Fission Yeast Centromeres Research Articles

Sequence-dependent heterochromatin formation in the human malaria parasite Plasmodium falciparum

The human malaria parasite Plasmodium falciparum represses transcription of the gene encoding AP2-G, which is the master regulator of germ cell differentiation, via heterochromatin condensation following histone H3 lysine 9 trimethylation (H3K9me3). Although H3K9me3-marked heterochromatin is typically constitutive and its establishment depends on the RNA interference (RNAi) pathway in fission yeast centromeres, malaria parasites lack molecular members essential for RNAi. We developed a strategy to assess heterochromatin establishment on artificial chromosomes introduced into P. falciparum. We show that a particular DNA sequence in the AP2-G promoter is able to induce de novo H3K9me3 nucleosome deposition. In addition, we also found that the AP2-G promoter contains a distinct element required in maintenance of the repression memory. Thus, we speculate that malaria parasites have evolutionarily acquired a sequence-dependent establishment system of non-constitutive, i.e. facultative, H3K9me3-marked heterochromatin.

Establishment of centromere identity is dependent on nuclear spatial organization

The establishment of centromere-specific CENP-A chromatin is influenced by epigenetic and genetic processes. Central domain sequences from fission yeast centromeres are preferred substrates for CENP-ACnp1 incorporation, but their use is context dependent, requiring adjacent heterochromatin. CENP-ACnp1 overexpression bypasses heterochromatin dependency, suggesting that heterochromatin ensures exposure to conditions or locations permissive for CENP-ACnp1 assembly. Centromeres cluster around spindle-pole bodies (SPBs). We show that heterochromatin-bearing minichromosomes localize close to SPBs, consistent with this location promoting CENP-ACnp1 incorporation. We demonstrate that heterochromatin-independent de novo CENP-ACnp1 chromatin assembly occurs when central domain DNA is placed near, but not far from, endogenous centromeres or neocentromeres. Moreover, direct tethering of central domain DNA at SPBs permits CENP-ACnp1 assembly, suggesting that the nuclear compartment surrounding SPBs is permissive for CENP-ACnp1 incorporation because target sequences are exposed to high levels of CENP-ACnp1 and associated assembly factors. Thus, nuclear spatial organization is a key epigenetic factor that influences centromere identity.

Correction for Carlsten et al., "Mediator Promotes CENP-A Incorporation at Fission Yeast Centromeres".

Volume 32, no. 19, p. 4035–4043, 2012, https://doi.org/10.1128/MCB.00374-12. Page 4037, Fig. 1F, right panel: The bottom left image duplicates the bottom center image by mistake. The dg row should appear as shown below.

DNA replication machinery prevents Rad52-dependent single-strand annealing that leads to gross chromosomal rearrangements at centromeres

Homologous recombination between repetitive sequences can lead to gross chromosomal rearrangements (GCRs). At fission yeast centromeres, Rad51-dependent conservative recombination predominantly occurs between inverted repeats, thereby suppressing formation of isochromosomes whose arms are mirror images. However, it is unclear how GCRs occur in the absence of Rad51 and how GCRs are prevented at centromeres. Here, we show that homology-mediated GCRs occur through Rad52-dependent single-strand annealing (SSA). The rad52-R45K mutation, which impairs SSA activity of Rad52 protein, dramatically reduces isochromosome formation in rad51 deletion cells. A ring-like complex Msh2–Msh3 and a structure-specific endonuclease Mus81 function in the Rad52-dependent GCR pathway. Remarkably, mutations in replication fork components, including DNA polymerase α and Swi1/Tof1/Timeless, change the balance between Rad51-dependent recombination and Rad52-dependent SSA at centromeres, increasing Rad52-dependent SSA that forms isochromosomes. Our results uncover a role of DNA replication machinery in the recombination pathway choice that prevents Rad52-dependent GCRs at centromeres.

Transcriptional silencing of centromere repeats by heterochromatin safeguards chromosome integrity.

The centromere region of chromosomes consists of repetitive DNA sequences, and is, therefore, one of the fragile sites of chromosomes in many eukaryotes. In the core region, the histone H3 variant CENP-A forms centromere-specific nucleosomes that are required for kinetochore formation. In the pericentromeric region, histone H3 is methylated at lysine 9 (H3K9) and heterochromatin is formed. The transcription of pericentromeric repeats by RNA polymerase II is strictly repressed by heterochromatin. However, the role of the transcriptional silencing of the pericentromeric repeats remains largely unclear. Here, we focus on the chromosomal rearrangements that occur at the repetitive centromeres, and highlight our recent studies showing that transcriptional silencing by heterochromatin suppresses gross chromosomal rearrangements (GCRs) at centromeres in fission yeast. Inactivation of the Clr4 methyltransferase, which is essential for the H3K9 methylation, increased GCRs with breakpoints located in centromeric repeats. However, mutations in RNA polymerase II or the transcription factor Tfs1/TFIIS, which promotes restart of RNA polymerase II following its backtracking, reduced the GCRs that occur in the absence of Clr4, demonstrating that heterochromatin suppresses GCRs by repressing the Tfs1-dependent transcription. We also discuss how the transcriptional restart gives rise to chromosomal rearrangements at centromeres.

Centromere DNA Destabilizes H3 Nucleosomes to Promote CENP-A Deposition during the Cell Cycle

SummaryActive centromeres are defined by the presence of nucleosomes containing CENP-A, a histone H3 variant, which alone is sufficient to direct kinetochore assembly. Once assembled at a location, CENP-A chromatin and kinetochores are maintained at that location through a positive feedback loop where kinetochore proteins recruited by CENP-A promote deposition of new CENP-A following replication. Although CENP-A chromatin itself is a heritable entity, it is normally associated with specific sequences. Intrinsic properties of centromeric DNA may favor the assembly of CENP-A rather than H3 nucleosomes. Here we investigate histone dynamics on centromere DNA. We show that during S phase, histone H3 is deposited as a placeholder at fission yeast centromeres and is subsequently evicted in G2, when we detect deposition of the majority of new CENP-ACnp1. We also find that centromere DNA has an innate property of driving high rates of turnover of H3-containing nucleosomes, resulting in low nucleosome occupancy. When placed at an ectopic chromosomal location in the absence of any CENP-ACnp1 assembly, centromere DNA appears to retain its ability to impose S phase deposition and G2 eviction of H3, suggesting that features within centromere DNA program H3 dynamics. Because RNA polymerase II (RNAPII) occupancy on this centromere DNA coincides with H3 eviction in G2, we propose a model in which RNAPII-coupled chromatin remodeling promotes replacement of H3 with CENP-ACnp1 nucleosomes.

Heterochromatin and RNAi regulate centromeres by protecting CENP-A from ubiquitin-mediated degradation.

Centromere is a specialized chromatin domain that plays a vital role in chromosome segregation. In most eukaryotes, centromere is surrounded by the epigenetically distinct heterochromatin domain. Heterochromatin has been shown to contribute to centromere function, but the precise role of heterochromatin in centromere specification remains elusive. Centromeres in most eukaryotes, including fission yeast (Schizosaccharomyces pombe), are defined epigenetically by the histone H3 (H3) variant CENP-A. In contrast, the budding yeast Saccharomyces cerevisiae has genetically-defined point centromeres. The transition between regional centromeres and point centromeres is considered as one of the most dramatic evolutionary events in centromere evolution. Here we demonstrated that Cse4, the budding yeast CENP-A homolog, can localize to centromeres in fission yeast and partially substitute fission yeast CENP-ACnp1. But overexpression of Cse4 results in its localization to heterochromatic regions. Cse4 is subject to efficient ubiquitin-dependent degradation in S. pombe, and its N-terminal domain dictates its centromere distribution via ubiquitination. Notably, without heterochromatin and RNA interference (RNAi), Cse4 fails to associate with centromeres. We showed that RNAi-dependent heterochromatin mediates centromeric localization of Cse4 by protecting Cse4 from ubiquitin-dependent degradation. Heterochromatin also contributes to the association of native CENP-ACnp1 with centromeres via the same mechanism. These findings suggest that protection of CENP-A from degradation by heterochromatin is a general mechanism used for centromere assembly, and also provide novel insights into centromere evolution.

Centromere Stability: The Replication Connection.

The fission yeast centromere, which is similar to metazoan centromeres, contains highly repetitive pericentromere sequences that are assembled into heterochromatin. This is required for the recruitment of cohesin and proper chromosome segregation. Surprisingly, the pericentromere replicates early in the S phase. Loss of heterochromatin causes this domain to become very sensitive to replication fork defects, leading to gross chromosome rearrangements. This review examines the interplay between components of DNA replication, heterochromatin assembly, and cohesin dynamics that ensures maintenance of genome stability and proper chromosome segregation.

Remarkable Evolutionary Plasticity of Centromeric Chromatin.

Centromeres were familiar to cell biologists in the late 19th century, but for most eukaryotes the basis for centromere specification has remained enigmatic. Much attention has been focused on the cenH3 (CENP-A) histone variant, which forms the foundation of the centromere. To investigate the DNA sequence requirements for centromere specification, we applied a variety of epigenomic approaches, which have revealed surprising diversity in centromeric chromatin properties. Whereas each point centromere of budding yeast is occupied by a single precisely positioned tetrameric nucleosome with one cenH3 molecule, the "regional" centromeres of fission yeast contain unphased presumably octameric nucleosomes with two cenH3s. In Caenorhabditis elegans, kinetochores assemble all along the chromosome at sites of cenH3 nucleosomes that resemble budding yeast point centromeres, whereas holocentric insects lack cenH3 entirely. The "satellite" centromeres of most animals and plants consist of cenH3-containing particles that are precisely positioned over homogeneous tandem repeats, but in humans, different α-satellite subfamilies are occupied by CENP-A nucleosomes with very different conformations. We suggest that this extraordinary evolutionary diversity of centromeric chromatin architectures can be understood in terms of the simplicity of the task of equal chromosome segregation that is continually subverted by selfish DNA sequences.

Mitotic Nuclear Envelope Breakdown and Spindle Nucleation Are Controlled by Interphase Contacts between Centromeres and the Nuclear Envelope

Faithful genome propagation requires coordination between nuclear envelope (NE) breakdown, spindle formation, and chromosomal events. The conserved linker of nucleoskeleton and cytoskeleton (LINC) complex connects fission yeast centromeres and the centrosome, across the NE, during interphase. During meiosis, LINC connects the centrosome with telomeres rather than centromeres. We previously showed that loss of telomere-LINC contacts compromises meiotic spindle formation. Here, we define the precise events regulated by telomere-LINC contacts and address the analogous possibility that centromeres regulate mitotic spindle formation. We develop conditionally inactivated LINC complexes in which the conserved SUN-domain protein Sad1 remains stable but severs interphase centromere-LINC contacts. Strikingly, the loss of such contacts abolishes spindle formation. We pinpoint the defect to a failure in the partial NE breakdown required for centrosome insertion into the NE, a step analogous to mammalian NE breakdown. Thus, interphase chromosome-LINC contacts constitute a cell-cycle control device linking nucleoplasmic and cytoplasmic events.

Inner Kinetochore Protein Interactions with Regional Centromeres of Fission Yeast.

Centromeres of the fission yeast Schizosaccharomyces pombe lack the highly repetitive sequences that make most other "regional" centromeres refractory to analysis. To map fission yeast centromeres, we applied H4S47C-anchored cleavage mapping and native and cross-linked chromatin immunoprecipitation with paired-end sequencing. H3 nucleosomes are nearly absent from the central domain, which is occupied by centromere-specific H3 (cenH3 or CENP-A) nucleosomes with two H4s per particle that are mostly unpositioned and are more widely spaced than nucleosomes elsewhere. Inner kinetochore proteins CENP-A, CENP-C, CENP-T, CENP-I, and Scm3 are highly enriched throughout the central domain except at tRNA genes, with no evidence for preferred kinetochore assembly sites. These proteins are weakly enriched and less stably incorporated in H3-rich heterochromatin. CENP-A nucleosomes protect less DNA from nuclease digestion than H3 nucleosomes, while CENP-T protects a range of fragment sizes. Our results suggest that CENP-T particles occupy linkers between CENP-A nucleosomes and that classical regional centromeres differ from other centromeres by the absence of CENP-A nucleosome positioning.

Anniversary of the discovery/isolation of the yeast centromere by Clarke and Carbon.

The first centromere was isolated 35 years ago by Louise Clarke and John Carbon from budding yeast. They embarked on their journey with rudimentary molecular tools (by today's standards) and little knowledge of the structure of a chromosome, much less the nature of a centromere. Their discovery opened up a new field, as centromeres have now been isolated from fungi and numerous plants and animals, including mammals. Budding yeast and several other fungi have small centromeres with short, well-defined sequences, known as point centromeres, whereas regional centromeres span several kilobases up to megabases and do not seem to have DNA sequence specificity. Centromeres are at the heart of artificial chromosomes, and we have seen the birth of synthetic centromeres in budding and fission yeast and mammals. The diversity in centromeres throughout phylogeny belie conserved functions that are only beginning to be understood.

Sequence features and transcriptional stalling within centromere DNA promote establishment of CENP-A chromatin.

Centromere sequences are not conserved between species, and there is compelling evidence for epigenetic regulation of centromere identity, with location being dictated by the presence of chromatin containing the histone H3 variant CENP-A. Paradoxically, in most organisms CENP-A chromatin generally occurs on particular sequences. To investigate the contribution of primary DNA sequence to establishment of CENP-A chromatin in vivo, we utilised the fission yeast Schizosaccharomyces pombe. CENP-ACnp1 chromatin is normally assembled on ∼10 kb of central domain DNA within these regional centromeres. We demonstrate that overproduction of S. pombe CENP-ACnp1 bypasses the usual requirement for adjacent heterochromatin in establishing CENP-ACnp1 chromatin, and show that central domain DNA is a preferred substrate for de novo establishment of CENP-ACnp1 chromatin. When multimerised, a 2 kb sub-region can establish CENP-ACnp1 chromatin and form functional centromeres. Randomization of the 2 kb sequence to generate a sequence that maintains AT content and predicted nucleosome positioning is unable to establish CENP-ACnp1 chromatin. These analyses indicate that central domain DNA from fission yeast centromeres contains specific information that promotes CENP-ACnp1 incorporation into chromatin. Numerous transcriptional start sites were detected on the forward and reverse strands within the functional 2 kb sub-region and active promoters were identified. RNAPII is enriched on central domain DNA in wild-type cells, but only low levels of transcripts are detected, consistent with RNAPII stalling during transcription of centromeric DNA. Cells lacking factors involved in restarting transcription—TFIIS and Ubp3—assemble CENP-ACnp1 on central domain DNA when CENP-ACnp1 is at wild-type levels, suggesting that persistent stalling of RNAPII on centromere DNA triggers chromatin remodelling events that deposit CENP-ACnp1. Thus, sequence-encoded features of centromeric DNA create an environment of pervasive low quality RNAPII transcription that is an important determinant of CENP-ACnp1 assembly. These observations emphasise roles for both genetic and epigenetic processes in centromere establishment.

The CENP-A N-Tail Confers Epigenetic Stability to Centromeres via the CENP-T Branch of the CCAN in Fission Yeast

In most eukaryotes, centromeres are defined epigenetically by presence of the histone H3 variant CENP-A [1-3]. CENP-A-containing chromatin recruits the constitutive centromere-associated network (CCAN) of proteins, which in turn directs assembly of the outer kinetochore to form microtubule attachments and ensure chromosome segregation fidelity [4-6]. Whereas the mechanisms that load CENP-A at centromeres are being elucidated, the functions of its divergent N-terminal tail remain enigmatic [7-12]. Here, we employ the well-studied fission yeast centromere [13-16] to investigate the function of the CENP-A (Cnp1) N-tail. We show that alteration of the N-tail does not affect Cnp1 loading at centromeres, outer kinetochore formation, or spindle checkpoint signaling but nevertheless elevates chromosome loss. N-tail mutants exhibited synthetic lethality with an altered centromeric DNA sequence, with rare survivors harboring chromosomal fusions in which the altered centromere was epigenetically inactivated. Elevated centromere inactivation was also observed for N-tail mutants with unaltered centromeric DNA sequences. N-tail mutants specifically reduced localization of the CCAN proteins Cnp20/CENP-T and Mis6/CENP-I, but not Cnp3/CENP-C. Overexpression of Cnp20/CENP-T suppressed defects in an N-tail mutant, suggesting a link between reduced CENP-T recruitment and the observed centromere inactivation phenotype. Thus, the Cnp1 N-tail promotes epigenetic stability of centromeres in fission yeast, at least in part via recruitment of the CENP-T branch of the CCAN.

A systematic genetic screen identifies new factors influencing centromeric heterochromatin integrity in fission yeast.

BackgroundHeterochromatin plays important roles in the regulation and stability of eukaryotic genomes. Both heterochromatin components and pathways that promote heterochromatin assembly, including RNA interference, RNAi, are broadly conserved between the fission yeast Schizosaccharomyces pombe and humans. As a result, fission yeast has emerged as an important model system for dissecting mechanisms governing heterochromatin integrity. Thus far, over 50 proteins have been found to contribute to heterochromatin assembly at fission yeast centromeres. However, previous studies have not been exhaustive, and it is therefore likely that further factors remain to be identified.ResultsTo gain a more complete understanding of heterochromatin assembly pathways, we have performed a systematic genetic screen for factors required for centromeric heterochromatin integrity. In addition to known RNAi and chromatin modification components, we identified several proteins with previously undescribed roles in heterochromatin regulation. These included both known and newly characterised splicing-associated proteins, which are required for proper processing of centromeric transcripts by the RNAi pathway, and COP9 signalosome components Csn1 and Csn2, whose role in heterochromatin assembly can be explained at least in part by a role in the Ddb1-dependent degradation of the heterochromatin regulator Epe1.ConclusionsThis work has revealed new factors involved in RNAi-directed heterochromatin assembly in fission yeast. Our findings support and extend previous observations that implicate components of the splicing machinery as a platform for RNAi, and demonstrate a novel role for the COP9 signalosome in heterochromatin regulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0481-4) contains supplementary material, which is available to authorized users.

Schizosaccharomyces pombe centromere protein Mis19 links Mis16 and Mis18 to recruit CENP‐A through interacting with NMD factors and the SWI/SNF complex

CENP-A is a centromere-specific variant of histone H3 that is required for accurate chromosome segregation. The fission yeast Schizosaccharomyces pombe and mammalian Mis16 and Mis18 form a complex essential for CENP-A recruitment to centromeres. It is unclear, however, how the Mis16-Mis18 complex achieves this function. Here, we identified, by mass spectrometry, novel fission yeast centromere proteins Mis19 and Mis20 that directly interact with Mis16 and Mis18. Like Mis18, Mis19 and Mis20 are localized at the centromeres during interphase, but not in mitosis. Inactivation of Mis19 in a newly isolated temperature-sensitive mutant resulted in CENP-A delocalization and massive chromosome missegregation, whereas Mis20 was dispensable for proper chromosome segregation. Mis19 might be a bridge component for Mis16 and Mis18. We isolated extragenic suppressor mutants for temperature-sensitive mis18 and mis19 mutants and used whole-genome sequencing to determine the mutated sites. We identified two groups of loss-of-function suppressor mutations in non-sense-mediated mRNA decay factors (upf2 and ebs1), and in SWI/SNF chromatin-remodeling components (snf5, snf22 and sol1). Our results suggest that the Mis16-Mis18-Mis19-Mis20 CENP-A-recruiting complex, which is functional in the G1-S phase, may be counteracted by the SWI/SNF chromatin-remodeling complex and non-sense-mediated mRNA decay, which may prevent CENP-A deposition at the centromere.

DNA replication components as regulators of epigenetic inheritance—lesson from fission yeast centromere

Genetic information stored in DNA is accurately copied and transferred to subsequent generations through DNA replication. This process is accomplished through the concerted actions of highly conserved DNA replication components. Epigenetic information stored in the form of histone modifications and DNA methylation, constitutes a second layer of regulatory information important for many cellular processes, such as gene expression regulation, chromatin organization, and genome stability. During DNA replication, epigenetic information must also be faithfully transmitted to subsequent generations. How this monumental task is achieved remains poorly understood. In this review, we will discuss recent advances on the role of DNA replication components in the inheritance of epigenetic marks, with a particular focus on epigenetic regulation in fission yeast. Based on these findings, we propose that specific DNA replication components function as key regulators in the replication of epigenetic information across the genome.

A systematic genetic screen identifies new factors influencing centromeric heterochromatin integrity in fission yeast

Heterochromatin plays important roles in the regulation and stability of eukaryotic genomes. Both heterochromatin components and pathways that promote heterochromatin assembly, including RNA interference, RNAi, are broadly conserved between the fission yeast Schizosaccharomyces pombe and humans. As a result, fission yeast has emerged as an important model system for dissecting mechanisms governing heterochromatin integrity. Thus far, over 50 proteins have been found to contribute to heterochromatin assembly at fission yeast centromeres. However, previous studies have not been exhaustive, and it is therefore likely that further factors remain to be identified. To gain a more complete understanding of heterochromatin assembly pathways, we have performed a systematic genetic screen for factors required for centromeric heterochromatin integrity. In addition to known RNAi and chromatin modification components, we identified several proteins with previously undescribed roles in heterochromatin regulation. These included both known and newly characterised splicing-associated proteins, which are required for proper processing of centromeric transcripts by the RNAi pathway, and COP9 signalosome components Csn1 and Csn2, whose role in heterochromatin assembly can be explained at least in part by a role in the Ddb1-dependent degradation of the heterochromatin regulator Epe1. This work has revealed new factors involved in RNAi-directed heterochromatin assembly in fission yeast. Our findings support and extend previous observations that implicate components of the splicing machinery as a platform for RNAi, and demonstrate a novel role for the COP9 signalosome in heterochromatin regulation.

Histone deacetylases govern heterochromatin in every phase

Deacetylation of histone tails has been shown to play a role during heterochromatin formation, but the precise mechanism of action has not been understood. Complementary results presented in two recent articles in The EMBO Journal (Alper et al , 2013; Buscaino et al , 2013) together reveal how histone deacetylases (HDACs) affect the various phases of heterochromatin formation: establishment, maintenance and spreading. Heterochromatin, condensed chromatin that is transcriptionally inactive, plays important roles in epigenetic regulation of gene expression and other chromosomal functions, such as chromosome segregation and telomere maintenance. Fission yeast is a good model system to analyse heterochromatin because of the similarity of its heterochromatin structure to other eukaryotes and the ease of genetic analysis. In fact, this organism has contributed greatly to our understanding of the importance of RNA interference (RNAi) during heterochromatin formation. Heterochromatin is defined by methylation of lysine 9 of histone H3 (H3K9me) and hypo‐acetylation of histones. H3K9me provides the binding sites for heterochromatin protein 1, HP1 (Swi6 in fission yeast, Nakayama et al , 2001). Since deacetylation of histone tails by HDACs generally correlates with the repression of transcription at euchromatic regions, a similar function of HDACs is expected at heterochromatin. Indeed, the HDAC Clr3 is required for efficient repression of …

Centromeric motion facilitates the mobility of interphase genomic regions in fission yeast

Dispersed genetic elements, such as retrotransposons and Pol-III-transcribed genes, including tRNA and 5S rRNA, cluster and associate with centromeres in fission yeast through the function of condensin. However, the dynamics of these condensin-mediated genomic associations remains unknown. We have examined the 3D motions of genomic loci including the centromere, telomere, rDNA repeat locus, and the loci carrying Pol-III-transcribed genes or long-terminal repeat (LTR) retrotransposons in live cells at as short as 1.5-second intervals. Treatment with carbendazim (CBZ), a microtubule-destabilizing agent, not only prevents centromeric motion, but also reduces the mobility of the other genomic loci during interphase. Further analyses demonstrate that condensin-mediated associations between centromeres and the genomic loci are clonal, infrequent and transient. However, when associated, centromeres and the genomic loci migrate together in a coordinated fashion. In addition, a condensin mutation that disrupts associations between centromeres and the genomic loci results in a concomitant decrease in the mobility of the loci. Our study suggests that highly mobile centromeres pulled by microtubules in cytoplasm serve as 'genome mobility elements' by facilitating physical relocations of associating genomic regions.