Sequence Features and Transcriptional Stalling within Centromere DNA Promote Establishment of CENP-A Chromatin

Summary

The kinetochore directs the separation of chromosomes and is assembled at a special region of the chromosome—the centromere. DNA is wrapped around particles called nucleosomes, which contain histone proteins. The nucleosomes at centromeres are specialized, and contain the centromere-specific histone CENP-A. CENP-A nucleosomes form the platform upon which the kinetochore is built. Thus, CENP-A and centromere function go hand-in-hand. How the cell ensures that CENP-A is deposited at centromeres and not elsewhere is not well understood. We investigated the role that DNA sequence plays in defining centromere function in fission yeast. Our observations suggest that it is not the DNA sequence per se that is important for attracting CENP-A, but rather, the particular environment that the sequence creates. During transcription of centromeric DNA, RNA polymerase (RNAPII) appears to get stuck or stalled. Particular proteins—such as TFIIS and Ubp3—are known to help restart RNAPII so it can continue transcribing. We found that when cells lack Ubp3 or TFIIS, CENP-A becomes deposited on centromere sequences. We propose that persistent stalling of RNAPII on centromere DNA attracts factors that help deposit CENP-A. This study highlights the influence of DNA sequence in creating an attractive environment for CENP-A assembly.

Introduction

Centromeres are the chromosomal sites upon which kinetochores are assembled to ensure accurate segregation of sister chromatids into daughter cells. Most kinetochores are built upon a specialized type of chromatin in which canonical histone H3 is replaced by the histone variant CENP-A. Although the centromere-kinetochore complex performs conserved essential functions, and kinetochore proteins are generally conserved [1], centromeric DNA is not conserved, even between related species, and a huge variety of centromere sequences and structures exist [2–5]. The point centromeres of budding yeast consist of 125 bp of DNA and utilize an essential centromere-specific DNA binding protein [6]. At the other extreme, the nematode, Caenorhabditis elegans, has holocentric centromeres, in which kinetochore proteins assemble at multiple loci along each chromosome arm [7,8]. The majority of centromeres studied to date are regional. Centromeres in various plant and animal species are composed of arrays of different types of satellite, repetitive sequences and transposable elements, for instance, human centromeres encompass several megabases of tandem repetitive arrays of alpha-satellite sequence [9–11]. Fission yeast centromeres represent another type of regional centromere, in which a unique central core of 4–7 kb is flanked by inverted repeat elements and blocks of relatively large repeat units, resulting in centromeres of 40–120 kb [12]. Even the centromeres of different chromosomes in individual species are not necessarily homologous; each Candida albicans centromere has a unique central core, whilst chicken and potato each utilize both repeat-rich and unique sequence centromeres [13–15]. Thus, functional centromeres are assembled on diverse types of sequences in different organisms and it remains unknown if there is a universal fundamental property that defines centromeric sequences.

Abundant evidence indicates that centromeres are epigenetically regulated [16]. Although rare, neocentromeres have been observed in many species, forming on DNA sequences that do not normally possess centromere function and share no sequence homology with normal centromeres [17]. The best-characterized example in human is the neocentromere in 10q25 on the long arm of chromosome 10 that arose upon deletion of the centromere and loss of the entire alpha satellite array [18]. In S. pombe, neocentromeres form in close proximity to telomeres following the engineered deletion of a centromere [19]. Conversely, centromeres can be inactivated on dicentric human chromosomes despite the continued presence of alpha-satellite sequence at both centromeric loci [20]. In S. pombe one centromere on a dicentric chromosome can be inactivated by mechanisms such as heterochromatinisation or formation of a domain of histone hypoacetylation [21]. These and numerous other examples demonstrate that centromeric sequences are neither necessary nor sufficient for kinetochore assembly.

The histone H3 variant, CENP-A acts as the epigenetic mark that specifies centromere identity [22–24]. CENP-A is found only at active centromeres, including neocentromeres, and is absent at inactivated centromeres. The forced recruitment of CENP-A either by directly tethering CENP-A or its chaperone (HJURP) to a non-centromeric locus leads to the accumulation of CENP-A and kinetochore proteins at that location [24,25]. It is thought that continued deposition of CENP-A at centromere regions through cell and organism generations involves a self-propagation mechanism in which CENP-A chromatin, or features of the kinetochore which is assembled upon it, are recognized and attract additional CENP-A [26,27].

In most organisms there is no obligate coupling of sequence and CENP-A assembly, yet kinetochores are normally assembled upon particular centromeric sequences in any given species [4]. This suggests that centromeric sequences possess underlying properties that promote CENP-A incorporation. Alternatively, the preponderance of particular sequences at centromeres could be driven by properties of CENP-A chromatin or kinetochores themselves [28]. However, centromeric DNA allows the de novo assembly of functional centromeres following its introduction into cells in many organisms. Alpha satellite arrays are able to direct the de novo assembly of centromeres when introduced into certain cell lines as naked DNA [29,30]. De novo assembly of centromeres also occurs when centromeric DNA from S. pombe is introduced into cells. However, de novo establishment does not seem to be a universal property: despite promiscuous neocentromere formation in C. albicans, transformation with bone fide centromeric sequences does not result in kinetochore assembly[13]. At the other extreme, many sequences introduced into the holocentric organism C. elegans appear able to assemble CENP-A chromatin [31,32]. Thus, the relationship between centromeric sequence and the establishment and maintenance of CENP-A chromatin is enigmatic.

Transcription has received a lot of attention as a possible contributor to assembly of CENP-A chromatin. Transcripts emanating from centromeric regions have been detected in many organisms, including maize, human, rice, budding yeast, fission yeast and tammar wallaby [33–38]. Interfering with the chromatin status or transcriptional properties of centromeric repeats affects maintenance of CENP-A chromatin and segregation function on human artificial chromosomes (HACs) [39,40]. RNA Polymerase II (RNAPII) has been detected at mitotic mammalian centromeres where it may influence centromere function [38]. In fission yeast, transient H2B ubiquitylation may loosen centromeric chromatin to promote transcription and CENP-A^Cnp1 incorporation and defective reassembly of H3 chromatin behind elongating RNAPII aids CENP-A^Cnp1 incorporation [41,42]. However, although there are numerous tantalising hints that transcriptional activity contributes to centromere function or identity, much remains to be understood [33,38,43,44].

Here we investigate the contribution of DNA sequence to the establishment of CENP-A chromatin in fission yeast, an organism in which epigenetic mechanisms clearly influence centromere identity. Normally proximal heterochromatin is required to facilitate establishment of CENP-A^Cnp1 chromatin on centromere central domain sequences [45,46]. We show that this requirement can be bypassed by overexpression of CENP-A^Cnp1 and that central domain DNA is a preferred substrate for establishment of CENP-A^Cnp1 chromatin. We find that there is functional redundancy within the central domain but that sub-regions are non-equivalent in their ability to establish CENP-A^Cnp1 chromatin. Analysis of a 2 kb region capable of directing CENP-A^Cnp1 assembly indicates that it contains numerous transcriptional start sites, along with promoter elements, and that relatively high levels of RNAPII are recruited, despite low levels of transcripts produced, consistent with the presence of stalled RNAPII. Our observations suggest that redundant sequence features in the centromere central domain create a unique transcriptional environment that is permissive for CENP-A^Cnp1 establishment. Consistent with this, defective transcriptional elongation where stalled RNAPII is increased promotes the establishment of CENP-A^Cnp1 chromatin.

Results

Elevated CENP-A^Cnp1 levels bypass the requirement for heterochromatin in establishing CENP-A^Cnp1 chromatin

In wild-type fission yeast cells, de novo CENP-A^Cnp1 chromatin establishment on circular plasmid-based minichromosomes requires an outer repeat or tethered Clr4 histone H3K9 methyltransferase to form a block of heterochromatin in close proximity to central domain DNA from centromeres [45,46]. CENP-A^Cnp1 can also be deposited at other non-centromeric locations in the genome when it is overexpressed, however the level incorporated at these ectopic sites is much lower than that detected at natural centromeres [41,47]. To determine whether central domain DNA is a preferential substrate for the establishment of CENP-A^Cnp1 chromatin, plasmid pMcc2 bearing 8.5 kb of central domain from cen2 (imr2-cc2-imr2) sequence, but no heterochromatic outer repeat sequences, was transformed into cells expressing additional GFP-CENP-A^Cnp1 (Fig. 1A).

All strains used have 6 kb of cen2 central domain DNA replaced with 5.5 kb of cen1 central domain DNA (cc2Δ::cc1—Fig. 1A, S1 Fig.) so that only 2.5 kb of normal cen2 central domain DNA remains at this modified cen2 (imr2L, regions J, K, R; Fig. 1A). The resulting deletion of fragments L-Q from the cen2 central domain allows detailed and specific analysis of 6 kb of central domain DNA when borne by plasmid-based minichromosomes. Quantitative chromatin immunoprecipitation assays (qChIP) shows that CENP-A^Cnp1 chromatin does not assemble on regions L, M N, O or P when a plasmid (pMcc2) containing the 8.5 kb cc2 sequence, but lacking heterochromatin, was transformed into wild-type cells [45]. However, when pMcc2 was transformed into cells over-expressing CENP-A^Cnp1 (hi-CENP-A^Cnp1; ∼15 fold more than wild-type cells [41]), CENP-A^Cnp1 and the kinetochore proteins CENP-C^Cnp3 and CENP-K^Sim4 were easily detected over the central domain of pMcc2 by qChIP (Fig. 1B-E). Importantly, these centromeric proteins were enriched on centromeric DNA but not on the plasmid backbone, indicating that CENP-A^Cnp1 chromatin assembles specifically on central domain DNA from centromeres (Fig. 1). The relative level of enrichment of CENP-A^Cnp1 and the other kinetochore proteins on different parts of pMcc2 suggests all proteins are distributed uniformly across this plasmid-borne central domain (Fig. 1B-D). Furthermore, the levels of histone H3 associated with the L-P regions of pMcc2 were reduced in cells expressing additional CENP-A^Cnp1 compared to control cells (Fig. 1E). We conclude that H3 chromatin is normally assembled on central domain DNA on pMcc2 in wild-type cells but CENP-A^Cnp1 chromatin assembles instead when pMcc2 is placed in hi-CENP-A^Cnp1 cells.

CENP-A^Cnp1 can assemble on pre-chromatinised substrates and is trans-generationally inherited

To determine whether CENP-A^Cnp1 can become established on plasmids that are already assembled in chromatin, the pMcc2 plasmid was transformed into cells expressing wt-CENP-A^Cnp1 levels and subsequently crossed with hi-CENP-A^Cnp1 cells. qChIP analyses indicate that CENP-A^Cnp1 is initially absent from pMcc2 in the wt-CENP-A^Cnp1 parental strain and then becomes assembled in CENP-A^Cnp1 chromatin when transferred into the hi-CENP-A^Cnp1 environment, indicating that plasmid-borne cc2 initially assembled in normal (H3) chromatin can be converted to CENP-A^Cnp1 chromatin (Fig. 2A).

In addition, a copy of cc2 (8.5 kb) was inserted on the arm of the 530 kb Ch16 linear minichromosome which carries a complete cen3 [48] (Ch16-cc2; Fig. 2B). When the expression of additional GFP-CENP-A^Cnp1 was repressed (0h+T), no CENP-A^Cnp1 was detected on cc2. However, when GFP-CENP-A^Cnp1 was induced (48h-T) both CENP-A^Cnp1 and CENP-C^Cnp3 were detected on cc2 (Fig. 2B). Thus, cc2 borne on a linear minichromosome can be converted from a pre-chromatinised state to a CENP-A^Cnp1 state. Moreover, colony colour assays indicate that hi-CENP-A^Cnp1 expression induces increased loss of Ch16-cc2, which is consistent with a second functional kinetochore being formed at cc2 on Ch16 (Fig. 2C). Thus, Ch16-cc2 behaves as an inducible dicentric chromosome controlled by CENP-A^Cnp1 levels.

It is possible that high levels of CENP-A^Cnp1 are continuously required to maintain CENP-A^Cnp1 on pMcc2, or alternatively, once established, CENP-A^Cnp1 and kinetochore proteins may persist even when CENP-A^Cnp1 is returned to wild-type levels (wt-CENP-A^Cnp1). To investigate the maintenance of CENP-A^Cnp1 chromatin, pMcc2 was first transformed into hi-CENP-A^Cnp1 cells to allow the assembly of centromeric chromatin and subsequently these pMcc2-containing cells were crossed with wt-CENP-A^Cnp1 cells to transfer the pMcc2 plasmid into cells expressing wild-type CENP-A^Cnp1 levels. ChIP analyses show that CENP-A^Cnp1 persisted on the pMcc2 in this wild-type background (Fig. 2D). Western analysis of extracts from Parental, F1 and F2 cells confirmed that GFP-CENP-A^Cnp1 was lost in F1 and F2 cells (Fig. 2D). Thus, CENP-A^Cnp1 chromatin behaves as a true epigenetic entity in that once established it carries its own efficient propagation mechanism, persisting even though the original stimulus has been removed. More remarkably, this CENP-A^Cnp1 chromatin is maintained through 2 rounds of meiosis and at least 50 mitotic divisions. Thus central domain sequences are particularly receptive to the establishment and maintenance of CENP-A^Cnp1 chromatin.

Central domain sequence and length affect de novo CENP-A^Cnp1 deposition

To determine if specific regions from the central domain of cen2 are required to establish CENP-A^Cnp1 chromatin, plasmids bearing different sub-fragments from cc2 were transformed into wt-CENP-A^Cnp1 or hi-CENP-A^Cnp1 cells (Fig. 3). We used an unbiased approach to divide the 8.5 kb cc2 into 1 kb regions (J-R). Deletion of 1 kb from the centre of cc2 (N) does not affect CENP-A^Cnp1 establishment (pΔN; Fig. 3A, compare with pMcc2, Fig. 1B). Notably, CENP-A^Cnp1 incorporation on a plasmid carrying identical centromeric DNA as pΔN but with the right half (O-R) inverted relative to the native sequence, was less efficient (pΔN-rev; Fig. 3B). Thus, the relative orientation of central domain sequences within cc2 influences the degree of CENP-A^Cnp1 deposition; directionality or the juxtaposition of certain sequences may be important for promoting CENP-A^Cnp1 incorporation. The central CENP-A^Cnp1 domain at endogenous fission yeast centromeres is composed of inverted imr repeats that flank the central core. ChIP analyses demonstrated that, in a plasmid-based establishment assay, the imr repeats are dispensable for de novo CENP-A^Cnp1 incorporation on the remaining central domain sequences (pΔimr; Fig. 3C).

Deletion of additional regions (LMN) of cc2 markedly decreased the efficiency of CENP-A^Cnp1 incorporation relative to pMcc2 and pΔN (Fig. 3D; compare Fig. 1B, Fig. 3A), suggesting that either the LM region is critical for promoting CENP-A^Cnp1 incorporation or that the overall reduced centromeric DNA length diminishes CENP-A^Cnp1 deposition (Fig. 3D). However, further investigation using plasmids bearing smaller cc2 fragments suggests that the specific sequences present have a more significant influence on CENP-A^Cnp1 deposition than the overall length of cc2 DNA present (Fig. 3E,F). For example, pΔJM and pΔNR differ by only 500 bp, however, pΔJM incorporated substantially more CENP-A^Cnp1 than pΔNR (Fig. 3F). We conclude that specific sequences from the central domain of fission yeast centromeres, combined with their overall length, promote the efficient de novo assembly of CENP-A^Cnp1 chromatin.

A 2 kb region of centromeric DNA is sufficient to direct de novo CENP-A^Cnp1 chromatin assembly

It is possible that shorter fragments of centromere DNA from within the central domain can actively promote CENP-A^Cnp1 assembly but that because longer total lengths are required to stabilise incorporated CENP-A^Cnp1 the activity of shorter fragments cannot be detected. To address this possibility we selected two distinct sequences from the central domain of cen2 for analyses. The 2 kb OP region was present on all the pMcc2 derivatives with which we detected significant CENP-A^Cnp1 incorporation following transformation into hi-CENP-A^Cnp1 cells (Fig. 3). In addition, ChIP-seq analysis indicates particularly high CENP-A^Cnp1 nucleosome occupancy within OP at endogenous cen2 [49 and Fig. 4A]. In contrast, the 2 kb LM region appears to be dispensable for de novo CENP-A^Cnp1 assembly on pMcc2 derived plasmids and exhibits low CENP-A^Cnp1 nucleosome occupancy (Fig. 3, Fig. 4A).

Initial tests showed that neither OP (p1xOP) nor LM (p1xLM) sequences alone were capable of inducing significant de novo CENP-A^Cnp1 incorporation when introduced into hi-CENP-A^Cnp1 cells (Fig. 4B). This finding is consistent with a minimal length of central domain DNA being required for stable CENP-A^Cnp1 chromatin assembly and retention. To satisfy this apparent length requirement, the OP and the LM fragments were multimerised as tandem repeats to create 3xOP and 3xLM (p3xOP, p3xLM; Fig. 4C). Remarkably, when transformed into hi-CENP-A^Cnp1 cells no CENP-A^Cnp1 was detectable on p3xOP whereas p3xLM allowed a reasonable level of CENP-A^Cnp1 incorporation (Fig. 4C). This suggests that in isolation the OP region is unable to promote CENP-A^Cnp1 deposition even though in the context of an entire central domain it normally accepts CENP-A^Cnp1 and ends up with high CENP-A^Cnp1 nucleosome occupancy (Fig. 4A). We note that the removal of the LM region from the central domain of pMcc2 derived plasmids greatly reduced the level of CENP-A^Cnp1 incorporated on OP (compare pΔN Fig. 3A with pΔLMN Fig. 3D). Thus, in contrast to OP, the LM region appears to have the ability to induce CENP-A^Cnp1 deposition. To directly test this possibility, a single copy of LM was placed adjacent to two tandem copies of OP (pLM-2xOP) and transformed into hi-CENP-A^Cnp1 cells. High levels of CENP-A^Cnp1 were detected on both the LM and OP regions of pLM-2xOP (Fig. 4D), thus the LM region has an innate ability to stimulate CENP-A^Cnp1 deposition on the OP region. A different arrangement of the same sequences (pOPLMOP) also attracted CENP-A^Cnp1 in hi-CENP-A^Cnp1 cells (S2 Fig.). These analyses indicate that the 2 kb LM sequence contains all the features that are required to promote and accept CENP-A^Cnp1 assembly, and thus LM defines a 2 kb region of S. pombe centromeric sequence that allows the de novo assembly of CENP-A^Cnp1 chromatin.

The 2 kb LM element is sufficient to form functional centromeres

Plasmids bearing an entire central core domain flanked by outer heterochromatin repeats assemble functional centromeres when transformed into wild-type cells [45,50]. To determine if the 2 kb LM region imparts centromere function, a plasmid carrying the 3xLM tandem repeat adjacent to a 5 kb outer repeat heterochromatin forming element (pH-3xLM) was transformed into wild-type cells expressing CENP-A^Cnp1 at normal levels (Fig. 5A). The establishment of functional centromeres in the resulting transformants was monitored by an ade6-based colony colour sectoring assay [51]. Minichromosomes carrying full-length cc2 and 5 kb of outer repeat heterochromatin were able to establish functional centromeres upon transformation (Fig. 5B and S2 Fig.). pH-3xLM and pH-LM-2xOP transformants also established functional centromeres, but at lower frequency than pH-cc2 (Fig. 5B and S2 Fig.). Differences in the ability of various constructs to form functional centromeres may reflect the particular configuration of sequences in individual minichromosomes. In contrast, pH-3xOP (3xOP flanked by heterochromatin) was unable to establish functional centromeres. Thus the LM sequence in a 3x tandem array, flanked by heterochromatin, is sufficient to form functional centromeres. ChIP analyses confirmed that kinetochores were assembled on pH-3xLM since CENP-A^Cnp1 and the kinetochore proteins CENP-C^Cnp3 and CENP-K^Sim4 were enriched over the LM sequences at levels comparable to endogenous centromeres (Fig. 5C). We conclude that the LM sequence within pH-3xLM not only promotes incorporation of CENP-A^Cnp1 into chromatin but also supports the assembly of a functional centromere.

Features within centromeric central domain DNA are required to promote CENP-A^Cnp1 chromatin establishment

Nucleosome occupancy is known to be influenced by a combination of DNA sequence and the action of chromatin remodelers [52]. Primary DNA sequence itself influences nucleosome occupancy since DNA sequences with a high GC content and periodic dinucleotide patterns, that are devoid of poly(dA:dT) sequences, are strongly favored for nucleosome occupancy because of biophysical constraints that allow such sequences to wrap more easily around nucleosomes. These constraints have led to the development of algorithms that predict the probability of nucleosome occupancy [53,54]. In common with centromeres of many organisms, fission yeast centromeric DNA is AT-rich with a higher frequency of poly(dA:dT) tracts. It is therefore possible that H3 nucleosomes have a lower affinity for such sequences whereas CENP-A nucleosomes may be unperturbed by such AT rich DNA. To examine the underlying sequence specificity within centromeric DNA that favours the deposition of CENP-A^Cnp1 nucleosomes, the sequence of LM DNA was altered by randomisation using a 5 bp sliding window throughout the entire 2 kb element. This generated a synthetic LM sequence (SynR-LM) that is 62.6% identical to the wild-type LM sequence, retaining the same AT content and dinucleotide periodicity, and thus the same predicted nucleosome occupancy as the wild-type LM element (Fig. 6A) [55].

Synthesised SynR-LM assembled as a 3xSynR-LM tandem array was placed in the same plasmid backbone as p3xLM to generate p3xSynR-LM. p3xSynR-LM was transformed into wt-CENP-A^Cnp1 and hi-CENP-A^Cnp1 cells. In contrast to p3xLM, CENP-A^Cnp1 was not detectable on the shuffled LM sequence of pSynR-LM (Fig. 6B, compare with Fig. 4B). These analyses demonstrate that preservation of nucleotide composition (AT-content, dinucleotide periodicity) and predicted nucleosome occupancy within an altered centromeric DNA is not sufficient to allow CENP-A^Cnp1 deposition. The fact that the natural 2 kb LM sequence is active whereas the artificial SynR-LM is inactive reveals that the primary sequence of wild-type centromeric LM DNA encodes properties that somehow allow its recognition in vivo and consequent de novo assembly of CENP-A^Cnp1 chromatin.

Centromeric DNA produces an unusual transcriptional environment

Upon transformation into cells innate features within 3xLM sequence must allow it to be either immediately assembled in CENP-A^Cnp1 chromatin, or, initially assembled in H3 chromatin with subsequent remodelling that exchanges canonical H3 for CENP-A^Cnp1. The process of transcription is obviously accompanied by chromatin remodelling and non-coding transcripts synthesised from within the central CENP-A^Cnp1 domains of fission yeast centromeres have been detected [37,42]. The transcription of central domain DNA might influence the assembly of CENP-A^Cnp1 chromatin. In cells expressing CENP-A^Cnp1 at wild-type levels, plasmid-borne central domain sequences are assembled in H3 rather than CENP-A^Cnp1 chromatin (Fig. 1E). Higher levels of RNAPII are detected on plasmid-borne central domain sequences (pMcc2) introduced into wild-type cells than when cc2 is assembled in CENP-A^Cnp1 chromatin on pMcc2 or at endogenous centromeres (Fig. 7A, S3 Fig.). Although relatively high levels of RNAPII associate with the pMcc2 central domain when assembled as H3 chromatin in wild-type cells (10–30% of levels at act1⁺) (Fig. 1E, Fig. 7A), the level of transcripts emanating from the central domain is very low (<0.1% of act1⁺), even when analysed in exosome defective cells (dis3–54; Fig. 7B). Thus, although ample RNAPII is recruited to the central domain of pMcc2 few transcripts are generated, suggesting that transcriptional stalling occurs.

To map transcriptional start sites (TSSs) within the LM and OP regions, 5’ RACE was performed on RNA extracted from dis3–54 exosome mutant cells harbouring p3xLM or p3xOP (Fig. 7C, S4 Fig.). Many TSSs were identified within LM and OP, suggesting that these regions contain several promoters (Fig. 7C). 200 bp regions from both LM and OP were tested for their ability to drive production of β-galactosidase when placed upstream of a lacZ reporter in fission yeast and as shown in Fig. 7D, the regions displayed promoter activity. Mutated or inverted versions of promoter region M2 did not promote transcription of LacZ (S4 Fig.). Whilst most regions of LM and OP exhibit promoter activity that is lower than that of nmt81 control promoter, it is notable that region-O1 and region-P2 from OP have equivalent and 10-fold higher activity, respectively (Fig. 7D). It is possible that the higher promoter activity possessed by some regions of OP may affect its ability to establish CENP-A^Cnp1. We surmise that the central domain from cen2 is peppered with promoters that can drive the production of transcripts on both strands. Their relative arrangement along with the strength and pattern of transcription may affect CENP-A^Cnp1 incorporation.

CENP-A^Cnp1 establishment is enhanced in mutants that increase RNAPII stalling

The progression of RNAPII is impeded by obstacles such as nucleosomes, DNA damage, bound proteins and by sequences that are intrinsically difficult to transcribe, causing transcriptional pausing, stalling or arrest [56]. RNAPII-associated proteins ease the passage of RNAPII through such impediments, contributing to the processivity of the polymerase [57]. TFIIS facilitates transcriptional elongation of stalled/backtracked RNAPII by stimulating cleavage of nascent transcripts [58–60]. Upon stalling an elongating RNAPII becomes mono- then poly-ubiquitylated on the largest Rpb1 subunit. A rescue pathway involving de-ubiquitylation by the ubiquitin hydrolase Ubp3 is deployed to restart stalled RNAPII [56,61].

Our analyses suggest that the central domain chromatin landscape contains numerous promoters on both strands and multiple TSSs. In addition, long poly(dA:dT) tracts are likely to be an intrinsically problematic sequence for RNAPII transcription and present a barrier to RNAPII elongation [62,63]. We reasoned that mutants that are defective in the response to transcriptional stalling might influence the ability of the central domain to become assembled in CENP-A^Cnp1 chromatin. To test this possibility, wild-type and TFIIS (tfs1Δ) mutant cells expressing hi-CENP-A^Cnp1 were transformed with pMcc2. Surprisingly, slightly increased levels of CENP-A^Cnp1 were detected on pMcc2 in the tfs1Δ mutant compared to wild-type cells, suggesting that loss of TFSII promotes CENP-A^Cnp1 deposition (S5 Fig.). Consistent with this, even when pMcc2 was transformed into tfs1Δ cells expressing wt-CENP-A^Cnp1 levels, CENP-A^Cnp1 was detected on the pMcc2 central domain (Fig. 8A). In order to determine whether the effect on CENP-A^Cnp1 establishment was specific to tfs1Δ or a general consequence of increased RNAPII stalling, we also investigated if loss of the ubiquitin hydrolase Ubp3, which normally rescues arrested RNAPII, affects CENP-A^Cnp1 deposition. Strikingly, CENP-A^Cnp1 was detected at high levels on central domain sequences in ubp3Δ cells transformed with pMcc2. CENP-A^Cnp1 was also detected on p3xLM, but not p3xOP in ubp3Δ (Fig. 8B, S6 Fig.). CENP-C^Cnp3 and CENP-K^Sim4 centromere proteins were also significantly enriched on pMcc2 in ubp3Δ cells (S8 Fig.). These effects were not due to increased abundance of CENP-A^Cnp1 in tfs1Δ or ubp3Δ cells as protein levels were similar to wild-type cells (S7 Fig.). In fact, a reduction in CENP-A^Cnp1 and CENP-C^Cnp3 levels was detected at endogenous centromeres in ubp3Δ, but not tfs1Δ cells (S8 Fig.). Tfs1 and Ubp3 were previously reported to modulate RNAi-independent heterochromatin assembly [64]. To test whether the effect on CENP-A^Cnp1 establishment in tfs1Δ or ubp3Δ cells could be due to spurious assembly of heterochromatin on pMcc2, H3K9me2 ChIP was performed. The level of H3K9me2 on pMcc2 in tfs1Δand ubp3Δ was similar to that on a negative control locus, act1⁺, and assembly of CENP-A^Cnp1 on pMcc2 in these mutants was not dependent on the H3K9-methyltransferase Clr4 (S9 Fig.). Thus, CENP-A^Cnp1 assembly on pMcc2 in the absence of TFIIS or Ubp3 does not result from induction by ectopic heterochromatin.

If lack of TFIIS or Ubp3 hinders transcriptional elongation, an increased level of RNAPII would be expected on affected chromatin templates. Elevated levels of Rpb1/RNAPII were detected on the central domain of pMcc2 in tfs1Δ (TFIIS) and ubp3Δ cells (Fig. 8C). In addition, increased levels of the elongation-specific Phospho-Ser2 form of RNAPII were observed on the central domain of pMcc2 in ubp3Δ cells, suggestive of failure to efficiently clear stalled RNAPII (Fig. 8D). Thus, two mutants, which perturb the progress of RNAPII elongation complexes in different ways, lead to deposition of CENP-A^Cnp1. These observations suggest that altering the transcriptional properties of the central domain chromatin through increased RNAPII stalling creates an environment that is permissive for establishment of CENP-A^Cnp1 chromatin in place of H3 chromatin.

Discussion

It is thought that once established, CENP-A chromatin has the ability to be ‘self-propagating’, and through the recruitment of factors that are themselves involved in deposition of CENP-A, it ensures its own maintenance [16,17,23,24,26,65]. Epigenetic inheritance can be defined as the propagation of a state in the absence of the initial inducer of that state. In this study, the inducer—overexpression of CENP-A^Cnp1—causes an event that would not normally occur, the assembly of CENP-A^Cnp1 chromatin on episomal centromeric DNA (pMcc2). When CENP-A^Cnp1-assembled pMcc2 is crossed from hi-CENP-A^Cnp1 cells into wt-CENP-A^Cnp1 cells, CENP-A^Cnp1 is propagated in the absence of the initial inducer through many generations and through meiosis. These observations further strengthen the evidence that CENP-A behaves as a bona fide epigenetic entity [24].

It is clear that both epigenetic and genetic factors influence CENP-A assembly. We have investigated the role of DNA sequence in establishment of CENP-A chromatin in fission yeast, an organism where analysis is not confounded by repetitive arrays of short satellite sequences. CENP-A^Cnp1 is normally restricted to the central domain of centromeres where it forms the basis for the kinetochore. Central domain DNA is a preferred substrate for establishment of CENP-A^Cnp1 chromatin upon overexpression, whilst other genomic loci do not support accumulation of high levels of CENP-A^Cnp1 [47], and even vector DNA adjacent to the central domain is not a good substrate. Conditions and mechanisms that influence assembly of CENP-A^Cnp1 on naïve plasmid DNA are also able to convert pre-chromatinised cc2 present on episomal plasmids or linear minichromosomes. What makes central domain DNA a preferred site for CENP-A^Cnp1 assembly? The lack of homology between cc2 and cc1/cc3 sequences suggests that it is not a simple case of specific sequence that is critical [66–68]. Our analyses indicate that there is functional redundancy within the central domain and no one particular sequence is either necessary or sufficient for CENP-A^Cnp1 establishment, consistent with previous findings [50]. Despite this redundancy it appears that there are inherent distinctions between different regions of cc2. The 2 kb sub-regions, LM and OP, are functionally non-equivalent and consistently behaved differently when challenged to assemble CENP-A^Cnp1 chromatin. LM is competent to establish centromeric chromatin upon CENP-A^Cnp1 overexpression, contains sufficient information to make a functional centromere when placed next to heterochromatin (pH-3xLM), and assembles CENP-A^Cnp1 chromatin in cells lacking Ubp3. On the other hand, the OP region fails to become assembled in CENP-A^Cnp1 chromatin in all these situations, yet can accept CENP-A^Cnp1 when adjacent to one copy of LM, which apparently acts as an initiator. The ability of LM, but not OP, to substitute for full-length cc2 sequence indicates that not all sequences are equivalent and LM must contain all information necessary to make this region permissive for CENP-A^Cnp1 establishment. It is possible that the observed higher promoter activity observed in the OP region (Fig. 7D) prevents stabilisation of CENP-A^Cnp1 nucleosomes on this sequence.

In common with many organisms, the central domain of S. pombe centromeres is AT rich and this property might contribute to the propensity of centromeric DNA to attract CENP-A [5,68]. S. pombe central domain DNA has an AT content of 72% (genome average of 64%), as does the establishment competent LM sequence. However, other regions that alone fail to support CENP-A^Cnp1 establishment have a similar AT content, such as OP (71% AT) and intergenic regions (72% AT). Moreover, randomisation of the LM sequence resulted in SynR-LM that, even with identical nucleotide composition (72% AT), was incompetent for CENP-A^Cnp1 establishment. Thus, high AT content alone, even when it mimics natural nucleosome positioning predictions, is not a defining factor in CENP-A^Cnp1 assembly. Together our observations indicate that rather than there being a specific critical sequence, central domain sequences encode unique properties capable of triggering or promoting the establishment of CENP-A^Cnp1 chromatin.

Transcription-coupled remodelling is associated with the deposition of histone variants and could potentially contribute to the assembly of CENP-A chromatin [69,70]. However, the simple act of transcription cannot be sufficient to provide specificity to the deposition of CENP-A. Our observations suggest that the transcriptional landscape of the centromeric central domain is unusual: scattered promoters of various strengths resulting in pervasive low quality transcription and numerous TSSs on both strands, in conjunction with poly(dA:dT) tracts that are inherently difficult to transcribe are likely to cause collision between convergently transcribing RNAPIIs and pile-ups at difficult sequences [63,71]. The relatively high density of RNAPII on pMcc2 contrasts with very low levels of transcripts (Fig. 7), consistent with inefficient progress of transcription by RNAPII on cc2, and many stalled elongation complexes. In addition, long tracts of poly(dA:dT) are known to disfavour nucleosome assembly, consistent with the apparently wide spacing of nucleosomes at endogenous centromeres [49,72]. These regions may be de facto nucleosome free regions, similar to those at promoters, allowing cryptic initiation of transcription to occur [72,73]. The randomized synthetic sequence SynR-LM that is a poor substrate for CENP-A^Cnp1 deposition has similar long A tracts, but transcription-related sequence-sensitive elements—such as promoters and transcription factor binding sites—would be destroyed. Thus, the central domain, due to its sequence-encoded properties, may produce a distinctive chromatin and transcriptional environment.

CENP-A^Cnp1 chromatin does not assemble de novo on cc2 sequence alone in wild-type cells expressing normal CENP-A^Cnp1 levels [45]. Instead, we envisage that the unique transcriptional chromatin environment created by the cc2 sequence renders it permissive for CENP-A^Cnp1 establishment, but that establishment occurs only if other favourable conditions exist. CENP-A^Cnp1 is preferentially incorporated on these central domain sequences upon overexpression, when adjacent to heterochromatin, and in the absence of factors that usually enhance transcriptional elongation. Any explanation of CENP-A^Cnp1 chromatin establishment on central domain DNA must also account for how CENP-A^Cnp1 is incorporated instead of H3. Serine 2 in the CTD heptad repeat of Rpb1 is phosphorylated in elongating RNAPII, and this Ser2P-Rbp1/RNAPII becomes ubiquitylated upon stalling [74–76]. The ubiquitin hydrolase Ubp3 normally acts as a proof-reading activity to prevent degradation of stalled but rescuable RNAPII [56,61]. Absence of Ubp3 compromises the processing of stalled RNAPII, resulting in the accumulation of ubiquitylated Ser2P-Rbp1/RNAPII complexes. We propose that such modifications contribute to the distinctive status of central domain chromatin, leading to recruitment of factors that promote CENP-A^Cnp1 deposition (Fig. 8E). Alternatively, it may create an environment in which H3 nucleosomes are efficiently turned over/evicted, whereas CENP-A^Cnp1 nucleosomes are poorly evicted specifically in the context of stalled RNAPII. In cells lacking Ubp3, severe or prolonged stalling, even with normal levels of CENP-A^Cnp1, would provide extended opportunities for CENP-A^Cnp1 recruitment, or poor eviction of CENP-A^Cnp1 during prolonged stalling. TFIIS promotes transcriptional elongation by cleaving nascent transcripts in the context of stalled/backtracked RNAPII [57,58,77]. Although the effects of TFIIS deletion are more subtle than lack of Ubp3, the accumulation of RNAPII correlates with assembly CENP-A^Cnp1 chromatin, supporting a mechanism where persistent RNAPII stalling within central domain triggers remodelling that results in CENP-A^Cnp1 deposition.

In this model, when naïve central domain DNA (pMcc2) is introduced into wild-type cells, transient stalling occurs but it is efficiently cleared with the aid of factors such as TFIIS and Ubp3 (Fig. 8E). Because in wild-type cells CENP-A^Cnp1 levels are extremely low compared to histone H3 there would be little opportunity for CENP-A^Cnp1 to gain access to cc2, and with efficient clearing of stalled RNAPII, CENP-A^Cnp1 would fail to accumulate in cc2 [78]. CENP-A^Cnp1 overexpression would increase the probability of interaction with the transiently stalled RNAPII in central domain chromatin, increasing the likelihood of recruitment. Alternatively, increased access coupled with poor eviction would lead to CENP-A^Cnp1 accumulation. In addition, CENP-A^Cnp1 nucleosomes themselves, which have distinct N-terminal tails that lack the conserved lysine residues of H3 whose modification aids transcription, are likely to present a greater barrier to transcription than H3 nucleosomes [79]. Thus, once incorporated, CENP-A^Cnp1 nucleosomes might exacerbate the poor transcriptional elongation, creating conditions permissive for recruitment of more CENP-A^Cnp1 in a self-perpetuating system. Longer regions of central domain DNA would have greater probability of triggering stalling events and thus be more likely to initiate the incorporation of CENP-A^Cnp1. In the context of this model, heterochromatin could promote establishment of CENP-A^Cnp1 chromatin on adjacent cc2 sequence by drawing plasmids to sites of endogenous heterochromatin such as the spindle pole body where they would encounter a higher concentration of CENP-A^Cnp1 than non-heterochromatinized plasmids located in the nuclear interior [80]. Alternatively, heterochromatin-associated chromatin modifying activities may influence transcriptional elongation by RNAPII within cc2, causing enhanced stalling and deposition of CENP-A^Cnp1[41].

Following establishment of CENP-A chromatin and kinetochore assembly, transcription could play a proof-reading role that evicts H3 deposited at centromeres during S phase [81]. Indeed, transcription and RNAPII have been detected at centromeres in mammalian cells and transcription/RNAPII may play a role in centromere integrity [33,34,38]. Transcription of human α-satellite arrays introduced as HACs is known to occur. Although CENP-A assembly is compatible with targeting of mild transcriptional activators, targeting of a strong transcriptional activator is deleterious [30,38,82]. Thus transcription and/or the transcription-coupled histone modifications detected at centromeres may promote CENP-A deposition at mammalian centromeres.

In conclusion, we show that the sequence of fission yeast centromere central domain DNA is important only in so far as it encodes for certain properties that contribute to the region’s unusual chromatin and transcriptional landscape. Establishment of CENP-A^Cnp1 chromatin is driven by these sequence-encoded properties that when combined with the presence of nearby heterochromatin, overexpressed CENP-A^Cnp1 or increased RNAPII stalling, tips the balance in favour CENP-A^Cnp1 chromatin assembly. It seems likely that a similar combination of factors, which together favour CENP-A incorporation, must also contribute to the formation of neocentromeres at novel chromosomal locations.

Materials and Methods

Cell growth and manipulation

Standard genetic and molecular techniques were followed. Fission yeast methods were as described [83]. Fission yeast strains are listed in Table 1. Minichromosomes used in this study were transformed by electroporation. Transformants were selected by growth on PMG—ura—ade at 32°C. As circular minichromosomes lack heterochromatin and therefore centromeric cohesion, plasmids were maintained in cells by selection in medium lacking adenine and uracil. 3 independent colonies from each transformation were analysed for the presence of kinetochore proteins by chromatin immunoprecipitation (ChIP).

Centromere plasmids and minichromosomes

Plasmids bearing centromere fragments contained a minimal ars1 element to ensure efficient replication in S. pombe, in addition to selectable markers sup3-5 (complements ade6-704), ura4⁺ and KAN^R. 8.5 kb of central domain DNA (cc2 plus inner part of imr2L and imr2R) was cloned into the multiple cloning site as a SalI-NcoI fragment to create pMcc2. Various sub-fragments of cc2 (J-Q) were amplified by PCR and cloned into the multiple cloning site as BamHI/BglII fragments. 5.6 kb of heterochromatin-forming outer repeat sequence was inserted adjacent to central domain sequences to test ability to form functional centromeres.

A plasmid, pMC28, bearing cc2, a KAN resistance marker and an inverted ura4 sequence was constructed from pMcc2. Linearisation of the plasmid at NotI within the inverted ura4 sequence allowed integration at ura4⁺ located on the arm of Ch16-m23:ura4⁺. Ch16-m23: ura4⁺ is a derivative of Ch16, a 530 kb minichromosome, itself derived from Chromosome III [48]. It also bears the ade6-216 allele which complements the ade6-210 allele present on endogenous Chromosome III by interallelic complementation. Integration of linearised pMC28 on Ch16-m23:ura4 allowed selection on the counter-selective drug 5-fluoro-orotic acid and G418 (KAN). Cells that lost the Ch16-m23:ura4::cc2-KAN (abbreviated as Ch16-cc2) became red on limiting adenine and were sensitive to G418. For growth in liquid, cells containing Ch16-cc2 were grown in media lacking adenine.

ChIP

ChIP was performed as previously described [84] using anti-CENP-A^cnp1 antibody, anti CENP-C^Cnp3 antibody, anti-CENP-K^Sim4 antibody, anti-H3 antibody (ab1791; Abcam,), anti-H3K9me2 antibody (T. Urano) and anti-total RNA polymerase II (4F8; 61081, Active Motif), anti-Rpb1-Ser2P (3E10; 61083, Active Motif) and analysed by qPCR. Primers are listed in Table 2. P-values were calculated by standard t-test on 3 replicates between wild-type and mutant; p<0.05 was considered significant.

Establishment assay

For the establishment assay, cells were transformed with minichromosomes (containing 5.6 kb of outer repeat sequence in addition to cc2 sequences), by electroporation with ∼200 ng of DNA and plated on selective medium. Resultant colonies were replicated onto rich medium containing limiting adenine. The presence of pale pink/white colonies indicates establishment of a functional centromere on the minichromosome. Establishment efficiency is calculated as percentage of these colonies divided by the total number of transformants. Colonies were streaked on limiting adenine plates to confirm the presence of sectoring that is indicative of centromere function.

Real-time PCR (qPCR)

Quantitative PCR reactions were carried out in 10 μl volume, with 5μl Light Cycler 480 SybrGreen Master Mix (Roche), 0.5μl each primer (10 μM) and 3μl ChIP or total template. The data were analysed using Light Cycler 480 Software 1.5 (Roche).

5’RACE-PCR and RT-PCR

5’RACE-PCR was performed as previously described [37]. In brief, RNA was isolated with RNeasy mini/midi kit (Qiagen) according to the manufacturer’s protocol. Poly(A) containing RNA was purified from 500 μg of total RNA by affinity purification with biotinylated oligo-dT using PolyATtract mRNA Isolation Systems (Promega). 5’RACE PCR was performed using SMARTer 5’/3’ RACE (Clontech) according to the manufacturer’s protocol. PCR products were then run on 1% agarose gel, purified and cloned into pGEM-T Easy vector (Promega) and subsequently sequenced. Reverse transcription reaction for 5’RACE and qRT-PCR was performed using Superscript III Reverse Transcriptase (Invitrogen) using RNA extracted from 3 independent colonies. For qRT-PCR, transcript levels were normalized over gDNA to take into account differences in copy number between plasmids and normalized relative to act1⁺.

LacZ assay

LacZ assay was performed as described [85]. pREP81X-LacZ was digested with XhoI and PstI and the nmt81 promoter upstream of LacZ replaced with sequences from centromere 2. Plasmids were transformed into wild-type and grown on minimal medium (n = 3).

Southern analysis

DNA was extracted as previously described [83]. The DNA was digested with BglII/SpeI or SphI/SpeI, run on a 1% agarose gel, blotted on nylon membrane (Hybond N, Amersham) and UV-crosslinked. The membrane was hybridized with DNA probes specific for central domain 1 or central domain 2. To make the probes, PCR products were used as template in the labelling reaction using High Prime (Roche). Primers sequences are listed in Table 2.

Western analysis

Western analysis was performed as described previously using anti-GFP antibody (Roche) and anti-H3 antibody (ab1794-abcam) [86]. The intensities of GFP and H3 signals were acquired using LICOR Odyssey Infrared Imaging System software (Li-COR Bioscience).

Supporting Information

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