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Background Cellular quiescence is a reversible differentiation state during which cells modify their gene expression program to inhibit metabolic functions and adapt to a new cellular environment. The epigenetic changes accompanying these alterations are not well understood. We used fission yeast cells as a model to study the regulation of quiescence. When these cells are starved for nitrogen, the cell cycle is arrested in G1, and the cells enter quiescence (G0). A gene regulatory program is initiated, including downregulation of thousands of genes—for example, those related to cell proliferation—and upregulation of specific genes—for example, autophagy genes—needed to adapt to the physiological challenge. These changes in gene expression are accompanied by a marked alteration of nuclear organization and chromatin structure. Results Here, we investigated the role of Leo1, a subunit of the conserved RNA polymerase-associated factor 1 (Paf1) complex, in the quiescence process using fission yeast as the model organism. Heterochromatic regions became very dynamic in fission yeast in G0 during nitrogen starvation. The reduction of heterochromatin in early G0 was correlated with reduced target of rapamycin complex 2 (TORC2) signaling. We demonstrated that cells lacking Leo1 show reduced survival in G0. In these cells, heterochromatic regions, including subtelomeres, were stabilized, and the expression of many genes, including membrane transport genes, was abrogated. TOR inhibition mimics the effect of nitrogen starvation, leading to the expression of subtelomeric genes, and this effect was suppressed by genetic deletion of leo1. Conclusions We identified a protein, Leo1, necessary for survival during quiescence. Leo1 is part of a conserved protein complex, Paf1C, linked to RNA polymerase II. We showed that Leo1, acting downstream of TOR, is crucial for the dynamic reorganization of chromosomes and the regulation of gene expression during cellular quiescence. Genes encoding membrane transporters are not expressed in quiescent leo1 mutant cells, and cells die after 2 weeks of nitrogen starvation. Taken together, our results suggest that Leo1 is essential for the dynamic regulation of heterochromatin and gene expression during cellular quiescence. Electronic supplementary material The online version of this article (10.1186/s13072-019-0292-7) contains supplementary material, which is available to authorized users.

The Helicase-related protein 3 (Hrp3), an ATP-dependent chromatin remodeling enzyme from the CHD family, is crucial for maintaining global nucleosome occupancy in Schizosaccharomyces pombe (S. pombe). Although the ATPase domain of Hrp3 is essential for chromatin remodeling, the contribution of non-ATPase domains of Hrp3 is still unclear. Here, we investigated the role of non-ATPase domains using in vitro methods. In our study, we expressed and purified recombinant S. pombe histone proteins, reconstituted them into histone octamers, and assembled nucleosome core particles. Using reconstituted nucleosomes and affinity-purified wild type and mutant Hrp3 from S. pombe we created a homogeneous in vitro system to evaluate the ATP hydrolyzing capacity of truncated Hrp3 proteins. We found that all non-ATPase domain deletions (∆chromo, ∆SANT, ∆SLIDE, and ∆coupling region) lead to reduced ATP hydrolyzing activities in vitro with DNA or nucleosome substrates. Only the coupling region deletion showed moderate stimulation of ATPase activity with the nucleosome. Interestingly, affinity-purified Hrp3 showed co-purification with all core histones suggesting a strong association with the nucleosomes in vivo. However, affinity-purified Hrp3 mutant with SANT and coupling regions deletion showed complete loss of interactions with the nucleosomes, while SLIDE and chromodomain deletions reduced Hrp3 interactions with the nucleosomes. Taken together, nucleosome association and ATPase stimulation by DNA or nucleosomes substrate suggest that the enzymatic activity of Hrp3 is fine-tuned by unique contributions of all four non-catalytic domains.

Histone lysine methylations have primarily been linked to selective recruitment of reader or effector proteins that subsequently modify chromatin regions and mediate genome functions. Here, we describe a divergent role for histone H4 lysine 20 mono-methylation (H4K20me1) and demonstrate that it directly facilitates chromatin openness and accessibility by disrupting chromatin folding. Thus, accumulation of H4K20me1 demarcates highly accessible chromatin at genes, and this is maintained throughout the cell cycle. In vitro, H4K20me1-containing nucleosomal arrays with nucleosome repeat lengths (NRL) of 187 and 197 are less compact than unmethylated (H4K20me0) or trimethylated (H4K20me3) arrays. Concordantly, and in contrast to trimethylated and unmethylated tails, solid-state NMR data shows that H4K20 mono-methylation changes the H4 conformational state and leads to more dynamic histone H4-tails. Notably, the increased chromatin accessibility mediated by H4K20me1 facilitates gene expression, particularly of housekeeping genes. Altogether, we show how the methylation state of a single histone H4 residue operates as a focal point in chromatin structure control. While H4K20me1 directly promotes chromatin openness at highly transcribed genes, it also serves as a stepping-stone for H4K20me3-dependent chromatin compaction., The effect of histone H4 lysine 20 methylation (H4K20me) on chromatin accessibility are not well established. Here the authors show how H4K20 methylation regulates chromatin structure and accessibility to ensure precise transcriptional outputs through the cell cycle using genome-wide approaches, in vitro biophysical assays, and NMR.

Background The histone 3 lysine 4 (H3K4) monomethylase KMT2C is mutated across several cancer types; however, the effects of mutations on epigenome organization, gene expression, and cell growth are not clear. A frequently recurring mutation in colorectal cancer (CRC) with microsatellite instability is a single nucleotide deletion within the exon 38 poly-A(9) repeat (c.8390delA) which results in frameshift preceding the functional carboxy-terminal SET domain. To study effects of KMT2C expression in CRC cells, we restored one allele to wild type KMT2C in the two CRC cell lines RKO and HCT116, which both are homozygous c.8390delA mutant. Results Gene editing resulted in increased KMT2C expression, increased H3K4me1 levels, altered gene expression profiles, and subtle negative effects on cell growth, where higher dependence and stronger effects of KMT2C expression were observed in RKO compared to HCT116 cells. Surprisingly, we found that the two RKO and HCT116 CRC cell lines have distinct baseline H3K4me1 epigenomic profiles. In RKO cells, a flatter genome-wide H3K4me1 profile was associated with more increased H3K4me1 deposition at enhancers, reduced cell growth, and more differential gene expression relative to HCT116 cells when KMT2C was restored. Profiling of H3K4me1 did not indicate a highly specific regulation of gene expression as KMT2C-induced H3K4me1 deposition was found globally and not at a specific enhancer sub-set in the engineered cells. Although we observed variation in differentially regulated gene sets between cell lines and individual clones, differentially expressed genes in both cell lines included genes linked to known cancer signaling pathways, estrogen response, hypoxia response, and aspects of immune system regulation. Conclusions Here, KMT2C restoration reduced CRC cell growth and reinforced genome-wide H3K4me1 deposition at enhancers; however, the effects varied depending upon the H3K4me1 status of KMT2C deficient cells. Results indicate that KMT2C inactivation may promote colorectal cancer development through transcriptional dysregulation in several pathways with known cancer relevance.

The methylation of histone H3 at lysine 9 (H3K9me), performed by the methyltransferase Clr4/SUV39H, is a key event in heterochromatin assembly. In fission yeast, Clr4, together with the ubiquitin E3 ligase Cul4, forms the Clr4 methyltransferase complex (CLRC), whose physiological targets and biological role are currently unclear. Here, we show that CLRC-dependent H3 ubiquitylation regulates Clr4's methyltransferase activity. Affinity-purified CLRC ubiquitylates histone H3, and mass spectrometric and mutation analyses reveal that H3 lysine 14 (H3K14) is the preferred target of the complex. Chromatin immunoprecipitation analysis shows that H3K14 ubiquitylation (H3K14ub) is closely associated with H3K9me-enriched chromatin. Notably, the CLRC-mediated H3 ubiquitylation promotes H3K9me by Clr4, suggesting that H3 ubiquitylation is intimately linked to the establishment and/or maintenance of H3K9me. These findings demonstrate a cross-talk mechanism between histone ubiquitylation and methylation that is involved in heterochromatin assembly.

Histone H2B monoubiquitylation (H2Bub1) is tightly linked to RNA polymerase II transcription elongation, and is also directly implicated in DNA replication and repair. Loss of H2Bub1 is associated with defects in cell cycle progression, but how these are related to its various functions, and the underlying mechanisms involved, is not understood. Here we describe a role for H2Bub1 in the regulation of replication-dependent histone genes in the fission yeast Schizosaccharomyces pombe. H2Bub1 activates histone genes indirectly by suppressing antisense transcription of ams2+—a gene encoding a GATA-type transcription factor that activates histone genes and is required for assembly of centromeric chromatin. Mutants lacking the ubiquitylation site in H2B or the H2B-specific E3 ubiquitin ligase Brl2 had elevated levels of ams2+ antisense transcripts and reduced Ams2 protein levels. These defects were reversed upon inhibition of Cdk9—an ortholog of the kinase component of positive transcription elongation factor b (P-TEFb)—indicating that they likely resulted from aberrant transcription elongation. Reduced Cdk9 activity also partially rescued chromosome segregation phenotypes of H2Bub1 mutants. In a genome-wide analysis, loss of H2Bub1 led to increased antisense transcripts at over 500 protein-coding genes in H2Bub1 mutants; for a subset of these, including several genes involved in chromosome segregation and chromatin assembly, antisense derepression was Cdk9-dependent. Our results highlight antisense suppression as a key feature of cell cycle-dependent gene regulation by H2Bub1, and suggest that aberrant transcription elongation may underlie the effects of H2Bub1 loss on cell cycle progression.

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.

Analysis of large-scale interphase genome positioning with reference to a nuclear landmark has recently been studied using sequencing-based single cell approaches. However, these approaches are dependent upon technically challenging, time consuming and costly high throughput sequencing technologies, requiring specialized bioinformatics tools and expertise. Here, we propose a novel, affordable and robust microscopy-based single cell approach, termed Topokaryotyping, to analyze and reconstruct the interphase positioning of genomic loci relative to a given nuclear landmark, detectable as banding pattern on mitotic chromosomes. This is accomplished by proximity-dependent histone labeling, where biotin ligase BirA fused to nuclear envelope marker Emerin was coexpressed together with Biotin Acceptor Peptide (BAP)-histone fusion followed by (i) biotin labeling, (ii) generation of mitotic spreads, (iii) detection of the biotin label on mitotic chromosomes and (iv) their identification by karyotyping. Using Topokaryotyping, we identified both cooperativity and stochasticity in the positioning of emerin-associated chromatin domains in individual cells. Furthermore, the chromosome-banding pattern showed dynamic changes in emerin-associated domains upon physical and radiological stress. In summary, Topokaryotyping is a sensitive and reliable technique to quantitatively analyze spatial positioning of genomic regions interacting with a given nuclear landmark at the single cell level in various experimental conditions.

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.

// Magnus Tobiasson 1, * , Hani Abdulkadir 1, * , Andreas Lennartsson 2 , Shintaro Katayama 2 , Francesco Marabita 3, 4 , Ayla De Paepe 1 , Mohsen Karimi 1 , Kaarel Krjutskov 2, 5, 6 , Elisabet Einarsdottir 2, 5 , Michael Grovdal 1 , Monika Jansson 1 , Asmaa Ben Azenkoud 1 , Lina Corddedu 2 , Soren Lehmann 1, 7 , Karl Ekwall 2 , Juha Kere 2, 5 , Eva Hellstrom-Lindberg 1, * and Johanna Ungerstedt 1, * 1 Center for Hematology and Regenerative Medicine, Department of Medicine Huddinge, Division of Hematology Karolinska Institutet, Karolinska University Hospital Huddinge, Huddinge, Sweden 2 Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden 3 Unit of Computational Medicine, Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden 4 National Bioinformatics Infrastructure Sweden, Stockholm, Sweden 5 Molecular Neurology Research Program, University of Helsinki, and Folkhalsan Institute of Genetics, Helsinki, Finland 6 Competence Centre on Health Technologies, Tartu, Estonia 7 Department of Medical Sciences, Uppsala University, Uppsala, Sweden * These authors have contributed equally to this work Correspondence to: Magnus Tobiasson, email: johanna.ungerstedt@ki.se Keywords: MDS, azacitidine, epigenetics, DNA methylation, histone modifications Received: November 09, 2016 Accepted: February 01, 2017 Published: February 28, 2017 ABSTRACT Azacitidine (Aza) is first-line treatment for patients with high-risk myelodysplastic syndromes (MDS), although its precise mechanism of action is unknown. We performed the first study to globally evaluate the epigenetic effects of Aza on MDS bone marrow progenitor cells assessing gene expression (RNA seq), DNA methylation (Illumina 450k) and the histone modifications H3K18ac and H3K9me3 (ChIP seq). Aza induced a general increase in gene expression with 924 significantly upregulated genes but this increase showed no correlation with changes in DNA methylation or H3K18ac, and only a weak association with changes in H3K9me3. Interestingly, we observed activation of transcripts containing 15 endogenous retroviruses (ERVs) confirming previous cell line studies. DNA methylation decreased moderately in 99% of all genes, with a median β-value reduction of 0.018; the most pronounced effects seen in heterochromatin. Aza-induced hypomethylation correlated significantly with change in H3K9me3. The pattern of H3K18ac and H3K9me3 displayed large differences between patients and healthy controls without any consistent pattern induced by Aza. We conclude that the marked induction of gene expression only partly could be explained by epigenetic changes, and propose that activation of ERVs may contribute to the clinical effects of Aza in MDS.

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 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.

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.

Nucleosome composition actively contributes to chromatin structure and accessibility. Cells have developed mechanisms to remove or recycle histones, generating a landscape of differentially aged nucleosomes. This study aimed to create a high-resolution, genome-wide map of nucleosome turnover in Schizosaccharomyces pombe. The recombination-induced tag exchange (RITE) method was used to study replication-independent nucleosome turnover through the appearance of new histone H3 and the disappearance or preservation of old histone H3. The genome-wide location of histones was determined by chromatin immunoprecipitation–exonuclease methodology (ChIP-exo). The findings were compared with diverse chromatin marks, including histone variant H2A.Z, post-translational histone modifications, and Pol II binding. Finally, genome-wide mapping of the methylation states of H4K20 was performed to determine the relationship between methylation (mono, di, and tri) of this residue and nucleosome turnover. Our analysis showed that histone recycling resulted in low nucleosome turnover in the coding regions of active genes, stably expressed at intermediate levels. High levels of transcription resulted in the incorporation of new histones primarily at the end of transcribed units. H4K20 was methylated in low-turnover nucleosomes in euchromatic regions, notably in the coding regions of long genes that were expressed at low levels. This transcription-dependent accumulation of histone methylation was dependent on the histone chaperone complex FACT. Our data showed that nucleosome turnover is highly dynamic in the genome and that several mechanisms are at play to either maintain or suppress stability. In particular, we found that FACT-associated transcription conserves histones by recycling them and is required for progressive H4K20 methylation.

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.

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.

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.

Azacitidine induces profound genome-wide hypomethylation in primary myelodysplastic bone marrow cultures but may also reduce histone acetylation

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.

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-ACnp1 deposition in fission yeast. Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-ACnp1 incorporation at non-centromeric sites. FACT has little or no effect on CENP-ACnp1 assembly at endogenous centromeres where CENP-ACnp1 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-ACnp1 at specific loci, including subtelomeric regions, where CENP-ACnp1 is preferentially assembled. Moreover, defective Clr6-CII function allows the de novo assembly of CENP-ACnp1 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-ACnp1 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-ACnp1 chromatin and associated kinetochores. These findings have important implications for genetic and epigenetic processes involved in centromere specification., Author Summary Centromeres are the chromosomal locations at which kinetochores, the machinery that directs chromosome segregation, are assembled. In most eukaryotes, centromere location is epigenetically determined, meaning that the underlying DNA sequence does not dictate where they are formed. The genome is packaged in particles called nucleosomes, composed of histone proteins. Centromere DNA is wrapped around unusual nucleosomes that differ from those elsewhere in the genome because the histone H3-like CENP-A replaces the normal histone H3 component. We used fission yeast to investigate where CENP-ACnp1 nucleosomes are formed in cells containing excess CENP-ACnp1 and how the formation of these non-centromeric CENP-A nucleosomes is controlled. H3 nucleosomes are disassembled and reassembled during transcription by RNA polymerase II (RNAPII). We show that the normal process of reassembling robust H3 chromatin on RNAPII genes is required to prevent CENP-ACnp1 assembly in its place. Centromeres allow a low level of RNAPII transcription, and our analyses suggest that DNA sequences and chromatin contexts at centromeres may limit the activities required to stabilize and reassemble H3 chromatin during transcription in order to promote the establishment of CENP-ACnp1 chromatin and associated kinetochores.

Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology, and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-ACnp1 at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays, we show that Top3, unlike Top1 and Top2, is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-ACnp1 occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-ACnp1 at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-ACnp1 in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology, which in turn affects the dynamics of CENP-ACnp1 nucleosomes., Author Summary Centromeres are unique regions on eukaryotic chromosomes that are essential for chromosome segregation at mitosis and meiosis. Centromere identity and function depends on the presence of specialized chromatin with nucleosomes containing the centromere-specific histone H3 variant CENP-A. Assembly and disassembly of nucleosomes have previously been shown to involve a family of enzymes known as DNA topoisomerases. We show that centromeres are unique in that they are associated with high levels of Top3, but low levels of Top1 and Top2, suggesting that Top3 is particularly important for centromeric DNA topology. Impaired function of Top3 or its partner Rqh1 results in chromosome segregation defects and increased levels of CENP-ACnp1 at centromeres. This role in limiting the levels of CENP-ACnp1 at centromeres is independent of the established role for the Top3-Rqh1 complex in homologous recombination. Therefore, we hypothesize that the Top3-Rqh1 complex exerts this effect by regulating centromere DNA topology, which in turn affects CENP-ACnp1 nucleosome dynamics. Specific removal of negative supercoiling by Top3 could directly have a negative effect on assembly of CENP-ACnp1 nucleosomes with left-handed negative wrapping of DNA and/or act indirectly by inhibiting transcription-coupled CENP-ACnp1 assembly. Alternatively, Top3 may be a factor that promotes formation of CENP-ACnp1 hemisomes with right-handed wrapping of DNA over conventional octamers. This suggests a new role for the Top3-Rqh1 complex at centromeres and may contribute to the understanding of the structural and functional specification of centromeres.

The histone-encoding genes in Schizosaccharomyces pombe were physically mapped by hybridisation to filters containing cosmid and P1 genomic libraries. The H2A.2 gene and the H2A.1-H2B.1 gene pair mapped between the ade6 and rikI genes on chromosome III. The three H4-H3 gene pairs were mapped to three different regions by a H4.1 probe. Southern analysis of clones from each region revealed the positions of the three H4-H3 gene pairs. H4.1-H3.1 was localised to chromosome I between the mei2 and rad1 genes; H4.2-H3.2 mapped between rad3 and cdc2 on chromosome II; H4.3-H3.3 was localised to a region between the nuc1 and puc1 genes on chromosome II.

Cytogenetically normal acute myeloid leukemia (CN-AML) compose between 40% and 50% of all adult acute myeloid leukemia (AML) cases. In this clinically diverse group, molecular aberrations, such as FLT3-ITD, NPM1, and CEBPA mutations, recently have added to the prognostic accuracy. Aberrant DNA methylation is a hallmark of cancer, including AML. We investigated in total 118 CN-AML samples in a test and a validation cohort for genome-wide promoter DNA methylation with Illumina Methylation Bead arrays and compared them with normal myeloid precursors and global gene expression. IDH and NPM1 mutations were associated with different methylation patterns (P = .0004 and .04, respectively). Genome-wide methylation levels were elevated in IDH-mutated samples (P = .006). We observed a negative impact of DNA methylation on transcription. Genes targeted by Polycomb group (PcG) proteins and genes associated with bivalent histone marks in stem cells showed increased aberrant methylation in AML (P < .0001). Furthermore, high methylation levels of PcG target genes were independently associated with better progression-free survival (odds ratio = 0.47, P = .01) and overall survival (odds ratio = 0.36, P = .001). In summary, genome-wide methylation patterns show preferential methylation of PcG targets with prognostic impact in CN-AML.

The chromosomes of eukaryotes are organized into structurally and functionally discrete domains. This implies the presence of insulator elements that separate adjacent domains, allowing them to maintain different chromatin structures. We show that the Fun30 chromatin remodeler, Fft3, is essential for maintaining a proper chromatin structure at centromeres and subtelomeres. Fft3 is localized to insulator elements and inhibits euchromatin assembly in silent chromatin domains. In its absence, euchromatic histone modifications and histone variants invade centromeres and subtelomeres, causing a mis-regulation of gene expression and severe chromosome segregation defects. Our data strongly suggest that Fft3 controls the identity of chromatin domains by protecting these regions from euchromatin assembly., Author Summary Active and inactive chromatin domains are often juxtaposed along the chromosome arms, and this demands mechanisms that separate them apart. This is performed by insulators that block the spreading of chromatin domains beyond their natural borders. Here, we show that an ATP-dependent chromatin remodeler, Fission yeast fun thirty (Fft3), is localized at known insulator elements and protects centromeric and subtelomeric chromatin domains. When Fft3 is absent, euchromatin invades the centromere and subtelomeres, causing a change in histone modification, incorrect incorporation of histone variants, mis-regulation of gene expression, and severe chromosome segregation defects. We conclude that Fft3 controls the identity of these chromatin domains by shielding them from euchromatin.

The multiprotein Mediator complex is an important regulator of RNA polymerase II-dependent genes in eukaryotic cells. In contrast to the situation in many other eukaryotes, the conserved Med15 protein is not a stable component of Mediator isolated from fission yeast. We here demonstrate that Med15 exists in a protein complex together with Hrp1, a CHD1 ATP-dependent chromatin-remodeling protein. The Med15-Hrp1 subcomplex is not a component of the core Mediator complex but can interact with the L-Mediator conformation. Deletion of med15(+) and hrp1(+) causes very similar effects on global steady-state levels of mRNA, and genome-wide analyses demonstrate that Med15 associates with a distinct subset of Hrp1-bound gene promoters. Our findings therefore indicate that Mediator may directly influence histone density at regulated promoters.

Eukaryotic genomes are repetitively packaged into chromatin by nucleosomes, however they are regulated by the differences between nucleosomes, which establish various chromatin states. Local chromatin cues direct the inheritance and propagation of chromatin status via self-reinforcing epigenetic mechanisms. Replication-independent histone exchange could potentially perturb chromatin status if histone exchange chaperones, such as Swr1C, loaded histone variants into wrong sites. Here we show that in Schizosaccharomyces pombe, like Saccharomyces cerevisiae, Swr1C is required for loading H2A.Z into specific sites, including the promoters of lowly expressed genes. However S. pombe Swr1C has an extra subunit, Msc1, which is a JumonjiC-domain protein of the Lid/Jarid1 family. Deletion of Msc1 did not disrupt the S. pombe Swr1C or its ability to bind and load H2A.Z into euchromatin, however H2A.Z was ectopically found in the inner centromere and in subtelomeric chromatin. Normally this subtelomeric region not only lacks H2A.Z but also shows uniformly lower levels of H3K4me2, H4K5, and K12 acetylation than euchromatin and disproportionately contains the most lowly expressed genes during vegetative growth, including many meiotic-specific genes. Genes within and adjacent to subtelomeric chromatin become overexpressed in the absence of either Msc1, Swr1, or paradoxically H2A.Z itself. We also show that H2A.Z is N-terminally acetylated before, and lysine acetylated after, loading into chromatin and that it physically associates with the Nap1 histone chaperone. However, we find a negative correlation between the genomic distributions of H2A.Z and Nap1/Hrp1/Hrp3, suggesting that the Nap1 chaperones remove H2A.Z from chromatin. These data describe H2A.Z action in S. pombe and identify a new mode of chromatin surveillance and maintenance based on negative regulation of histone variant misincorporation., Author Summary Chromatin is based on a repetitive structural unit called the nucleosome. However, the regulatory properties of chromatin are mediated by the differences between nucleosomes, due to post-translational modifications or the inclusion of histone variants. These differences are maintained by inheritance through cis-acting epigenetic mechanisms. Here we describe a case where the local character of chromatin is not only determined by cis-acting mechanisms but also negatively regulated in trans. The case involves loading of the histone H2A variant, H2A.Z, into chromatin. We show that H2A.Z in the yeast Schizosaccharomyces pombe is mainly found in genes at the first transcribed nucleosome and is inserted into this nucleosome by the Swr1C remodeling machine. However, Swr1C has a regulatory subunit, Msc1, which is not required for H2A.Z promoter loading but prevents H2A.Z occupancy in the inner centromere and subtelomeric regions. These two specialized regions are neither eu- nor heterochromatin and share certain characteristics, which may predispose them to the aberrant inclusion of H2A.Z and the requirement for trans regulation by Msc1.

The centromere is the region of the chromosome where the kinetochore forms. Kinetochores are the attachment sites for spindle microtubules that separate duplicated chromosomes in mitosis and meiosis. Kinetochore formation depends on a special chromatin structure containing the histone H3 variant CENP-A. The epigenetic mechanisms that maintain CENP-A chromatin throughout the cell cycle have been studied extensively but little is known about the mechanism that targets CENP-A to naked centromeric DNA templates. In a recent report published in Science, such de novo centromere assembly of CENP-A is shown to be dependent on heterochromatin and the RNA interference pathway.

Epe1 is a JmjC domain protein that antagonizes heterochromatization in Schizosaccharomyces pombe. Related JmjC domain proteins catalyze a histone demethylation reaction that depends on Fe(II) and α-ketoglutarate. However, no detectable demethylase activity is associated with Epe1, and its JmjC domain lacks conservation of Fe(II)-binding residues. We report that Swi6 recruits Epe1 to heterochromatin and that overexpression of epe1+, like mutations in silencing genes or overexpression of swi6+, upregulates expression of certain genes. A significant overlap was observed between the lists of genes that are upregulated by overexpression of epe1+ and those that are upregulated by mutations in histone deacetylase genes. However, most of the common genes are not regulated by Clr4 histone methyltransferase. This suggests that Epe1 interacts with the heterochromatin assembly pathway at the stage of histone deacetylation. Mutational inactivation of Epe1 downregulates ∼12% of S. pombe genes, and the list of these genes overlaps significantly with the lists of genes that are upregulated by mutations in silencing genes and genes that are hyperacetylated at their promoter regions in clr6-1 mutants. We propose that an interplay between the repressive HDACs activity and Epe1 helps to regulate gene expression in S. pombe.

This article discusses the advances made in epigenetic research using the model organism fission yeast Schizosaccharomyces pombe. S. pombe has been used for epigenetic research since the discovery of position effect variegation (PEV). This is a phenomenon in which a transgene inserted within heterochromatin is variably expressed, but can be stably inherited in subsequent cell generations. PEV occurs at centromeres, telomeres, ribosomal DNA (rDNA) loci, and mating-type regions of S. pombe chromosomes. Heterochromatin assembly in these regions requires enzymes that modify histones and the RNA interference (RNAi) machinery. One of the key histone-modifying enzymes is the lysine methyltransferase Clr4, which methylates histone H3 on lysine 9 (H3K9), a classic hallmark of heterochromatin. The kinetochore is assembled on specialized chromatin in which histone H3 is replaced by the variant CENP-A. Studies in fission yeast have contributed to our understanding of the establishment and maintenance of CENP-A chromatin and the epigenetic activation and inactivation of centromeres.

We have used oligonucleotide tiling arrays to construct genome-wide high-resolution histone acetylation maps for fission yeast. The maps are corrected for nucleosome density and reveal surprisingly uniform patterns of modifications for five different histone acetylation sites. We found that histone acetylation and methylation patterns are generally polar, i.e. they change as a function of distance from the ATG codon. A typical fission yeast gene shows a distinct peak of histone acetylation around the ATG and gradually decreased acetylation levels in the coding region. The patterns are independent of gene length but dependent on the gene expression levels. H3K9Ac shows a stronger peak near the ATG and is more reduced in the coding regions of genes with high expression compared with genes with low expression levels. H4K16Ac is strongly reduced in coding regions of highly expressed genes. A second microarray platform was used to confirm the 5' to 3' polarity effects observed with tiling microarrays. By comparing coding region histone acetylation data in HDAC mutants and wild type, we found that hos2 affects primarily the 5' regions, sir2 and clr6 affect middle regions, and clr6 affects 3' regions. Thus, mechanisms involving different HDACs modulate histone acetylation levels to maintain a 5' to 3' polarity within the coding regions.

We have conducted a genomewide investigation into the enzymatic specificity, expression profiles, and binding locations of four histone deacetylases (HDACs), representing the three different phylogenetic classes in fission yeast (Schizosaccharomyces pombe). By directly comparing nucleosome density, histone acetylation patterns and HDAC binding in both intergenic and coding regions with gene expression profiles, we found that Sir2 (class III) and Hos2 (class I) have a role in preventing histone loss; Clr6 (class I) is the principal enzyme in promoter-localized repression. Hos2 has an unexpected role in promoting high expression of growth-related genes by deacetylating H4K16Ac in their open reading frames. Clr3 (class II) acts cooperatively with Sir2 throughout the genome, including the silent regions: rDNA, centromeres, mat2/3 and telomeres. The most significant acetylation sites are H3K14Ac for Clr3 and H3K9Ac for Sir2 at their genomic targets. Clr3 also affects subtelomeric regions which contain clustered stress- and meiosis-induced genes. Thus, this combined genomic approach has uncovered different roles for fission yeast HDACs at the silent regions in repression and activation of gene expression.

Centromeres of fission yeast are arranged with a central core DNA sequence flanked by repeated sequences. The centromere-associated histone H3 variant Cnp1 (SpCENP-A) binds exclusively to central core DNA, while the heterochromatin proteins and cohesins bind the surrounding outer repeats. CHD (chromo-helicase/ATPase DNA binding) chromatin remodeling factors were recently shown to affect chromatin assembly in vitro. Here, we report that the CHD protein Hrp1 plays a key role at fission yeast centromeres. The hrp1Delta mutant disrupts silencing of the outer repeats and central core regions of the centromere and displays chromosome segregation defects characteristic for dysfunction of both regions. Importantly, Hrp1 is required to maintain high levels of Cnp1 and low levels of histone H3 and H4 acetylation at the central core region. Hrp1 interacts directly with the centromere in early S-phase when centromeres are replicated, suggesting that Hrp1 plays a direct role in chromatin assembly during DNA replication.

Here, epigenetic regulation of centromeric chromatin in fission yeast (Schizosaccharomyces pombe) is reviewed, focussing on the role of histone modifications and the link to RNA interference (RNAi). Fission yeast centromeres are organized into two structurally and functionally distinct domains, both of which are required for centromere function. The central core domain anchors the kinetochore structure while the flanking heterochromatin domain is important for sister centromere cohesion. The chromatin structure of both domains is regulated epigenetically. In the central core domain, the histone H3 variant Cnp1(CENP-A) plays a key role. In the flanking heterochromatin domain, histones are kept underacetylated by the histone deacetylases (HDACs) Clr3, Clr6 and Sir2, and methylated by Clr4 methyltransferase (HMTase) to create a specific binding site for the Swi6 protein. Swi6 then directly mediates cohesin binding to the centromeric heterochromatin. Recently, a surprising link was made between heterochromatin formation and RNAi.

The mating-type region of the fission yeast Schizosaccharomyces pombe comprises three loci: mat1, mat2-P and mat3-M. mat1 is expressed and determines the mating type of the cell. mat2-P and mat3-M are two storage cassettes located in a 17 kb heterochromatic region with features identical to those of mammalian heterochromatin. Mutations in the swi6+, clr1+, clr2+, clr3+, clr4+ and clr6+ genes were obtained in screens for factors necessary for silencing the mat2-P-mat3-M region. swi6+ encodes a chromodomain protein, clr3+ and clr6+ histone deacetylases, and clr4+ a histone methyltransferase. Here, we describe the cloning and characterization of clr2+. The clr2+ gene encodes a 62 kDa protein with no obvious sequence homologs. Deletion of clr2+ not only affects transcriptional repression in the mating-type region, but also centromeric silencing and silencing of a PolII-transcribed gene inserted in the rDNA repeats. Using chromatin immunoprecipitation, we show that Clr2 is necessary for histone hypoacetylation in the mating-type region, suggesting that Clr2 acts upstream of histone deacetylases to promote transcriptional silencing.

Epigenetic regulation consists of a multitude of different modifications that determine active and inactive states of chromatin. Conditions such as cell differentiation or exposure to environmental stress require concerted changes in gene expression. To interpret epigenomics data, a spectrum of different interconnected datasets is needed, ranging from the genome sequence and positions of histones, together with their modifications and variants, to the transcriptional output of genomic regions. Here we present a tool, Podbat (Positioning database and analysis tool), that incorporates data from various sources and allows detailed dissection of the entire range of chromatin modifications simultaneously. Podbat can be used to analyze, visualize, store and share epigenomics data. Among other functions, Podbat allows data-driven determination of genome regions of differential protein occupancy or RNA expression using Hidden Markov Models. Comparisons between datasets are facilitated to enable the study of the comprehensive chromatin modification system simultaneously, irrespective of data-generating technique. Any organism with a sequenced genome can be accommodated. We exemplify the power of Podbat by reanalyzing all to-date published genome-wide data for the histone variant H2A.Z in fission yeast together with other histone marks and also phenotypic response data from several sources. This meta-analysis led to the unexpected finding of H2A.Z incorporation in the coding regions of genes encoding proteins involved in the regulation of meiosis and genotoxic stress responses. This incorporation was partly independent of the H2A.Z-incorporating remodeller Swr1. We verified an Swr1-independent role for H2A.Z following genotoxic stress in vivo. Podbat is open source software freely downloadable from www.podbat.org, distributed under the GNU LGPL license. User manuals, test data and instructions are available at the website, as well as a repository for third party-developed plug-in modules. Podbat requires Java version 1.6 or higher.

In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) and both up- and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1(+) gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1Delta strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase, deacetylase and chromatin remodelling activities in the regulation of gene expression.

The post-translational modifications of histones are key to the modulation of chromatin structure. Distinct patterns of modifications established by histone-modifying enzymes control diverse chromosomal processes. Here, we report the purification and molecular characterization of the fission yeast Clr6 histone deacetyl ase involved in higher order chromatin assembly. We show that a chromodomain protein Alp13, which belongs to the conserved MRG protein family linked to cellular senescence in humans, is associated with Clr6. In addition, Clr6 interacts with homologs of the mammalian transcriptional co-repressors Sin3, Pst1 and Pst2, and a WD40 repeat-containing protein, Prw1. Alp13, Pst2 and Prw1 form a stable complex with Clr6 in the nucleus. Deletion of any of these factors causes progressive loss of viability and sensitivity to DNA-damaging agents, and impairs condensation/resolution of chromosomes during mitosis. This is accompanied by hyperacetylation of histones and a reduction in histone H3 Ser10 phosphorylation, which correlates with chromosome condensation during mitosis. These results link the MRG family protein Alp13 to histone deacetylation, and suggest that Clr6 and its associated factors are essential for fundamental chromosomal events.