ABSTRACT
The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.
Subject(s)
Carrier Proteins/metabolism , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Kinetochores/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Carrier Proteins/genetics , Cell Cycle , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/analysis , Mutation , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/analysis , Schizosaccharomyces pombe Proteins/geneticsABSTRACT
Specialized chromatin containing CENP-A nucleosomes instead of H3 nucleosomes is found at all centromeres. However, the mechanisms that specify the locations at which CENP-A chromatin is assembled remain elusive in organisms with regional, epigenetically regulated centromeres. It is known that normal centromeric DNA is transcribed in several systems including the fission yeast, Schizosaccharomyces pombe. Here, we show that factors which preserve stable histone H3 chromatin during transcription also play a role in preventing promiscuous CENP-A(Cnp1) deposition in fission yeast. Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-A(Cnp1) incorporation at non-centromeric sites. FACT has little or no effect on CENP-A(Cnp1) assembly at endogenous centromeres where CENP-A(Cnp1) is normally assembled. In contrast, Clr6 complex II (Clr6-CII; equivalent to Rpd3S) histone deacetylase function has a more subtle impact on the stability of transcribed H3 chromatin and acts to prevent the ectopic accumulation of CENP-A(Cnp1) at specific loci, including subtelomeric regions, where CENP-A(Cnp1) is preferentially assembled. Moreover, defective Clr6-CII function allows the de novo assembly of CENP-A(Cnp1) chromatin on centromeric DNA, bypassing the normal requirement for heterochromatin. Thus, our analyses show that alterations in the process of chromatin assembly during transcription can destabilize H3 nucleosomes and thereby allow CENP-A(Cnp1) to assemble in its place. We propose that normal centromeres provide a specific chromatin context that limits reassembly of H3 chromatin during transcription and thereby promotes the establishment of CENP-A(Cnp1) chromatin and associated kinetochores. These findings have important implications for genetic and epigenetic processes involved in centromere specification.
Subject(s)
Aminopeptidases/genetics , Centromere/genetics , Chromosomal Proteins, Non-Histone/genetics , Molecular Chaperones/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/genetics , Transcription, Genetic , Cell Cycle Proteins/genetics , Centromere/metabolism , Chromatin Assembly and Disassembly , Chromosomal Proteins, Non-Histone/metabolism , DNA/genetics , Epigenesis, Genetic , Heterochromatin/genetics , Histones/genetics , Kinetochores , Nucleosomes/genetics , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
Inactivating mutations in the breast cancer susceptibility gene BRCA2 cause gross chromosomal rearrangements. Chromosome structural instability in the absence of BRCA2 is thought to result from defective homology-directed DNA repair. Here, we show that BRCA2 links the fidelity of telomere maintenance with genetic integrity. Absence of BRCA2 resulted in signs of dysfunctional telomeres, such as telomere shortening, erosions, and end fusions in proliferating mouse fibroblasts. BRCA2 localized to the telomeres in S phase in an ATR-dependent manner, and its absence resulted in the accumulation of common fragile sites, particularly at the G-rich lagging strand, and increased the telomere sister chromatid exchange in unchallenged cells. The incidence of common fragile sites and telomere sister chromatid exchange increased markedly after treatment with replication inhibitors. Congruently, telomere-induced foci were frequently observed in the absence of Brca2, denoting activation of the DNA damage response and abnormal chromosome end joining. These telomere end fusions constituted a significant portion of chromosome aberrations in Brca2-deficient cells. Our results suggest that BRCA2 is required for telomere homeostasis and may be particularly important for the replication of G-rich telomeric lagging strands.
Subject(s)
BRCA2 Protein/metabolism , Fibroblasts/metabolism , Homeostasis/physiology , S Phase/physiology , Telomere/metabolism , Animals , BRCA2 Protein/genetics , Cells, Cultured , Chromosome Aberrations , DNA Damage/physiology , Fibroblasts/cytology , Humans , Mice , Mice, Knockout , Sister Chromatid Exchange/physiology , Telomere/geneticsABSTRACT
The histone H3 variant CENP-A is the most favored candidate for an epigenetic mark that specifies the centromere. In fission yeast, adjacent heterochromatin can direct CENP-A(Cnp1) chromatin establishment, but the underlying features governing where CENP-A(Cnp1) chromatin assembles are unknown. We show that, in addition to centromeric regions, a low level of CENP-A(Cnp1) associates with gene promoters where histone H3 is depleted by the activity of the Hrp1(Chd1) chromatin-remodeling factor. Moreover, we demonstrate that noncoding RNAs are transcribed by RNA polymerase II (RNAPII) from CENP-A(Cnp1) chromatin at centromeres. These analyses reveal a similarity between centromeres and a subset of RNAPII genes and suggest a role for remodeling at RNAPII promoters within centromeres that influences the replacement of histone H3 with CENP-A(Cnp1).
Subject(s)
Centromere/metabolism , Chromatin Assembly and Disassembly/physiology , Chromosomal Proteins, Non-Histone , Promoter Regions, Genetic/physiology , RNA, Fungal/biosynthesis , Schizosaccharomyces pombe Proteins , Schizosaccharomyces/metabolism , Centromere/genetics , Heterochromatin/genetics , Heterochromatin/metabolism , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA, Fungal/genetics , Schizosaccharomyces/genetics , Transcription, Genetic/physiologyABSTRACT
The ability of elongating RNA polymerase II (RNAPII) to regulate the nucleosome barrier is poorly understood because we do not know enough about the involved factors and we lack a conceptual framework to model this process. Our group recently identified the conserved Fun30/SMARCAD1 family chromatin-remodeling factor, Fun30Fft3, as being critical for relieving the nucleosome barrier during RNAPII-mediated elongation, and proposed a model illustrating how Fun30Fft3 may contribute to nucleosome disassembly during RNAPII-mediated elongation. Here, we present a model that describes nucleosome dynamics during RNAPII-mediated elongation in mathematical terms and addresses the involvement of Fun30Fft3 in this process.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Gene Expression Regulation, Fungal , Nucleosomes/metabolism , RNA Polymerase II/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Algorithms , DNA Helicases/metabolism , Humans , Models, Biological , Nucleosomes/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Schizosaccharomyces/genetics , Transcription Factors/metabolism , Transcriptional ActivationABSTRACT
Methylation of histone H3 has been linked to the assembly of higher-order chromatin structures. Very recently, several examples, including the Schizosaccharomyces pombe mating-type region, chicken beta-globin locus, and inactive X-chromosome, revealed that H3-Lys9-methyl (Me) is associated with silent chromatin while H3-Lys4-Me is prominent in active chromatin. Surprisingly, it was shown that homologs of Drosophila Su(var)3-9 specifically methylate the Lys9 residue of histone H3. Here, to identify putative enzymes responsible for destabilization of heterochromatin, we screened genes whose overexpressions disrupt silencing at the silent mat3 locus in fission yeast. Interestingly, we identified two genes, rhp6(+) and ubcX(+) (ubiquitin-conjugating enzyme participating in silencing), both of which encode ubiquitin-conjugating enzymes. Their overexpression disrupted silencing at centromeres and telomeres as well as at mat3. Additionally, the overexpression interfered with centromeric function, as confirmed by elevated minichromosome loss and antimicrotubule drug sensitivity. On the contrary, deletion of rhp6(+) or ubcX(+) enhanced silencing at all heterochromatic regions tested, indicating that they are negative regulators of silencing. More importantly, chromatin immunoprecipitation showed that their overexpression alleviated the level of H3-Lys9-Me while enhancing the level of H3-Lys4-Me at the silent regions. On the contrary, their deletions enhanced the level of H3-Lys9-Me while alleviating that of H3-Lys4-Me. Taken together, the data suggest that two ubiquitin-conjugating enzymes, Rhp6 and UbcX, affect methylation of histone H3 at silent chromatin, which then reconfigures silencing.
Subject(s)
Drosophila Proteins , Heterochromatin/metabolism , Ligases/metabolism , Schizosaccharomyces/genetics , Ubiquitin-Conjugating Enzymes , Ubiquitin/metabolism , Amino Acid Sequence , Centromere/metabolism , Gene Silencing , Genes, Fungal , Genes, Mating Type, Fungal , Heterochromatin/genetics , Histones/metabolism , Methylation , Molecular Sequence Data , Mutation , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Sequence AlignmentABSTRACT
Recently, a histone H3 variant in Drosophila and humans, the H3.3 protein, was shown to replace canonical H3 in active chromatin in a replication-independent (RI) manner. In the fission yeast Schizosaccharomyces pombe, there exists a single form of H3, which is equivalent to H3.3 and is thought to participate in both replication-independent (RI) and replication-coupled (RC) nucleosome assembly. In this study, we show that RI deposition of H3 at heterochromatic regions is consistently lower than that at a gene-free euchromatic region, and deletion of the conserved heterochromatin-specific proteins Swi6 or Clr4 markedly increases RI deposition at heterochromatic regions such as the silent mating-type loci or centromeres. These results clearly show that RI deposition of H3 occurs preferentially in euchromatic regions. We also observed that RI deposition of H3 could be increased at the thi3(+) gene when transcription is induced, indicating transcription further facilitates RI deposition of H3. Taken together, these observations demonstrate that selective deposition of histone H3.3 at transcriptionally active chromatin by the RI assembly pathway is conserved in fission yeast and, thus, our data support an essential role of histone H3 replacement in maintaining active chromatin among diverse eukaryotic organisms ranging from fission yeast to humans.
Subject(s)
Chromatin/metabolism , Histones/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/genetics , Chromatin/chemistry , Chromatin Immunoprecipitation , DNA Replication , Euchromatin/chemistry , Euchromatin/metabolism , Heterochromatin/chemistry , Heterochromatin/metabolism , Promoter Regions, Genetic , RNA Polymerase II/metabolism , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics , Transcriptional ActivationABSTRACT
The centromere is the chromosomal locus at which the kinetochore is assembled to direct chromosome segregation. The histone H3 variant, centromere protein A (CENP-A), is known to epigenetically mark active centromeres, but the mechanism by which CENP-A propagates at the centromere, replacing histone H3, remains poorly understood. Using fission yeast, here we show that the Ino80 adenosine triphosphate (ATP)-dependent chromatin-remodeling complex, which removes histone H3-containing nucleosomes from associated chromatin, promotes CENP-ACnp1 chromatin assembly at the centromere in a redundant manner with another chromatin-remodeling factor Chd1Hrp1. CENP-ACnp1 chromatin actively recruits the Ino80 complex to centromeres to elicit eviction of histone H3-containing nucleosomes. Artificial targeting of Ino80 subunits to a non-centromeric DNA sequence placed in a native centromere enhances the spreading of CENP-ACnp1 chromatin into the non-centromeric DNA. Based on these results, we propose that CENP-ACnp1 chromatin employs the Ino80 complex to mediate the replacement of histone H3 with CENP-ACnp1, and thereby reinforces itself.The histone variant CENP-A marks active centromeres and replaces H3 at centromeres through a poorly understood mechanism. Here, the authors provide evidence that the chromatin remodeller Ino80 promotes CENP-A chromatin assembly at the centromere in fission yeast.
Subject(s)
Centromere/metabolism , Histones/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/genetics , 3-Isopropylmalate Dehydrogenase/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Centromere/genetics , Chromatin Assembly and Disassembly/physiology , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Epigenesis, Genetic , Histones/genetics , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Saccharomyces cerevisiae Proteins/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
Previous studies have revealed that nucleosomes impede elongation of RNA polymerase II (RNAPII). Recent observations suggest a role for ATP-dependent chromatin remodellers in modulating this process, but direct in vivo evidence for this is unknown. Here using fission yeast, we identify Fun30Fft3 as a chromatin remodeller, which localizes at transcribing regions to promote RNAPII transcription. Fun30Fft3 associates with RNAPII and collaborates with the histone chaperone, FACT, which facilitates RNAPII elongation through chromatin, to induce nucleosome disassembly at transcribing regions during RNAPII transcription. Mutants, resulting in reduced nucleosome-barrier, such as deletion mutants of histones H3/H4 themselves and the genes encoding components of histone deacetylase Clr6 complex II suppress the defects in growth and RNAPII occupancy of cells lacking Fun30Fft3. These data suggest that RNAPII utilizes the chromatin remodeller, Fun30Fft3, to overcome the nucleosome barrier to transcription elongation.
Subject(s)
Chromatin Assembly and Disassembly , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Nucleosomes/metabolism , RNA Polymerase II/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromatin/genetics , Chromosomal Proteins, Non-Histone/genetics , Gene Expression Regulation, Fungal , Histones/genetics , Histones/metabolism , Models, Genetic , Nucleosomes/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , Transcription, GeneticABSTRACT
Taz1p is the fission yeast orthologue of human TRF2, a telomeric repeat-binding protein. Delta(taz1) mutants are defective in telomeric silencing, telomere length control, and meiotic recombination events. A recent report demonstrated that the human Rap1p homolog (hRap1) is recruited to telomere by interaction with TRF2, arguing that the telomere control mechanism of higher eukaryotes is distinct from that of the budding yeast. Taz1p showed a significant similarity to human TRF2, but not with the budding yeast Rap1p (scRap1p). This suggests that Taz1p and TRF2 share common features in telomere regulation. To assess the roles of Taz1p in telomere-related functions in detail, we attempted to identify a protein(s) that interacts with Taz1p by using two-hybrid screening. Interestingly, the sequence analysis of a positive clone revealed a perfect match with a Rap1 homolog in S. pombe (spRap1), which showed a significant homology with scRap1p and hRap1p. Here we show that the spRap1 deficiency in haploid cells is viable, which results in increased telomere length regulation, disruption of telomere silencing, and aberrant meiosis (like the delta(taz1) mutant). This suggests that spRap1p might be recruited to the telomere by Taz1p and play crucial roles in telomere function. Interestingly, the delta(rap1) mutants in fission yeast are defective only for telomere silencing. Therefore, the role of spRap1p may be distinct from that of scRap1p, which is involved in the silencing at both the telomere and mating type locus. Our data, therefore, suggest that the regulation mechanisms of telomere in fission yeast resemble that of higher eukaryotic cells rather than the budding yeast.
Subject(s)
Gene Silencing , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/genetics , Telomere-Binding Proteins/metabolism , Telomere/metabolism , Base Sequence , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Molecular Sequence Data , Schizosaccharomyces/chemistry , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/genetics , Telomere-Binding Proteins/genetics , Two-Hybrid System TechniquesABSTRACT
The histone H3 variant, CENP-A, is normally assembled upon canonical centromeric sequences, but there is no apparent obligate coupling of sequence and assembly, suggesting that centromere location can be epigenetically determined. To explore the tolerances and constraints on CENP-A deposition we investigated whether certain locations are favoured when additional CENP-A(Cnp1) is present in fission yeast cells. Our analyses show that additional CENP-A(Cnp1) accumulates within and close to heterochromatic centromeric outer repeats, and over regions adjacent to rDNA and telomeres. The use of minichromosome derivatives with unique DNA sequences internal to chromosome ends shows that telomeres are sufficient to direct CENP-A(Cnp1) deposition. However, chromosome ends are not required as CENP-A(Cnp1) deposition also occurs at telomere repeats inserted at an internal locus and correlates with the presence of H3K9 methylation near these repeats. The Ccq1 protein, which is known to bind telomere repeats and recruit telomerase, was found to be required to induce H3K9 methylation and thus promote the incorporation of CENP-A(Cnp1) near telomere repeats. These analyses demonstrate that at non-centromeric chromosomal locations the presence of heterochromatin influences the sites at which CENP-A is incorporated into chromatin and, thus, potentially the location of centromeres.
Subject(s)
Chromosomal Proteins, Non-Histone/genetics , Repetitive Sequences, Nucleic Acid/genetics , Schizosaccharomyces pombe Proteins/genetics , Telomere-Binding Proteins/genetics , Telomere/genetics , Blotting, Western , Centromere/genetics , Centromere/metabolism , Chromatin/genetics , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Fungal/genetics , Chromosomes, Fungal/metabolism , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Gene Expression Profiling , Gene Expression Regulation, Fungal , Heterochromatin/genetics , Heterochromatin/metabolism , Histones/metabolism , Lysine/metabolism , Methylation , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Telomere/metabolism , Telomere-Binding Proteins/metabolismABSTRACT
Several studies have suggested that SUMO may participate in the regulation of heterochromatin, but direct evidence is lacking. Here, we present a direct link between sumoylation and heterochromatin stability. SUMO deletion impaired silencing at heterochromatic regions and induced histone H3 Lys4 methylation, a hallmark of active chromatin in fission yeast. Our findings showed that the SUMO-conjugating enzyme Hus5/Ubc9 interacted with the conserved heterochromatin proteins Swi6, Chp2 (a paralog of Swi6), and Clr4 (H3 Lys9 methyltransferase). Moreover, chromatin immunoprecipitation (ChIP) revealed that Hus5 was highly enriched in heterochromatic regions in a heterochromatin-dependent manner, suggesting a direct role of Hus5 in heterochromatin formation. We also found that Swi6, Chp2, and Clr4 themselves can be sumoylated in vivo and defective sumoylation of Swi6 or Chp2 compromised silencing. These results indicate that Hus5 associates with heterochromatin through interactions with heterochromatin proteins and modifies substrates whose sumoylations are required for heterochromatin stability, including heterochromatin proteins themselves.
Subject(s)
Chromosomal Instability , Gene Silencing , Heterochromatin/metabolism , Schizosaccharomyces/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Histone-Lysine N-Methyltransferase , Methyltransferases/genetics , Methyltransferases/metabolism , Protein Binding , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/chemistry , Small Ubiquitin-Related Modifier Proteins/genetics , Two-Hybrid System Techniques , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
A study of gene silencing within the mating-type region of fission yeast defines two distinct pathways responsible for the establishment of heterochromatin assembly. One is RNA interference-dependent and acts on centromere-homologous repeats (cenH). The other is a stochastic Swi6 (the fission yeast HP1 homolog)-dependent mechanism that is not fully understood. Here we find that activating transcription factor (Atf1) and Pcr1, the fission yeast bZIP transcription factors homologous to human ATF-2, are crucial for proper histone deacetylation of both H3 and H4. This deacetylation is a prerequisite for subsequent H3 lysine 9 methylation and Swi6-dependent heterochromatin assembly across the rest of the silent mating-type (mat) region lacking the RNA interference-dependent cenH repeat. Moreover, Atf1 and Pcr1 can form complexes with both a histone deacetylase, Clr6, and Swi6, and clr6 mutations affected the H3/H4 acetylation patterns, similar to the atf1 and pcr1 deletion mutant phenotypes at the endogenous mat loci and at the ctt1+ promoter region surrounding ATF/CRE-binding site. These data suggest that Atf1 and Pcr1 participate in an early step essential for heterochromatin assembly at the mat locus and silencing of transcriptional targets of Atf1. Furthermore, a phosphorylation event catalyzed by the conserved mitogen-activated protein kinase pathway is important for regulation of heterochromatin silencing by Atf1 and Pcr1. These findings suggest a role for the mitogen-activated protein kinase pathway and histone deacetylase in Swi6-based heterochromatin assembly.