LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 Methylation and Gene Expression in Human Fibroblasts.

(1) Background: The LEO1 (Left open reading frame 1) protein is a conserved subunit of the PAF1C complex (RNA polymerase II-associated factor 1 complex). PAF1C has well-established mechanistic functions in elongation of transcription and RNA processing. We previously showed, in fission yeast, that LEO1 controls histone H3K9 methylation levels by affecting the turnover of histone H3 in chromatin, and that it is essential for the proper regulation of gene expression during cellular quiescence. Human fibroblasts enter a reversible quiescence state upon serum deprivation in the growth media. Here we investigate the function of LEO1 in human fibroblasts. (2) Methods: We knocked out the LEO1 gene using CRISPR/Cas9 methodology in human fibroblasts and verified that the LEO1 protein was undetectable by Western blot. We characterized the phenotype of the ΔLEO1 knockout cells with FACS analysis and cell growth assays. We used RNA-sequencing using spike-in controls to measure gene expression and spike-in controlled ChIP-sequencing experiments to measure the histone modification H3K9me2 genome-wide. (3) Results: Gene expression levels are altered in quiescent cells, however factors controlling chromatin and gene expression changes in quiescent human cells are largely unknown. The ΔLEO1 knockout fibroblasts are viable but have reduced metabolic activity compared to wild-type cells. ΔLEO1 cells showed a slower entry into quiescence and a different morphology compared to wild-type cells. Gene expression was generally reduced in quiescent wild-type cells. The downregulated genes included genes involved in cell proliferation. A small number of genes were upregulated in quiescent wild-type cells including several genes involved in ERK1/ERK2 and Wnt signaling. In quiescent ΔLEO1 cells, many genes were mis-regulated compared to wild-type cells. This included genes involved in Calcium ion transport and cell morphogenesis. Finally, spike-in controlled ChIP-sequencing experiments demonstrated that the histone modification H3K9me2 levels are globally increased in quiescent ΔLEO1 cells. (4) Conclusions: Thus, LEO1 is important for proper entry into cellular quiescence, control of H3K9me2 levels, and gene expression in human fibroblasts.

An essential role for the Ino80 chromatin remodeling complex in regulation of gene expression during cellular quiescence.

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.

Histone H4 lysine 20 mono-methylation directly facilitates chromatin openness and promotes transcription of housekeeping genes.

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 Role of Non-Catalytic Domains of Hrp3 in Nucleosome Remodeling.

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.

High-Throughput Flow Cytometry Combined with Genetic Analysis Brings New Insights into the Understanding of Chromatin Regulation of Cellular Quiescence.

Cellular quiescence is a reversible differentiation state when cells are changing the gene expression program to reduce metabolic functions and adapt to a new cellular environment. When fission yeast cells are deprived of nitrogen in the absence of any mating partner, cells can reversibly arrest in a differentiated G0-like cellular state, called quiescence. This change is accompanied by a marked alteration of nuclear organization and a global reduction of transcription. Using high-throughput flow cytometry combined with genetic analysis, we describe the results of a comprehensive screen for genes encoding chromatin components and regulators that are required for the entry and the maintenance of cellular quiescence. We show that the histone acetylase and deacetylase complexes, SAGA and Rpd3, have key roles both for G0 entry and survival during quiescence. We reveal a novel function for the Ino80 nucleosome remodeling complex in cellular quiescence. Finally, we demonstrate that components of the MRN complex, Rad3, the nonhomologous end-joining, and nucleotide excision DNA repair pathways are essential for viability in G0.

Restoration of KMT2C/MLL3 in human colorectal cancer cells reinforces genome-wide H3K4me1 profiles and influences cell growth and gene expression.

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.

Abo1 is required for the H3K9me2 to H3K9me3 transition in heterochromatin.

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.

AML displays increased CTCF occupancy associated with aberrant gene expression and transcription factor binding.

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.

The CDK Pef1 and protein phosphatase 4 oppose each other for regulating cohesin binding to fission yeast chromosomes.

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.

Copy number of 8q24.3 drives HSF1 expression and patient outcome in cancer: an individual patient data meta-analysis.

BACKGROUND: The heat-shock transcription factor 1 (HSF1) has been linked to cell proliferation and survival in cancer and has been proposed as a biomarker for poor prognosis. Here, we assessed the role of HSF1 expression in relation to copy number alteration (CNA) and cancer prognosis. METHODS: Using 10,287 cancer genomes from The Cancer Genome Atlas and Cbioportal databases, we assessed the association of HSF1 expression with CNA and cancer prognosis. CNA of 8q24.3 was categorized as diploid (reference), deletion (fewer copies), gain (+ 1 copy) and amplification (≥ + 2 copies). Multivariate logistic regression modeling was used to assess 5-year survival among those with a first cancer diagnosis and complete follow-up data (N = 9568), categorized per anatomical location and histology, assessing interaction with tumor stage, and expressed as odds ratios and 95% confidence intervals. RESULTS: We found that only 54.1% of all tumors have a normal predicted 8q24.3 copy number and that 8q24.3 located genes including HSF1 are mainly overexpressed due to increased copies number of 8q24.3 in different cancers. The tumor of patients having respectively gain (+ 1 copy) and amplification (≥ + 2 copies) of 8q24.3 display a global increase of 5-year mortality (odds ratio = 1.98, 95% CI 1.22-3.21) and (OR = 2.19, 1.13-4.26) after full adjustment. For separate cancer types, tumor patients with 8q24.3 deletion showed a marked increase of 5-year mortality in uterine (OR = 4.84, [2.75-8.51]), colorectal (OR = 4.12, [1.15-14.82]), and ovarian (OR = 1.83, [1.39-2.41]) cancers; and decreased mortality in kidney cancer (OR = 0.41, [0.21-0.82]). Gain of 8q24.3 resulted in significant mortality changes in 5-year mortality for cancer of the uterus (OR = 3.67, [2.03-6.66]), lung (OR = 1.76, [1.24-2.51]), colorectal (OR = 1.75, [1.32-2.31]) cancers; and amplification for uterine (OR = 4.58, [1.43-14.65]), prostate (OR = 4.41 [3.41-5.71]), head and neck (OR = 2.68, [2.17-3.30]), and stomach (OR = 0.56, [0.36-0.87]) cancers. CONCLUSIONS: Here, we show that CNAs of 8q24.3 genes, including HSF1, are tightly linked to 8q24.3 copy number in tumor patients and can affect patient outcome. Our results indicate that the integration of 8q24.3 CNA detection may be a useful predictor for cancer prognosis.

Chromatin remodeler Fft3 plays a dual role at blocked DNA replication forks.

Here, we investigate the function of fission yeast Fun30/Smarcad1 family of SNF2 ATPase-dependent chromatin remodeling enzymes in DNA damage repair. There are three Fun30 homologues in fission yeast, Fft1, Fft2, and Fft3. We find that only Fft3 has a function in DNA repair and it is needed for single-strand annealing of an induced double-strand break. Furthermore, we use an inducible replication fork barrier system to show that Fft3 has two distinct roles at blocked DNA replication forks. First, Fft3 is needed for the resection of nascent strands, and second, it is required to restart the blocked forks. The latter function is independent of its ATPase activity.

H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly.

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.

Leo1 is essential for the dynamic regulation of heterochromatin and gene expression during cellular quiescence.

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.

Histone H2B Ubiquitylation Regulates Histone Gene Expression by Suppressing Antisense Transcription in Fission Yeast.

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.

Comprehensive profiling of the fission yeast transcription start site activity during stress and media response.

Fission yeast, Schizosaccharomyces pombe, is an attractive model organism for transcriptional and chromatin biology research. Such research is contingent on accurate annotation of transcription start sites (TSSs). However, comprehensive genome-wide maps of TSSs and their usage across commonly applied laboratory conditions and treatments for S. pombe are lacking. To this end, we profiled TSS activity genome-wide in S. pombe cultures exposed to heat shock, nitrogen starvation, hydrogen peroxide and two commonly applied media, YES and EMM2, using Cap Analysis of Gene Expression (CAGE). CAGE-based annotation of TSSs is substantially more accurate than existing PomBase annotation; on average, CAGE TSSs fall 50-75 bp downstream of PomBase TSSs and co-localize with nucleosome boundaries. In contrast to higher eukaryotes, dispersed TSS distributions are not common in S. pombe. Our data recapitulate known S. pombe stress expression response patterns and identify stress- and media-responsive alternative TSSs. Notably, alteration of growth medium induces changes of similar magnitude as some stressors. We show a link between nucleosome occupancy and genetic variation, and that the proximal promoter region is genetically diverse between S. pombe strains. Our detailed TSS map constitutes a central resource for S. pombe gene regulation research.

Topokaryotyping demonstrates single cell variability and stress dependent variations in nuclear envelope associated domains.

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 binding of Chp2's chromodomain to methylated H3K9 is essential for Chp2's role in heterochromatin assembly in fission yeast.

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.

Repression of Cell Differentiation by a cis-Acting lincRNA 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.