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Cellular quiescence is an important physiological state both in unicellular and multicellular eukaryotes. Quiescent cells are halted for proliferation and stop the cell cycle at the G0 stage. Using fission yeast as a model organism, we have previously found that several subunits of a conserved chromatin remodeling complex, Ino80C (INOsitol requiring nucleosome remodeling factor), are required for survival in quiescence. Here, we demonstrate that Ino80C has a key function in the regulation of gene expression in G0 cells. We show that null mutants for two Ino80C subunits, Iec1 and Ies2, a putative subunit Arp42, a null mutant for the histone variant H2A.Z, and a null mutant for the Inositol kinase Asp1 have very similar phenotypes in quiescence. These mutants show reduced transcription genome-wide and specifically fail to activate 149 quiescence genes, of which many are localized to the subtelomeric regions. Using spike in normalized ChIP-seq experiments, we show that there is a global reduction of H2A.Z levels in quiescent wild-type cells but not in iec1∆ cells and that a subtelomeric chromosome boundary element is strongly affected by Ino80C. Based on these observations, we propose a model in which Ino80C is evicting H2A.Z from chromatin in quiescent cells, thereby inactivating the subtelomeric boundary element, leading to a reorganization of the chromosome structure and activation of genes required to survive in quiescence.

CCTC-binding factor (CTCF) is a key regulator of gene expression through organization of the chromatin structure. Still, it is unclear how CTCF binding is perturbed in leukemia or in cancer in general. We studied CTCF binding by chromatin immunoprecipitation sequencing in cells from patients with acute myeloid leukemia (AML) and in normal bone marrow (NBM) in the context of gene expression, DNA methylation, and azacitidine exposure. CTCF binding was increased in AML compared with NBM. Aberrant CTCF binding was enriched for motifs for key myeloid transcription factors such as CEBPA, PU.1, and RUNX1. AML with TET2 mutations was characterized by a particularly strong gain of CTCF binding, highly enriched for gain in promoter regions, while AML in general was enriched for changes at enhancers. There was a strong anticorrelation between CTCF binding and DNA methylation. Gain of CTCF occupancy was associated with increased gene expression; however, the genomic location (promoter vs distal regions) and enrichment of motifs (for repressing vs activating cofactors) were decisive for the gene expression pattern. Knockdown of CTCF in K562 cells caused loss of CTCF binding and transcriptional repression of genes with changed CTCF binding in AML, as well as loss of RUNX1 binding at RUNX1/CTCF-binding sites. In addition, CTCF knockdown caused increased differentiation. Azacitidine exposure caused major changes in CTCF occupancy in AML patient cells, partly by restoring a CTCF-binding pattern similar to NBM. We conclude that AML displays an aberrant increase in CTCF occupancy that targets key genes for AML development and impacts gene expression.

At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A(Cnp1), which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation through not only the RNA interference (RNAi) machinery but also RNAi-independent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here demonstrate that Mediator directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator colocalizes with Pol II at centromeres, and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired via both the RNAi-dependent and -independent pathways, resulting in loss of CENP-A(Cnp1) from the core centromere, a defect in kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20Δ cells appears to directly block CENP-A(Cnp1) incorporation since inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function.

Cohesin has essential roles in chromosome structure, segregation and repair. Cohesin binding to chromosomes is catalyzed by the cohesin loader, Mis4 in fission yeast. How cells fine tune cohesin deposition is largely unknown. Here we provide evidence that Mis4 activity is regulated by phosphorylation of its cohesin substrate. A genetic screen for negative regulators of Mis4 yielded a CDK called Pef1, whose closest human homologue is CDK5. Inhibition of Pef1 kinase activity rescued cohesin loader deficiencies. In an otherwise wild-type background, Pef1 ablation stimulated cohesin binding to its regular sites along chromosomes while ablating Protein Phosphatase 4 had the opposite effect. Pef1 and PP4 control the phosphorylation state of the cohesin kleisin Rad21. The CDK phosphorylates Rad21 on Threonine 262. Pef1 ablation, non phosphorylatable Rad21-T262 or mutations within a Rad21 binding domain of Mis4 alleviated the effect of PP4 deficiency. Such a CDK/PP4 based regulation of cohesin loader activity could provide an efficient mechanism for translating cellular cues into a fast and accurate cohesin response.

Heterochromatin regulation is critical for genomic stability. Different H3K9 methylation states have been discovered, with distinct roles in heterochromatin formation and silencing. However, how the transition from H3K9me2 to H3K9me3 is controlled is still unclear. Here, we investigate the role of the conserved bromodomain AAA-ATPase, Abo1, involved in maintaining global nucleosome organisation in fission yeast. We identified several key factors involved in heterochromatin silencing that interact genetically with Abo1: histone deacetylase Clr3, H3K9 methyltransferase Clr4, and HP1 homolog Swi6. Cells lacking Abo1 cultivated at 30 °C exhibit an imbalance of H3K9me2 and H3K9me3 in heterochromatin. In abo1∆ cells, the centromeric constitutive heterochromatin has increased H3K9me2 but decreased H3K9me3 levels compared to wild-type. In contrast, facultative heterochromatin regions exhibit reduced H3K9me2 and H3K9me3 levels in abo1∆. Genome-wide analysis showed that abo1∆ cells have silencing defects in both the centromeres and subtelomeres, but not in a subset of heterochromatin islands in our condition. Thus, our work uncovers a role of Abo1 in stabilising directly or indirectly Clr4 recruitment to allow the H3K9me2 to H3K9me3 transition in heterochromatin.

International audience; Retrotransposons, the ancestors of retroviruses, have the potential for gene disruption and genomic takeover if not kept in check. Paradoxically, although host cells repress these elements by multiple mechanisms, they are transcribed and are even activated under stress conditions. Here, we describe a new mechanism of retrotransposon regulation through transcription start site (TSS) selection by altered nucleosome occupancy. We show that Fun30 chromatin remodelers cooperate to maintain a high level of nucleosome occupancy at retrotransposon-flanking long terminal repeat (LTR) elements. This enforces the use of a downstream TSS and the production of a truncated RNA incapable of reverse transcription and retrotransposition. However, in stressed cells, nucleosome occupancy at LTR elements is reduced, and the TSS shifts to allow for productive transcription. We propose that controlled retrotransposon transcription from a nonproductive TSS allows for rapid stress-induced activation, while preventing uncontrolled transposon activity in the genome.

The maintenance of open and repressed chromatin states is crucial for the regulation of gene expression. To study the genes involved in maintaining chromatin states, we generated a random mutant library in Schizosaccharomyces pombe and monitored the silencing of reporter genes inserted into the euchromatic region adjacent to the heterochromatic mating type locus. We show that Leo1–Paf1 [a subcomplex of the RNA polymerase II‐associated factor 1 complex (Paf1C)] is required to prevent the spreading of heterochromatin into euchromatin by mapping the heterochromatin mark H3K9me2 using high‐resolution genomewide ChIP (ChIP–exo). Loss of Leo1–Paf1 increases heterochromatin stability at several facultative heterochromatin loci in an RNAi‐independent manner. Instead, deletion of Leo1 decreases nucleosome turnover, leading to heterochromatin stabilization. Our data reveal that Leo1–Paf1 promotes chromatin state fluctuations by enhancing histone turnover.

Here, we review the role of sucrose nonfermenting (SNF2) family enzymes in blood cell development. The SNF2 family comprises helicase-like ATPases, originally discovered in yeast, that can remodel chromatin by changing chromatin structure and composition. The human genome encodes 30 different SNF2 enzymes. SNF2 family enzymes are often part of multisubunit chromatin remodeling complexes (CRCs), which consist of noncatalytic/auxiliary subunit along with the ATPase subunit. However, blood cells express a limited set of SNF2 ATPases that are necessary to maintain the pool of hematopoietic stem cells (HSCs) and drive normal blood cell development and differentiation. The composition of CRCs can be altered by the association of specific auxiliary subunits. Several auxiliary CRC subunits have specific functions in hematopoiesis. Aberrant expressions of SNF2 ATPases and/or auxiliary CRC subunit(s) are often observed in hematological malignancies. Using large-scale data from the International Cancer Genome Consortium (ICGC) we observed frequent mutations in genes encoding SNF2 helicase-like enzymes and auxiliary CRC subunits in leukemia. Hence, orderly function of SNF2 family enzymes is crucial for the execution of normal blood cell developmental program, and defects in chromatin remodeling caused by mutations or aberrant expression of these proteins may contribute to leukemogenesis.

Here we describe how mating-type tests are conducted in

In this introduction we discuss some basic genetic tools and techniques that are used with the fission yeast

Here we provide an ethyl methanesulfonate (EMS) mutagenesis protocol for

Here we describe the processing of

Here we provide methods for setting up standard crosses with

Here we describe procedures for the selection of diploid

RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.

Eukaryotic chromatin is remodelled by the evolutionarily conserved Snf2 family of enzymes in an ATP-dependent manner. Several Snf2 enzymes are part of CRCs (chromatin remodelling complexes). In the present review we focus our attention on the functions of Snf2 enzymes and CRCs in fission yeast. We discuss their molecular mechanisms and roles and in regulating gene expression, DNA recombination, euchromatin and heterochromatin structure.

The spatial organization of the genome inside the nucleus affects many nuclear processes, such as DNA replication, DNA repair, and gene transcription. In metazoans, the nuclear periphery harbors mainly repressed genes that associate with the nuclear lamina. This review discusses how peripheral positioning is connected to transcriptional regulation in yeasts. Tethering of reporter genes to the nuclear envelope was found to result in transcriptional silencing. Similarly, repression of the silent mating type loci and subtelomeric genes is influenced by their position close to the nuclear envelope. In contrast, active genes are bound by nucleoporins and inducible genes associate with the nuclear pore complex upon activation. Taken together, these results portray the nuclear envelope as a platform for transcriptional regulation, both through activation at nuclear pores and silencing at the nuclear envelope.

Acute myeloid leukemia (AML) is characterized by an impaired differentiation process leading to an accumulation of immature blasts in the blood. One feature of cytogenetically normal AML is alterations to the DNA methylome. We analyzed 57 AML patients with normal karyotype by using Illumina's 450k array and showed that aberrant DNA methylation is significantly altered at enhancer regions and that the methylation levels at specific enhancers predict overall survival of AML patients. The majority of sites that become differentially methylated in AML occur in regulatory elements of the human genome. Hypermethylation associates with enhancer silencing. In addition, chromatin immunoprecipitation sequencing analyses showed that a subset of hypomethylated sites correlate with enhancer activation, indicated by increased H3K27 acetylation. DNA hypomethylation is therefore not sufficient for enhancer activation. Some sites of hypomethylation occur at weak/poised enhancers marked with H3K4 monomethylation in hematopoietic progenitor cells. Other hypomethylated regions occur at sites inactive in progenitors and reflect the de novo acquisition of AML-specific enhancers. Altered enhancer dynamics are reflected in the gene expression of enhancer target genes, including genes involved in oncogenesis and blood cell development. This study demonstrates that histone variants and different histone modifications interact with aberrant DNA methylation and cause perturbed enhancer activity in cytogenetically normal AML that contributes to a leukemic transcriptome.

Early therapeutic decision-making is crucial in patients with higher-risk MDS where median survival is only around one year. Azacitidine prolongs survival for these patients (Fenaux et al, Lancet Oncology 2009) but clinically relevant biomarkers remain to be identified. We evaluated retrospectively, the impact of clinical parameters and mutational profiles in 134 consecutive patients treated with a median number of 7 cycles of Azacitidine (range 1-45), in accordance to European guidelines. The vast majority (n=114) had higher-risk disease i.e. MDS with IPSS int-2 or high, AML with multilinear dysplasia and 20-30% blasts or CMML-II. We combined an initial cohort from Karolinska University Hospital (n=89) with a validating cohort from King's College Hospital, London (n=45). Studied endpoints were response, as defined by the IWG criteria, and survival. Since prolonged survival is the main goal for this cohort of patients, we believe that survival is the most relevant endpoint, supported by the fact that even non-responding patients have a survival benefit from Azacitidine (Gore et al, Haematologica, 2013). While neither clinical parameters nor mutations had a significant impact on response rate, both karyotype and mutational profile were strongly associated with survival from the start of treatment, see Table 1 and Figure 1-2. IPSS high-risk cytogenetics was negatively associated with survival (median 20 vs 10 months; p Table 1. Variables associated with survival. Univariate analyses used the log-rank test. The cox model included all listed variables except response rate in a multivariate analyses. Estimated median survival (months) Univariate p-value Cox regression p-value Hazard ratio (95% CI) Response rate: CR / mCR vs PR/HI vs SD/PD 20 vs 20 vs 10 Figure 1. Kaplan-Meier estimated survival stratified for response and pre-treatment parameters Figure 1. Kaplan-Meier estimated survival stratified for response and pre-treatment parameters Figure 2. Forest plot indicating hazard ratio including confidence interval for all pre-treatment variables. The hazard ratios were retrieved using cox univariate regression models for each variable analyzed separately. Figure 2. Forest plot indicating hazard ratio including confidence interval for all pre-treatment variables. The hazard ratios were retrieved using cox univariate regression models for each variable analyzed separately. Figure 3. Kaplan-Meier estimated survival stratified for the two dominant predictors in the cox regression model: Adverse cytogenetics and histone modulator mutations Figure 3. Kaplan-Meier estimated survival stratified for the two dominant predictors in the cox regression model: Adverse cytogenetics and histone modulator mutations Disclosures McLornan: Novartis: Research Funding, Speakers Bureau. Jädersten:Celgene: Other: speakers fee. Kulasekararaj:Alexion: Consultancy. Mufti:Celgene: Consultancy, Other: Speakers fee. Hellström-Lindberg:Celgene Corporation: Research Funding.

The DNA glycosylase MutY homolog (Myh1) excises adenines misincorporated opposite guanines or 7,8-dihydro-8-oxo-guanines on DNA by base excision repair thereby preventing G:C to T:A mutations. Schizosaccharomyces pombe (Sp) Hst4 is an NAD + -dependent histone/protein deacetylase involved in gene silencing and maintaining genomic integrity. Hst4 regulates deacetylation of histone 3 Lys56 at the entry and exit points of the nucleosome core particle. Here, we demonstrate that the hst4 mutant is more sensitive to H 2 O 2 than wild-type cells. H 2 O 2 treatment results in an SpMyh1-dependent decrease in SpHst4 protein level and hyperacetylation of histone 3 Lys56. Furthermore, SpHst4 interacts with SpMyh1 and the cell cycle checkpoint Rad9-Rad1-Hus1 (9-1-1) complex. SpHst4, SpMyh1, and SpHus1 are physically bound to telomeres. Following oxidative stress, there is an increase in the telomeric association of SpMyh1. Conversely, the telomeric association of spHst4 is decreased. Deletion of SpMyh1 strongly abrogated telomeric association of SpHst4 and SpHus1. However, telomeric association of SpMyh1 is enhanced in hst4 Δ cells in the presence of chronic DNA damage. These results suggest that SpMyh1 repair regulates the functions of SpHst4 and the 9-1-1 complex in maintaining genomic stability.

The binding of heterochromatin protein 1 (HP1) to lysine 9-methylated histone H3 (H3K9me) is an essential step in heterochromatin assembly. Chp2, an HP1-family protein in the fission yeast Schizosaccharomyces pombe, is required for heterochromatic silencing. Chp2 recruits SHREC, a multifunctional protein complex containing the nucleosome remodeler Mit1 and the histone deacetylase Clr3. Although the targeting of SHREC to chromatin is thought to occur via two distinct modules regulated by the SHREC components Chp2 and Clr2, it is not clear how Chp2's chromatin binding regulates SHREC function. Here, we show that H3K9me binding by Chp2's chromodomain (CD) is essential for Chp2's silencing function and for SHREC's targeting to chromatin. Cells expressing a Chp2 mutant with defective H3K9me binding (Chp2-W199A) have a silencing defect, with a phenotype similar to that of chp2-null cells. Genetic analysis using a synthetic silencing system revealed that a Chp2 mutant and SHREC-component mutants had similar phenotypes, suggesting that Chp2's function also affects SHREC's chromatin binding. Size-exclusion chromatography of native protein complexes showed that Chp2-CD's binding of H3K9me3 ensures Clr3's chromatin binding, and suggested that SHREC's chromatin binding is mediated by separable functional modules. Interestingly, we found that the stability of the Chp2 protein depended on the Clr3 protein's histone deacetylase activity. Our findings demonstrate that Chp2's H3K9me binding is critical for SHREC function and that the two modules within the SHREC complex are interdependent.

DNA topoisomerases regulate the topological state of the DNA double helix and are key enzymes in the processes of DNA replication, transcription and genome stability. Using the fission yeast model Schizosaccharomyces pombe, we investigate genome wide how DNA topoisomerases I and II affect chromatin dynamics and gene expression in vivo. We show that topoisomerase I activity is directly required for efficient nucleosome disassembly at gene promoter regions. Lack of topoisomerase activity results in increased nucleosome occupancy, perturbed histone modifications and reduced transcription from these promoters. Strong correlative evidence suggests that topoisomerase I cooperates with the ATP-dependent chromatin remodeller Hrp1 in nucleosome disassembly. Our study links topoisomerase activity to the maintenance of open chromatin and regulating transcription in vivo.

Aims: To map histone modifications with unprecedented resolution both globally and locus-specifically, and to link modification patterns to gene expression. Materials & methods: Using correlations between quantitative mass spectrometry and chromatin immunoprecipitation/microarray analyses, we have mapped histone post-translational modifications in fission yeast (Schizosaccharomyces pombe). Results: Acetylations at lysine 9, 18 and 27 of histone H3 give the best positive correlations with gene expression in this organism. Using clustering analysis and gene ontology search tools, we identified promoter histone modification patterns that characterize several classes of gene function. For example, gene promoters of genes involved in cytokinesis have high H3K36me2 and low H3K4me2, whereas the converse pattern is found ar promoters of gene involved in positive regulation of the cell cycle. We detected acetylation of H4 preferentially at lysine 16 followed by lysine 12, 8 and 5. Our analysis shows that this H4 acetylation bias in the coding regions is dependent upon gene length and linked to gene expression. Our analysis also reveals a role for H3K36 methylation at gene promoters where it functions in a crosstalk between the histone methyltransferase Set2KMT3 and the histone deacetylase Clr6, which removes H3K27ac leading to repression of transcription. Conclusion: Histone modification patterns could be linked to gene expression in fission yeast.

The cell fate decision leading to gametogenesis requires the convergence of multiple signals on the promoter of a master regulator. In fission yeast, starvation-induced signaling leads to the transcriptional induction of the ste11 gene, which encodes the central inducer of mating and gametogenesis, known as sporulation. We find that the long intergenic non-coding (linc) RNA rse1 is transcribed divergently upstream of the ste11 gene. During vegetative growth, rse1 directly recruits a Mug187-Lid2-Set1 complex that mediates cis repression at the ste11 promoter through SET3C-dependent histone deacetylation. The absence of rse1 bypasses the starvation-induced signaling and induces gametogenesis in the presence of nutrients. Our data reveal that the remodeling of chromatin through ncRNA scaffolding of repressive complexes that is observed in higher eukaryotes is a conserved, likely very ancient mechanism for tight control of cell differentiation. Fauquenoy et al. report the first example of a yeast long intergenic non-coding RNA (lincRNA) that regulates the expression of the neighboring ste11 gene through scaffolding of a repressive complex. This constitutes an RNA-based mechanism of repression of cell differentiation.

The eukaryotic DNA is wrapped around histone octamers, which consist of four different histones, H2A, H2B, H3 and H4. The N-terminal tail of each histone is post-transcriptionally modified. The modification patterns constitute codes that regulate chromatin organisation and DNA utilization processes, including transcription. Recent progress in technology development has made it possible to perform systematic genome-wide studies of histone modifications. This helps immensely in deciphering the histone codes and their biological influence. In this review, we discuss the histone modification patterns found in genome-wide studies in different biological models and how they influence cell differentiation and carcinogenesis.

Histone acetylation and deacetylation are important for gene regulation. The histone acetyltransferase, Gcn5, is an activator of transcriptional initiation that is recruited to gene promoters. Here, we map genome-wide Gcn5 occupancy and histone H3K14ac at high resolution. Gcn5 is predominantly localized to coding regions of highly transcribed genes, where it collaborates antagonistically with the class-II histone deacetylase, Clr3, to modulate H3K14ac levels and transcriptional elongation. An interplay between Gcn5 and Clr3 is crucial for the regulation of many stress-response genes. Our findings suggest a new role for Gcn5 during transcriptional elongation, in addition to its known role in transcriptional initiation.

The majority of nuclear eukaryotic DNA is packaged into nucleosome cores where DNA is wrapped tightly around histone protein octamers. Such histone bound nucleosomal DNA is less accessible than the short linker DNA between nucleosome cores or the DNA in extended nucleosome free regions. Therefore, the positions of nucleosomes relative to a DNA sequence feature, like a transactivator binding site, a transcriptional start site or an origin of replication, can have profound effects on nuclear processes like transcription, replication, recombination and repair. Now that many DNA related processes are studied in a genome-wide manner, it is increasingly important to map the basic organization of their chromosomal DNA substrate, i.e., the positions of nucleosomes, on a genome-wide scale as well. To this end, the protection of nucleosomal DNA from digestion with micrococcal nuclease (MNase) is used as an assay for the presence of a nucleosome. The MNase protected DNA fragments, so called mononucleosomal DNA, can be mapped genome-wide by hybridization to microarrays. This method has been established for Saccharomyces cerevisiae, and we present here the adaptation of the method for Schizosaccharomyces pombe. As an independent method to validate genome-wide data for individual loci, we also include a protocol for the determination of locus specific nucleosome positioning by indirect end labeling.

The term epigenetics refers to heritable changes not encoded by DNA. The organization of DNA into chromatin fibers affects gene expression in a heritable manner and is therefore one mechanism of epigenetic inheritance. Large parts of eukaryotic genomes consist of constitutively highly condensed heterochromatin, important for maintaining genome integrity but also for silencing of genes within. Small RNA, together with factors typically associated with RNA interference (RNAi) targets homologous DNA sequences and recruits factors that modify the chromatin, commonly resulting in formation of heterochromatin and silencing of target genes. The scope of this review is to provide an overview of the roles of small RNA and the RNAi components, Dicer, Argonaute and RNA dependent polymerases in epigenetic inheritance via heterochromatin formation, exemplified with pathways from unicellular eukaryotes, plants and animals.

Heterochromatin formation at fission yeast centromeres is directed by RNA interference (RNAi). Noncoding transcripts derived from centromeric repeats are processed into small interfering RNAs (siRNAs) that direct the RNA-induced transcriptional silencing (RITS) effector complex to engage centromere transcripts, resulting in recruitment of the histone H3 lysine 9 methyltransferase Clr4, and hence silencing. We have found that defects in specific splicing factors, but not splicing itself, affect the generation of centromeric siRNAs and consequently centromeric heterochromatin integrity. Moreover, splicing factors physically associate with Cid12, a component of the RNAi machinery, and with centromeric chromatin, consistent with a direct role in RNAi. We propose that spliceosomal complexes provide a platform for siRNA generation and hence facilitate effective centromere repeat silencing.

The centromere is the DNA region that ensures genetic stability and is therefore of vital importance. Paradoxically, centromere proteins and centromeric structural domains are conserved despite that fact that centromere DNA sequences are highly variable and are not conserved. Remarkably, heritable states at the centromere can be propagated independent of the underlying centromeric DNA sequences. This review describes the epigenetic mechanisms governing centromere behavior, i.e., the mechanisms that control centromere assembly and propagation. A centromeric histone variant, CenH3, and histone modifications play key roles at centromeric chromatin. Histone modifications and RNA interference are important in assembly of pericentric heterochromatin structures. The molecular machinery that is directly involved in epigenetic control of centromeres is shared with regulation of gene expression. Nucleosome remodeling factors, histone chaperones, histone-modifying enzymes, transcription factors, and even RNA polymerase II itself control epigenetic states at centromeres.

Chromatin remodelling factors and histone chaperones were previously shown to cooperatively affect nucleosome assembly and disassembly processes in vitro. Here, we show that Schizosaccharomyces pombe CHD remodellers, the Hrp1 and Hrp3 paralogs physically interact with the histone chaperone Nap1. Genome-wide analysis of Hrp1, Hrp3 and Nap1 occupancy, combined with nucleosome density measurements revealed that the CHD factors and Nap1 colocalized in particular to promoter regions where they remove nucleosomes near the transcriptional start site. Hrp1 and Hrp3 also regulate nucleosome density in coding regions, where they have redundant roles to stimulate transcription. Previously, DNA replication-dependent and -independent nucleosome disassembly processes have been described. We found that nucleosome density increased in the hrp1 mutant in the absence of DNA replication. Finally, regions where nucleosome density increased in hrp1, hrp3 and nap1 mutants also showed nucleosome density and histone modification changes in HDAC and HAT mutants. Thus, this study revealed an important in vivo role for CHD remodellers and Nap1 in nucleosome disassembly at promoters and coding regions, which are linked to changes in histone acetylation.

The Saccharomyces cerevisiae Ssn6 and Tup1 proteins form a corepressor complex that is recruited to target genes by DNA-bound repressor proteins. Repression occurs via several mechanisms, including interaction with hypoacetylated N termini of histones, recruitment of histone deacetylases (HDACs), and interactions with the RNA polymerase II holoenzyme. The distantly related fission yeast, Schizosaccharomyces pombe, has two partially redundant Tup1-like proteins that are dispensable during normal growth. In contrast, we show that Ssn6 is an essential protein in S. pombe, suggesting a function that is independent of Tup11 and Tup12. Consistently, the group of genes that requires Ssn6 for their regulation overlaps but is distinct from the group of genes that depend on Tup11 or Tup12. Global chip-on-chip analysis shows that Ssn6 is almost invariably found in the same genomic locations as Tup11 and/or Tup12. All three corepressor subunits are generally bound to genes that are selectively regulated by Ssn6 or Tup11/12, and thus, the subunit specificity is probably manifested in the context of a corepressor complex containing all three subunits. The corepressor binds to both the intergenic and coding regions of genes, but differential localization of the corepressor within genes does not appear to account for the selective dependence of target genes on the Ssn6 or Tup11/12 subunits. Ssn6, Tup11, and Tup12 are preferentially found at genomic locations at which histones are deacetylated, primarily by the Clr6 class I HDAC. Clr6 is also important for the repression of corepressor target genes. Interestingly, a subset of corepressor target genes, including direct target genes affected by Ssn6 overexpression, is associated with the function of class II (Clr3) and III (Hst4 and Sir2) HDACs.

Background CHD1 and CHD2 chromatin remodeling enzymes play important roles in development, cancer and differentiation. At a molecular level, the mechanisms are not fully understood but include transcriptional regulation, nucleosome organization and turnover. Results Here we show human CHD1 and CHD2 enzymes co-occupy active chromatin regions associated with transcription start sites (TSS), enhancer like regions and active tRNA genes. We demonstrate that their recruitment is transcription-coupled. CHD1 and CHD2 show distinct binding profiles across active TSS regions. Depletion of CHD1 influences chromatin accessibility at TSS and enhancer-like chromatin regions. CHD2 depletion causes increased histone H3 and reduced histone variant H3.3 occupancy. Conclusions We conclude that transcription-coupled recruitment of CHD1 and CHD2 occurs at transcribed gene TSSs and at intragenic and intergenic enhancer-like sites. The recruitment of CHD1 and CHD2 regulates the architecture of active chromatin regions through chromatin accessibility and nucleosome disassembly. Electronic supplementary material The online version of this article (doi:10.1186/1756-8935-8-4) contains supplementary material, which is available to authorized users.

Heterochromatin underpins gene repression, genome integrity, and chromosome segregation. In the fission yeast Schizosaccharomyces pombe, conserved protein complexes effect heterochromatin formation via RNA interference-mediated recruitment of a histone H3 lysine 9 methyltransferase to cognate chromatin regions. To identify small molecules that inhibit heterochromatin formation, we performed an in vivo screen for loss of silencing of a dominant selectable kanMX reporter gene embedded within fission yeast centromeric heterochromatin. Two structurally unrelated compounds, HMS-I1 and HMS-I2, alleviated kanMX silencing and decreased repressive H3K9 methylation levels at the transgene. The decrease in methylation caused by HMS-I1 and HMS-I2 was observed at all loci regulated by histone methylation, including centromeric repeats, telomeric regions, and the mating-type locus, consistent with inhibition of the histone deacetylases (HDACs) Clr3 and/or Sir2. Chemical-genetic epistasis and expression profiles revealed that both compounds affect the activity of the Clr3-containing Snf2/HDAC repressor complex (SHREC). In vitro HDAC assays revealed that HMS-I1 and HMS-I2 inhibit Clr3 HDAC activity. HMS-I1 also alleviated transgene reporter silencing by heterochromatin in Arabidopsis and a mouse cell line, suggesting a conserved mechanism of action. HMS-I1 and HMS-I2 bear no resemblance to known inhibitors of chromatin-based activities and thus represent novel chemical probes for heterochromatin formation and function.

The centromere is the chromosomal region where the kinetochore forms. The kinetochore is a protein structure mediating contacts with the mitotic and meiotic spindle microtubule structures, enabling separation of chromosomes in mitosis and meiosis. Failure of the chromosome segregation machinery either by inactivation of the centromere or malfunction of the kinetochore or spindle results in chromosome mis-segregation and aneuploidy of the daughter cells. Aneuploidy is associated with birth defects or cancer. Genome stability can also be compromised by activation of extra centromeres (neocentromeres). Thus, to ensure euploidy and correct chromosome segregation, epigenetic activation of a single centromere per chromosome is required. The centromere is marked epigenetically by incorporation of the histone H3 variant CENP-A in the centromeric chromatin. In most organisms endogenous centromeres are surrounded and stabilized by adjacent heterochromatin, together constituting regional centromeres. In some organisms, such as budding yeast, point centromeres of a single nucleosome exist without pericentric heterochromatin. Yet, in other organisms, for example the worm C. elegans, the entire chromosome forms a holocentric centromere [1]. The centromere and kinetochore proteins are highly conserved in eukaryotes and the underlying DNA sequence is composed of complex repeats in most organisms. By contrast with the conserved proteins, the repetitive sequences are highly variable, both between species and individuals [2]. For technical reasons, endogenous human centromeres have been challenging to study and most of the mechanistic insights come from studies of human artificial chromosomes or centromeres in genetically amenable model organisms, such as budding and fission yeasts and the fruit fly. In fission yeast for example, the borders between CENP-A chromatin and the surrounding heterochromatin are clearly defined and the underlying DNA repeats are distinct. However, in human cells, the alpha satellite repeats make up the majority of the centromere DNA and heterochromatin is interspersed with CENP-A chromatin. Recent technical advances such as the delineation of the human centromere repeats, superresolution microscopy and the proximity ligation assay allow us to embark on more detailed examinations of the regulation of the human centromere and how it might contribute to disease. For an acentric chromosome fragment to be propagated through somatic cell divisions and through the germ line of a eukaryotic organism, a new centromere needs to form. A so-called ‘neocentromere’ is a functional centromere structure that forms at a nontypical genomic region and at DNA sequences that may differ from endogenous centromeres [3]. Such neocentromeres have been observed to spontaneously occur in several cancers characterized by genetically instability [4] and, occasionally, in cells that have gone through many cell divisions. Alternatively, the centromere formation can be studied experimentally by artificially removEpigenetic activation and inactivation of centromeres

RNA interference (RNAi) acts on long double-stranded RNAs (dsRNAs) in a variety of eukaryotes to generate small interfering RNAs that target homologous messenger RNA, resulting in their destruction. This process is widely used to 'knock-down' the expression of genes of interest to explore phenotypes(1-3). In plants(3-5), fission yeast(6-8), ciliates(9,10), flies(11) and mammalian cells(12,13), short interfering RNAs (siRNAs) also induce DNA or chromatin modifications at the homologous genomic locus, which can result in transcriptional silencing or sequence elimination(14). siRNAs may direct DNA or chromatin modification by siRNA - DNA interactions at the homologous locus(4,5). Alternatively, they may act by interactions between siRNA and nascent transcript(15,16). Here we show that in fission yeast ( Schizosaccharomyces pombe), chromatin modifications are only directed by RNAi if the homologous DNA sequences are transcribed. Furthermore, transcription by exogenous T7 polymerase is not sufficient. Ago1, a component of the RNAi effector RISC/RITS complex, associates with target transcripts and RNA polymerase II. Truncation of the regulatory carboxy-terminal domain (CTD) of RNApol II disrupts transcriptional silencing, indicating that, like other RNA processing events(17-19), RNAi-directed chromatin modification is coupled to transcription.

In the fission yeast Schizosaccharomyces pombe, three genes that function in the RNA interference (RNAi) pathway, ago1+, dcr1+, and rdp1+, have recently been shown to be important for timely formation of heterochromatin and accurate chromosome segregation. In the present study, we present evidence that null mutants for ago1+and dcr1+but not rdp1+, exhibit abnormal cytokinesis, cell cycle arrest deficiencies, and mating defects. Subsequent analyses showed that ago1+and dcr1+are required for regulated hyperphosphorylation of Cdc2 when encountering genotoxic insults. Because rdp1+is dispensable for this process, the functions of ago1+and dcr1+in this pathway are presumably independent of their roles in RNAi-mediated heterochromatin formation and chromosome segregation. This was further supported by the finding that ago1+is a multicopy suppressor of the S-M checkpoint deficiency and cytokinesis defects associated with loss of Dcr1 function, but not for the chromosome segregation defects of this mutant. Accordingly, we conclude that Dcr1-dependent production of small interfering RNAs is not required for enactment and/or maintenance of certain cell cycle checkpoints and that Ago1 and Dcr1 functionally diverge from Rdp1 to control cell cycle events in fission yeast. Finally, exogenous expression of hGERp95/EIF2C2/hAgo2, a human Ago1 homolog implicated in posttranscriptional gene silencing, compensated for the loss of ago1+function in S. pombe. This suggests that PPD proteins may also be important for regulation of cell cycle events in higher eukaryotes.

Completion of the fission yeast genome sequence has opened up possibilities for post-genomic approaches. We have constructed a DNA microarray for genome-wide gene expression analysis in fission yeast. The microarray contains DNA fragments, PCR-amplified from a genomic DNA template, that represent99% of the 5000 or so annotated fission yeast genes, as well as a number of control sequences. The GenomePRIDE software used attempts to design similarly sized DNA fragments corresponding to gene regions within single exons, near the 3'-end of genes that lack homology to other fission yeast genes. To validate the design and utility of the array, we studied expression changes after a 2 h temperature shift from 25 degrees C to 36 degrees C, conditions widely used when studying temperature-sensitive mutants. Obligingly, the vast majority of genes do not change more than two-fold, supporting the widely held view that temperature-shift experiments specifically reveal phenotypes associated with temperature-sensitive mutants. However, we did identify a small group of genes that showed a reproducible change in expression. Importantly, most of these corresponded to previously characterized heat-shock genes, whose expression has been reported to change after more extreme temperature shifts than those used here. We conclude that the DNA microarray represents a useful resource for fission yeast researchers as well as the broader yeast community, since it will facilitate comparison with the distantly related budding yeast, Saccharomyces cerevisiae. To maximize the utility of this resource, the array and its component parts are fully described in On-line Supplementary Information and are also available commercially.

The analysis of the Schizosaccharomyces pombe genome revealed the presence of 14 putative P-type ATPases. The clustering of ATPases resembles that of Saccharomyces cerevisiae, indicating that the main classes of pumps were already present before the split of the Archiascomycetes from the other Ascomycota. The overall amino acid identity between fission and budding yeast P-type ATPases is generally low (30-50%). This is similar to the fungus-plant and fungus-animal comparisons, suggesting that fungal ATPases underwent an extensive process of diversification. Unlike Sac. cerevisiae, fission yeast lacks Na(+)-ATPases, has a single heavy-metal ATPase and three ATPases of unknown specificity. The observed divergence within these fungi might reflect physiological differences, including adaptation to environmental stresses.

RNA interference is a form of gene silencing in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. Here we report a role for Dicer in chromosome segregation of fission yeast. Deletion of the Dicer ( dcr1 + ) gene caused slow growth, sensitivity to thiabendazole, lagging chromosomes during anaphase, and abrogated silencing of centromeric repeats. As Dicer in other species, Dcr1p degraded double-stranded RNA into ≈23 nucleotide fragments in vitro , and dcr1 Δ cells were partially rescued by expression of human Dicer, indicating evolutionarily conserved functions. Expression profiling demonstrated that dcr1 + was required for silencing of two genes containing a conserved motif.

Hrp3, a paralog of Hrp1, is a novel member of the CHD1 (chromo-helicase/ATPase-DNA binding 1) protein family of Schizosaccharomyces pombe. Although it has been considered that CHD1 proteins are required for chromatin modifications in transcriptional regulations, little is known about their roles in vivo. In this study, we examined the effects of Hrp3 on heterochromatin silencing using several S. pombe reporter strains. The phenotypic analysis revealed that hrp3(+) is not an essential gene for cell viability. However, Hrp3 is required for transcriptional repression at silence loci of mat3. A chromatin immunoprecipitation assay showed that Hrp3 directly associates with mat3 chromatin. Thus, our results strongly suggest that Hrp3 is involved in heterochromatin silencing and plays a direct role as a chromatin remodeling factor at mat3 in vivo.

Centromere function requires the proper coordination of several subfunctions, such as kinetochore assembly, sister chromatid cohesion, binding of kinetochore microtubules, orientation of sister kinetochores to opposite spindle poles, and their movement towards the spindle poles. Centromere structure appears to be organized in different, separable domains in order to accomplish these functions. Despite the conserved nature of centromere functions, the molecular genetic definition of the DNA sequences that form a centromere in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, in the fruit fly Drosophila melanogaster, and in humans has revealed little conservation at the level of centromere DNA sequences. Also at the protein level few centromere proteins are conserved in all of these four organisms and many are unique to the different organisms. The recent analysis of the centromere structure in the yeast S. pombe by electron microscopy and detailed immunofluorescence microscopy of Drosophila centromeres have brought to light striking similarities at the overall structural level between these centromeres and the human centromere. The structural organization of the centromere is generally multilayered with a heterochromatin domain and a central core/inner plate region, which harbors the outer plate structures of the kinetochore. It is becoming increasingly clear that the key factors for assembly and function of the centromere structure are the specialized histones and modified histones which are present in the centromeric heterochromatin and in the chromatin of the central core. Thus, despite the differences in the DNA sequences and the proteins that define a centromere, there is an overall structural similarity between centromeres in evolutionarily diverse eukaryotes.

In this introduction we discuss some basic genetic tools and techniques that are used with the fission yeast Schizosaccharomyces pombe. Genes commonly used for selection or as reporters are discussed, with an emphasis on genes that permit counterselection, intragenic complementation, or colony-color assays. S. pombe is most stable as a haploid organism. We describe its mating-type system, how to perform genetic crosses and methods for selecting and propagating diploids. We discuss the relative merits of tetrad dissection and random spore preparation in strain construction and genetic analyses. Finally, we present several types of mutant screens, with an evaluation of their respective strengths and limitations in the light of emerging technologies such as next-generation sequencing.

Here we describe how mating-type tests are conducted in Schizosaccharomyces pombe. Two methods can be employed: matings with h− and h+ tester strains and polymerase chain reaction (PCR) for mat1 content.

Here we describe procedures for the selection of diploid Schizosaccharomyces pombe. ade6-M210/ade6-M216 heteroallelic complementation is widely used to select for Ade+ diploids. Such diploids will readily sporulate when starved of nitrogen. For some investigations, stable diploids are preferable (e.g., for genetic complementation tests), and in these cases mating an h− strain with an h90 mat2-Pi-102 strain can be used to prevent sporulation. When ade6-M210/ade6-M216 mutations impact on, or show synthetic interactions with, the gene of interest, two different auxotrophic markers can be used to select complementation.

Here we describe the processing of Schizosaccharomyces pombe spores in batches (random spore analysis) or through tetrad dissections. Spores are usually prepared from matings between haploid strains (producing zygotic asci) or from sporulating diploids (producing azygotic asci). In random spore analysis, a snail enzyme preparation is used to digest the walls of asci to release free spores that are diluted and plated to form colonies. In tetrad dissection, a needle attached to a micromanipulator is used to pick asci and separate spores. Tetrad dissection has traditionally been the method of choice for genetic mapping and is very useful in the study of genetic interactions (e.g., suppressor analysis). It is also the preferred method for routine crosses because it ensures that every colony stems from a single spore. This can never be certain in random spore analysis.

Hematopoietic differentiation is governed by a complex regulatory program controlling the generation of different lineages of blood cells from multipotent hematopoietic stem cells. The transcriptional program that dictates hematopoietic cell fate and differentiation requires an epigenetic memory function provided by a network of epigenetic factors regulating DNA methylation, posttranslational histone modifications, and chromatin structure. Aberrant interactions between epigenetic factors and transcription factors cause perturbations in the blood cell differentiation program that result in various types of hematopoietic disorders. To elucidate the contributions of different epigenetic factors in human hematopoiesis, high-throughput cap analysis of gene expression was used to build transcription profiles of 199 epigenetic factors in a wide range of blood cells. Our epigenetic transcriptome analysis revealed cell type- (eg, HELLS and ACTL6A), lineage- (eg, MLL), and/or leukemia- (eg, CHD2, CBX8, and EPC1) specific expression of several epigenetic factors. In addition, we show that several epigenetic factors use alternative transcription start sites in different cell types. This analysis could serve as a resource for the scientific community for further characterization of the role of these epigenetic factors in blood development.

The organization of the genome into functional units, such as enhancers and active or repressed promoters, is associated with distinct patterns of DNA and histone modifications. The Encyclopedia of DNA Elements (ENCODE) project has advanced our understanding of the principles of genome, epigenome and chromatin organization, identifying hundreds of thousands of potential regulatory regions and transcription factor binding sites. Part of the ENCODE consortium, GENCODE, has annotated the human genome with novel transcripts including new noncoding RNAs and pseudogenes, highlighting transcriptional complexity. Many disease variants identified in genome-wide association studies are located within putative enhancer regions defined by the ENCODE project. Understanding the principles of chromatin and epigenome organization will help to identify new disease mechanisms, biomarkers and drug targets, particularly as ongoing epigenome mapping projects generate data for primary human cell types that play important roles in disease.

Functional centromeres are essential for proper cell division. Centromeres are established largely by epigenetic processes resulting in incorporation of the histone H3 variant CENP-A. Here, we demonstrate the direct involvement of H2B monoubiquitination, mediated by RNF20 in humans or Brl1 in Schizosaccharomyces pombe, in centromeric chromatin maintenance. Monoubiquinated H2B (H2Bub1) is needed for this maintenance, promoting noncoding transcription, centromere integrity and accurate chromosomal segregation. A transient pulse of centromeric H2Bub1 leads to RNA polymerase II-mediated transcription of the centromere's central domain, coupled to decreased H3 stability. H2Bub1-deficient cells have centromere cores that, despite their intact centromeric heterochromatin barriers, exhibit characteristics of heterochromatin, such as silencing histone modifications, reduced nucleosome turnover and reduced levels of transcription. In the H2Bub1-deficient cells, centromere functionality is hampered, thus resulting in unequal chromosome segregation. Therefore, centromeric H2Bub1 is essential for maintaining active centromeric chromatin.