assay Kit from Invitrogen (Carlsbad, CA). The samples were then diluted to give a final concentration of 2 pM and cluster generation was performed on the Illumina Cluster Station (Illumina, San Diego, CA) following manufacturer's instructions. The resulting flow cell was sequenced on the Illumina GA Classic for 36 cycles.
The sequences were extracted from .tiff image files using Firecrest and Bustard tools of the Illumina Genome Analyzer pipeline. These sequence reads were aligned to the human genome as reference sequence (NCBI v36.49 from ensembl) using Illumina's ELAND tool. Both 36 bp and 25 bp reads were used, along with unmasked and repeat-masked  human genome. Peaks in the read coverage from uniquely aligned reads were identified using the FindPeaks tool . Release version 3.1 of the tool was used with default parameters for binding site search, except the values for subpeaks as 0.2 and trim as 0.2. For non-overlapping sliding window bins of chromosome 5 data, we calculated the ratio of reads mapped (aligned) in the bin, to the total number of reads mapped on the chromosome. The difference of this ratio in the sample compared to control was calculated to identify enriched bins along the chromosome for that sample.
RNA was extracted from cells grown to log phase in SDC-uri or SD+FOA using the Epicentre MasterPure yeast RNA purification kit (Epicentre Biotechnologies, Madison, WI) according to manufacturer's instructions. cDNA was prepared with the BioRad iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA) with a combination of random hexamers and oligo (dT) primers. Real-time PCR was performed using the Roche LightCycler FastStart DNA MasterPLUS SYBR Green I kit (Roche Applied Science, Indianapolis, IN). Reactions were run on the Eppendorf Mastercycler ep realplex machine (Eppendorf, Westbury, NY). Primers used are listed in Table S2. A no RT negative control and melt curve analysis were performed on each run to ensure that no contaminating DNA or second products were amplified. Duplicate wells and 3 technical repeats were performed on cDNA from cen5Δ::URA3 Class A strains YJB9909 and YJB9916. ΔCt values were determined from the mean of the results of all technical and biological replicates (Figure 4C). Error bars show standard error of the mean (SEM).
Analysis of cen5Δ::URA3 transformants. A. PCR analysis of cen5Δ::URA3 transformants. Location of primer sets that amplify left (L) and right (R) borders of wild-type CEN5 and cen5Δ::URA3 alleles. Left and Right reactions each contained 3 primers (A (2002), B (1961), C (945) or X (1915),Y (1901), Z(944)). Primer sequences are listed in Table S2. Expected amplified fragment sizes from wild-type (L2, R2) and from the cen5Δ::URA3 allele (L1, R1) are indicated. Correct cen5Δ::URA3 transformants contain all four PCR products. B. PCR analysis of cen5Δ::NAT1 transformants. Location of primer sets that amplify left (L) and right (R) borders of wild-type CEN5 and cen5Δ::NAT1 alleles. Lanes 1, primers A+B; lanes 2, primers A+W; lanes 3, primers X+Y; lanes 4, primers V+X; lanes 5, primers A+B+W; and lanes 6, primers V+X+Y. Wild-type strain BWP17 was used as a negative control. C. Southern analysis of cen5Δ::NAT1 transformants. As in Figure 1C, DNA from cen5Δ::NAT1 (lane 1, YJB10805; lane 2, YJB10828) was digested with EcoRV and SacII, separated by agarose gel electrophoresis and probed for NAT1. The diagram indicates the position of restriction sites, expected sizes of restriction fragments, and probe locations. NAT1 was inserted on the short homolog in one strain (YJB10828), in the long homolog in the other (YJB10805). D. CHEF gel analysis of cen5Δ::NAT1 strains reveals no major karyotype alterations. Lane 1, cen5Δ::NAT1 (YJB10805); lane 2, cen5Δ::NAT1 (YJB10828). E. Growth curve analysis of strains homozygous for chromosome 5. Wild-type (RM10, black line) and sorbose-derived strains lacking CEN5 (YJB9907-6s, green; and YJB9907-3s, pink) or with intact CEN5 (YJB9726, cyan) were grown at 30 C. Note that number of cells in the initial culture influences the time when logarithmic division begins and differs between experiments; slope of the logarithmic phase of all curves was not significantly different between experiments (see Table 1).
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Chromatin immunoprecipitation of CEN5 and cen5Δ::URA3 DNA. A. Chromatin immunoprecipitation of the wild type homolog of Class A long cen5Δ strains. Contiguous ChIP PCR of CC5 and the flanking IR displayed as in Figure 7A for cen5Δ::URA3 strains that disrupted the long allele of CEN5. Class A strains were grown in SDC-uri (white) and then selected on SD+FOA (Gray). Class B strains were grown only on SDC-uri. Class A long allele strains represent the average of 4 extracts (two from YJB9916 and two from YJB9926). Class B long allele extracts were made from YJB9907 (two extracts) and YJB9929 (one extract). B. Chromatin immunoprecipitation of the wild type homolog of Class A short cen5Δ strains. Contiguous ChIP PCR of CC5 and the flanking IR displayed as in Figure 7A for cen5Δ::URA3 strains that disrupted the short (B) allele of CEN5. Class A strains were grown in SDC-uri (white) and then selected on SD+FOA (Gray). Class B strains were grown only on SDC-uri. Class A short allele strains represent the average of 4 extracts (two from YJB9909 and two from YJB9915). Class B short allele extracts were made from YJB9861 (two extracts). As in wild-type cells, CENP-ACse4p specifically associates with CC5. The LTR within CC5 (gray triangle) is detected only in extracts from strains in which the short homolog was deleted and the long (LTR-containing) homolog is retained. C. Chromatin immunoprecipitation of Class A long allele under selection and counterselection for URA3 expression. CENP-ACse4p associates with the LTR to the left of CEN5 when cen5Δ::URA3 has replaced CEN5 in the long allele (YJB9916). A representative contiguous ChIP PCR of cen5Δ::URA3 displayed as in Figure 7B but including the episemon LTR present on the long allele of CEN5 which was replaced with URA3 in strain YJB9916. ChIP was performed with cells grown in SDC-uri (white) or selected on 5-FOA (black). Similar results were obtained with YJB9926. D. Chromatin immunoprecipitation of Class B cen5Δ strains. CENP-ACse4p does not associate with URA3 or flanking DNA when Class B transformants are grown in SDC-uri. Contiguous ChIP PCR of Class B cen5D::URA3 strains (YJB9861 (white) and YJB9907 (black)) grown in SDC-uri, normalized and displayed as in Figure 7A.
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Centromeric DNA regions associated with CENP-ACse4p. Chromosome regions detected by ChIP-SEQ analysis of wild-type strain RM10 (red line) are compared, for each chromosome (listed at left) to regions previously reported as C. albicans centromere DNA, based on ChIP with primers spanning ~1 kb regions of the genome  (blue line). Lines are drawn to scale, chromosome coordinates for the regions are indicated at the ends of the lines and the length of the DNA associated with CENP-ACse4p at each centromere (in bp) is indicated to the right. Chr5 coordinate numbers also correspond to the position of the ChIP-SEQ peak in Figure 8B.
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PCR analysis of neoCEN regions in Class B strains. Regions analyzed for strains indicated on left are within the peaks identified in Figure 8B. Chromosomal coordinates and positions of the PCR products relative to ORFs (black) arrows are diagramed and primer pairs used are listed in Table S1. A. Analysis of YJB9907 and its derivatives. Top, YJB9907; bottom, cen5Δ::URA3 homozygous strains YJB9907-3s (grey bars) and YJB9907-6s (black bars). No CEN5 DNA is present in these strains. B. Analysis of YJB9929 and its derivatives. Top panel, YJB9907; middle panel, single colony #4 from YJB9929 grown in rich medium; Bottom panel, cen5Δ::URA3 homozygous strains YJB9929-1s (grey bars) and YJB9929-2s (black bars). No CEN5 DNA is present in these strains.
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Strains used in this study.
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Primers used in this study.
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We thank Keely Dulmage, Merima Helic and Carsten Paulson for technical help. We thank Karen E. S. Tang for analysis of gene expression near centromeres and Anja Forche for analysis of repeat organization near centromeres. We thank Bruce Eckloff and Sumit Middha at the Mayo Genomics Research Center for performing and analyzing the massively parallel high throughput ChIP-SEQ samples. We thank Sven Bergmann and Gil Hornung for providing growth curve analysis software prior to publication. We thank Robin Allshire, Laura Burrack, Duncan Clarke, Shin Enomoto, Anja Forche, Cheryl Gale, David Kirkpatrick, Paul Megee, P.T. Magee, B.B. Magee, Alison Pidoux, Nathan Springer and Amit Vas for helpful discussions and/or critical reading of the manuscript.
The authors have declared that no competing interests exist.
This work was supported by a MinnCResT fellowship (NIH T32-DE007288) to CK, a Microbial and Plant Genomics Institute Integrative fellowship to AS, a 3 M Graduate fellowship award to KB, a Minnesota Partnership for Biotechnology and Medical Genomics grant to Dr. David Smith and NIH R01 AI062427 and R01 AI075096 awards to JB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.