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Pairing of homologous chromosomes in meiosis is essential for sexual reproduction. We have previously demonstrated that the fission yeast sme2 RNA, a meiosis-specific long noncoding RNA (lncRNA), accumulates at the sme2 chromosomal loci and mediates their robust pairing in meiosis. However, the mechanisms underlying lncRNA-mediated homologous pairing have remained elusive. In this study, we identify conserved RNA-binding proteins that are required for robust pairing of homologous chromosomes. These proteins accumulate mainly at the sme2 and two other chromosomal loci together with meiosis-specific lncRNAs transcribed from these loci. Remarkably, the chromosomal accumulation of these lncRNA–protein complexes is required for robust pairing. Moreover, the lncRNA–protein complexes exhibit phase separation properties, since 1,6-hexanediol treatment reversibly disassembled these complexes and disrupted the pairing of associated loci. We propose that lncRNA–protein complexes assembled at specific chromosomal loci mediate recognition and subsequent pairing of homologous chromosomes., During meiosis, pairing of homologous chromosomes is critical for sexual reproduction. Here the authors reveal in S. pombe the role of lncRNA–protein complexes during the pairing of homologues chromosomes that assemble at specific chromosomal loci to mediate recognition of the pairs.

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.

In eukaryotic cells, there are two chromatin states, silenced and active, and the formation of a so-called boundary plays a critical role in demarcating these regions; however, the mechanisms underlying boundary formation are not well understood. In this study, we focused on S. cerevisiae ADA1, a gene previously shown to encode a protein with a robust boundary function. Ada1 is a component of the histone modification complex Spt-Ada-Gcn5-acetyltransferase (SAGA) and the SAGA-like (SLIK) complex, and it helps to maintain the integrity of these complexes. Domain analysis showed that four relatively small regions of Ada1 (Region I; 66-75 aa, II; 232-282 aa, III; 416-436 aa and IV; 476-488 aa) have a boundary function. Among these, Region II could form an intact SAGA complex, whereas the other regions could not. Investigation of cellular factors that interact with these small regions identified a number of proteasome-associated proteins. Interestingly, the boundary functions of Region II and Region III were affected by depletion of Ump1, a maturation and assembly factor of the 20S proteasome. These results suggest that the boundary function of Ada1 is functionally linked to proteasome processes and that the four relatively small regions in ADA1 form a boundary via different mechanisms.

In Saccharomyces cerevisiae, HMR/HML, telomeres and ribosomal DNA are heterochromatin-like regions in which gene transcription is prevented by the silent information regulator (Sir) complex. The Sir complex (Sir2, Sir3 and Sir4) can spread through chromatin from the silencer. Boundaries prevent Sir complex spreading, and we previously identified 55 boundary genes among all ~6,000 yeast genes. These boundary proteins can be distinguished into two types: those that activate transcription to prevent spreading of silencing, and those that prevent gene silencing by forming a boundary. We selected 44 transcription-independent boundary proteins from the 55 boundary genes by performing a one-hybrid assay and focused on GIC1 (GTPase interaction component 1). Gic1 is an effector of Cdc42, which belongs to the Rho family of small GTPases, and has not been reported to function in heterochromatin boundaries in vivo. We detected a novel boundary-forming activity of Gic1 at HMR-left and telomeric regions by conducting a chromatin immunoprecipitation assay with an anti-Sir3 antibody. We also found that Gic1 bound weakly to histones in two-hybrid analysis. Moreover, we performed domain analysis to identify domain(s) of Gic1 that are important for its boundary activity, and identified two minimum domains, which are located outside its Cdc42-binding domain.

Proteins in the nuclear envelope (NE) play a role in the dynamics and functions of the nucleus and of chromosomes during mitosis and meiosis. Mps3, a yeast NE protein with a conserved SUN domain, predominantly localizes on a yeast centrosome equivalent, spindle pole body (SPB), in mitotic cells. During meiosis, Mps3, together with SPB, forms a distinct multiple ensemble on NE. How meiosis-specific NE localization of Mps3 is regulated remains largely unknown. In this study, we found that a meiosis-specific component of the protein complex essential for sister chromatid cohesion, Rec8, binds to Mps3 during meiosis and controls Mps3 localization and proper dynamics on NE. Ectopic expression of Rec8 in mitotic yeast cells induced the formation of Mps3 patches/foci on NE. This required the cohesin regulator, WAPL ortholog, Rad61/Wpl1, suggesting that a meiosis-specific cohesin complex with Rec8 controls NE localization of Mps3. We also observed that two domains of the nucleoplasmic region of Mps3 are essential for NE localization of Mps3 in mitotic as well as meiotic cells. We speculate that the interaction of Mps3 with the meiosis-specific cohesin in the nucleoplasm is a key determinant for NE localization/function of Mps3.

In fission yeast, the formation of centromeric heterochromatin is induced through the RNA interference (RNAi)-mediated pathway. Some pre-mRNA splicing mutants (prp) exhibit defective formation of centromeric heterochromatin, suggesting that splicing factors play roles in the formation of heterochromatin, or alternatively that the defect is caused by impaired splicing of pre-mRNAs encoding RNAi factors. Herein, we demonstrate that the splicing factor spPrp16p is enriched at the centromere, and associates with Cid12p (a factor in the RNAi pathway) and the intron-containing dg ncRNA. Interestingly, removal of the dg intron, mutations of its splice sites, or replacement of the dg intron with an euchromatic intron significantly decreased H3K9 dimethylation. We also revealed that splicing of dg ncRNA is repressed in cells and its repression depends on the distance from the transcription start site to the intron. Inefficient splicing was also observed in other intron-containing centromeric ncRNAs, dh and antisense dg, and splicing of antisense dg ncRNA was repressed in the presence of the RNAi factors. Our results suggest that the introns retained in centromeric ncRNAs work as facilitators, co-operating with splicing factors assembled on the intron and serving as a platform for the recruitment of RNAi factors, in the formation of centromeric heterochromatin., Author summary Formation of centromeric heterochromatin is required for correct segregation of sister chromatids during mitosis. In fission yeast, formation of heterochromatin at centromeres is performed through the RNA interference (RNAi) system, which involves processing of noncoding RNAs transcribed from the centromeres. We found that the intron in the centromeric dg ncRNAs facilitates formation of centromeric heterochromatin in fission yeast. We showed that the splicing factor spPrp16p associates with the RNAi factor and intron-containing dg ncRNA. Removal of or mutations in the dg intron significantly decreased H3K9 dimethylation, suggesting that the intron and associated splicing factors serve as a platform for recruitment of RNAi factors. Inefficient splicing is a hallmark of intron-containing centromeric ncRNAs. Such repression of splicing seems to be important for facilitation of heterochromatin formation. Introns in euchromatic regions are removed by splicing to generate functional RNAs, whereas centromeric introns are retained in ncRNAs by splicing repression and play roles in gene silencing. Our findings shed light on the novel roles of introns in epigenetic regulation of gene expression and heterochromatin formation.

Histone H3K4 methylation has been linked to transcriptional activation. KDM5A (also known as RBP2 or JARID1A), a member of the KDM5 protein family, is an H3K4 demethylase, previously implicated in the regulation of transcription and differentiation. Here, we show that KDM5A is physically and functionally associated with two histone deacetylase complexes. Immunoaffinity purification of KDM5A confirmed a previously described association with the SIN3B-containing histone deacetylase complex and revealed an association with the nucleosome remodeling and deacetylase (NuRD) complex. Sucrose density gradient and sequential immunoprecipitation analyses further confirmed the stable association of KDM5A with these two histone deacetylase complexes. KDM5A depletion led to changes in the expression of hundreds of genes, two-thirds of which were also controlled by CHD4, the NuRD catalytic subunit. Gene ontology analysis confirmed that the genes commonly regulated by both KDM5A and CHD4 were categorized as developmentally regulated genes. ChIP analyses suggested that CHD4 modulates H3K4 methylation levels at the promoter and coding regions of target genes. We further demonstrated that the Caenorhabditis elegans homologues of KDM5 and CHD4 function in the same pathway during vulva development. These results suggest that KDM5A and the NuRD complex cooperatively function to control developmentally regulated genes.

Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that binds to lysine 9-methylated histone H3 (H3K9me), a hallmark of heterochromatin. Although HP1 phosphorylation has been described in several organisms, the biological implications of this modification remain largely elusive. Here we show that HP1's phosphorylation has a critical effect on its nucleosome binding properties. By in vitro phosphorylation assays and conventional chromatography, we demonstrated that casein kinase II (CK2) is the kinase primarily responsible for phosphorylating the N-terminus of human HP1α. Pull-down assays using in vitro-reconstituted nucleosomes showed that unmodified HP1α bound H3K9-methylated and H3K9-unmethylated nucleosomes with comparable affinity, whereas CK2-phosphorylated HP1α showed a high specificity for H3K9me3-modified nucleosomes. Electrophoretic mobility shift assays showed that CK2-mediated phosphorylation diminished HP1α's intrinsic DNA binding, which contributed to its H3K9me-independent nucleosome binding. CK2-mediated phosphorylation had a similar effect on the nucleosome-binding specificity of fly HP1a and S. pombe Swi6. These results suggested that HP1 phosphorylation has an evolutionarily conserved role in HP1's recognition of H3K9me-marked nucleosomes.

Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that binds lysine 9-methylated histone H3 (H3K9me), a hallmark of heterochromatin, and plays a crucial role in forming higher-order chromatin structures. HP1 has an N-terminal chromodomain and a C-terminal chromo shadow domain, linked by an unstructured hinge region. Although biochemical and structural studies have revealed each domain's properties, little is known about the mechanisms by which these domains cooperate to carry out HP1's function in forming higher-order chromatin structures. In this review, we summarize HP1's biochemical and structural properties and highlight the latest findings regarding HP1's interactions with nucleosomes.

The chromodomain of HP1α binds directly to lysine 9-methylated histone H3 (H3K9me). This interaction is enhanced by phosphorylation of serine residues in the N-terminal tail of HP1α by unknown mechanism. Here we show that phosphorylation modulates flexibility of HP1α’s N-terminal tail, which strengthens the interaction with H3. NMR analysis of HP1α’s chromodomain with N-terminal tail reveals that phosphorylation does not change the overall tertiary structure, but apparently reduces the tail dynamics. Small angle X-ray scattering confirms that phosphorylation contributes to extending HP1α’s N-terminal tail. Systematic analysis using deletion mutants and replica exchange molecular dynamics simulations indicate that the phosphorylated serines and following acidic segment behave like an extended string and dynamically bind to H3 basic residues; without phosphorylation, the most N-terminal basic segment of HP1α inhibits interaction of the acidic segment with H3. Thus, the dynamic string-like behavior of HP1α’s N-terminal tail underlies the enhancement in H3 binding due to phosphorylation.

Post-translational modifications of histones are critical not only for local regulation of gene expression, but also for higher-order structure of the chromosome and genome organization in general. These modifications enable a preset state to be maintained over subsequent generations and thus provide an epigenetic level of regulation. Heterochromatic regions of the genome are epigenetically regulated to maintain a ‘‘silent state’’ and protein coding genes inserted into these regions are subject to the same epigenetic silencing. The fission yeast Schizosaccharomyces pombe has well characterized regions of heterochromatin and has proven to be a powerful model for elucidation of epigenetic silencing mechanisms. Research in S. pombe led to the breakthrough discovery that epigenetic silencing is not solely a chromatin-driven transcriptional repression and that RNA interference of nascent transcripts can guide epigenetic silencing and associated histone modifications. Over the last 10 years, an eloquent integration of genetic and biochemical studies have greatly propelled our understanding of major players and effector complexes for regulation of RNAi-mediated epigenetic silencing in S. pombe. Here, we review recent research related to regulation of the epigenetic state in S. pombe heterochromatin, focusing specifically on the mechanisms by which transcription and RNA processing interact with the chromatin modification machinery to maintain the epigenetically silent state.

Silenced chromatin domains are restricted to specific regions. Eukaryotic chromosomes are organized into discrete domains delimited by domain boundaries. From approximately 6,000 genes in Saccharomyces cerevisiae, we previously isolated 55 boundary genes. In this study, we focus on the molecular function of one of boundary genes, YCR076C/FUB1 (function of boundary), whose function has not been clearly defined in vivo. Biochemical analysis of Fub1p revealed that it interacted with multiple subunits of the 20S proteasome core particle (20S CP). To further clarify the functional link between Fub1p and proteasome, several proteasome mutants were analyzed. Although only 20S CP subunits were isolated as Fub1p interactors, a genetic interaction was also observed for component of 19S regulatory particle (19S RP) suggesting involvement of Fub1p with the whole proteasome. We also analyzed the mechanism of boundary establishment by using proteasome composition factor-deficient strains. Deletion of pre9 and ump1, whose products have effects on the 20S CP, resulted in a decrease in boundary function. Domain analyses of Fub1p identified a minimum functional domain in the C terminus that was essential for boundary establishment and showed a limited sequence homology to the human PSMF1, which is known to inhibit proteasome activity. Finally, boundary assay showed that human PSMF1 also exhibited boundary establishment activity in yeast. Our results defined the functional correlation between Fub1p and PSMF1.

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.

Centromeres that are essential for faithful segregation of chromosomes consist of unique DNA repeats in many eukaryotes. Although recombination is under-represented around centromeres during meiosis, little is known about recombination between centromere repeats in mitotic cells. Here, we compared spontaneous recombination that occurs between ade6B/ade6X inverted repeats integrated at centromere 1 (cen1) or at a non-centromeric ura4 locus in fission yeast. Remarkably, distinct mechanisms of homologous recombination (HR) were observed in centromere and non-centromere regions. Rad51-dependent HR that requires Rad51, Rad54 and Rad52 was predominant in the centromere, whereas Rad51-independent HR that requires Rad52 also occurred in the arm region. Crossovers between inverted repeats (i.e. inversions) were under-represented in the centromere as compared to the arm region. While heterochromatin was dispensable, Mhf1/CENP-S, Mhf2/CENP-X histone-fold proteins and Fml1/FANCM helicase were required to suppress crossovers. Furthermore, Mhf1 and Fml1 were found to prevent gross chromosomal rearrangements mediated by centromere repeats. These data for the first time uncovered the regulation of mitotic recombination between DNA repeats in centromeres and its physiological role in maintaining genome integrity.

Heterochromatin is a structurally compacted region of chromosomes in which transcription and recombination are inactivated. DNA replication is temporally regulated in heterochromatin, but the molecular mechanism for regulation has not been elucidated. Among heterochromatin loci in Schizosaccharomyces pombe, the pericentromeric region and the silent mating-type (mat) locus replicate in early S phase, whereas the sub-telomeric region does not, suggesting complex mechanisms for regulation of replication in heterochromatic regions. Here, we show that Swi6, an S. pombe counterpart of heterochromatin protein 1 (HP1), is required for early replication of the pericentromeric region and the mat locus. Origin-loading of Sld3, which depends on Dfp1/Dbf4-dependent kinase Cdc7 (DDK), is stimulated by Swi6. An HP1-binding motif within Dfp1 is required for interaction with Swi6 in vitro and for early replication of the pericentromeric region and mat locus. Tethering of Dfp1 to the pericentromeric region and mat locus in swi6-deficient cells restores early replication of these loci. Our results show that a heterochromatic protein positively regulates initiation of replication in silenced chromatin by interacting with an essential kinase.

Heterochromatin protein 1 (HP1) recruits various effectors to heterochromatin for multiple functions, but its regulation is unclear. In fission yeast, a HP1 homolog Swi6 recruits SHREC, Epe1, and cohesin, which are involved in transcriptional gene silencing (TGS), transcriptional activation, and sister chromatid cohesion, respectively. We found that casein kinase II (CK2) phosphorylated Swi6. Loss of CK2-dependent Swi6 phosphorylation alleviated heterochromatic TGS without affecting heterochromatin structure. This was due to the inhibited recruitment of SHREC to heterochromatin, accompanied by an increase in Epe1. Interestingly, loss of phosphorylation did not affect cohesion. These results indicate that CK2-dependent Swi6 phosphorylation specifically controls TGS in heterochromatin.

MRG15, a mammalian protein related to the mortality factor MORF4, is required for cell proliferation and embryo survival. Our genetic analysis has revealed that the Caenorhabditis elegans ortholog MRG-1 serves similar roles. Maternal MRG-1 promotes embryo survival and is required for proliferation and immortality of the primordial germ cells (PGCs). As expected of a chromodomain protein, MRG-1 associates with chromatin. Unexpectedly, it is concentrated on the autosomes and not detectable on the X chromosomes. This association is not dependent on the autosome-enriched protein MES-4. Focusing on possible roles of MRG-1 in regulating gene expression, we determined that MRG-1 is required to maintain repression in the maternal germ line of transgenes on extrachromosomal arrays, and of several X-linked genes previously shown to depend on MES-4 for repression. MRG-1 is not required for PGCs to acquire transcriptional competence or for the turn-on of expression of several PGC-expressed genes (pgl-1, glh-1, glh-4 and nos-1). By contrast to this result in PGCs, MRG-1 is required for ectopic expression of those germline genes in somatic cells lacking the NuRD complex component MEP-1. We discuss how an autosome-enriched protein might repress genes on the X chromosome, promote PGC proliferation and survival, and influence the germ versus soma distinction.

Imbalances of gene expression in aneuploids, which contain an abnormal number of chromosomes, cause a variety of growth and developmental defects. Aneuploid cells of the fission yeast Schizosaccharomyces pombe are inviable, or very unstable, during mitotic growth. However, S. pombe haploid cells bearing minichromosomes derived from the chromosome 3 can grow stably as a partial aneuploid. To address biological consequences of aneuploidy, we examined the gene expression profiles of partial aneuploid strains using DNA microarray analysis. The expression of genes in disomic or trisomic cells was found to increase approximately in proportion to their copy number. We also found that some genes in the monosomic regions of partial aneuploid strains increased their expression level despite there being no change in copy number. This change in gene expression can be attributed to increased expression of the genes in the disomic or trisomic regions. However, even in an aneuploid strain that bears a minichromosome containing no protein coding genes, genes located within about 50 kb of the telomere showed similar increases in expression, indicating that these changes are not a secondary effect of the increased gene dosage. Examining the distribution of the heterochromoatin protein Swi6 using DNA microarray analysis, we found that binding of Swi6 within ~50 kb from the telomere occurred less in partial aneuploid strains compared to euploid strains. These results suggest that additional chromosomes in aneuploids could lead to imbalances in gene expression through changes in distribution of heterochromatin as well as in gene dosage.

Summary RNA interference (RNAi) is a conserved silencing mechanism that has widespread roles in RNA degradation, translational repression, and the epigenetic control of chromatin structure [1]. In fission yeast, heterochromatin assembly requires RNAi machinery and is initiated by small interference RNAs (siRNAs) derived from heterochromatic regions and by the RNA-induced transcriptional silencing (RITS) complex [2–7]. Although recent studies have been successful in uncovering the functions of effector complexes in the RNAi pathway [4, 5, 8–10], exactly how heterochromatic siRNAs are processed and function in assembling heterochromatin remains unclear. In this study we focused on a conserved ribonuclease, Eri1, which was originally identified as a negative regulator of RNAi in C. elegans [11], and show the importance of the Eri1 protein in RNAi-mediated heterochromatin assembly in fission yeast. Eri1 specifically degrades double-stranded siRNAs through two functional domains and represses the accumulation of cellular siRNAs in vivo. Deletion of eri1 + causes an increase in siRNAs associated with the RITS complex and enhances heterochromatic silencing, which is accompanied by increased levels of histone H3-K9 methylation and the Swi6 protein. Our findings suggest that the fission yeast Eri1 controls the accumulation of heterochromatic siRNAs and negatively regulates the RNAi-mediated heterochromatin assembly.

DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1(-/-) or Dnmt3a(-/-)Dnmt3b(-/-) ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome-wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein-1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases.

The chromodomain is a conserved motif that functions in the epigenetic control of gene expression. Here, we report the functional characterization of a chromodomain protein, Chp1, in the heterochromatin assembly in fission yeast. We show that Chp1 is a structural component of three heterochromatic regions-centromeres, the mating-type region, and telomeres-and that its localization in these regions is dependent on the histone methyltransferase Clr4. Although deletion of the chp1(+) gene causes centromere-specific decreases in Swi6 localization and histone H3-K9 methylation, we show that the role of Chp1 is not exclusive to the centromeres. We found that some methylation persists in native centromeric regions in the absence of Chp1, which is also true for the mating-type region and telomeres, and determined that Swi6 and Chp2 are critical to maintaining this residual methylation. We also show that Chp1 participates in the establishment of repressive chromatin in all three chromosomal regions. These results suggest that different heterochromatic regions share common structural properties, and that centromeric heterochromatin requires Chp1-mediated establishment steps differently than do other heterochromatic regions.

Heterochromatin is a functionally important chromosomal component, especially at centromeres. In fission yeast, conserved heterochromatin-specific modifications of the histone H3 tail, involving deacetylation of Lys 9 and Lys 14 and subsequent methylation of Lys 9, promote the recruitment of a heterochromatin protein, Swi6, a homolog of the Drosophila heterochromatin protein 1. However, the primary determinants of the positioning of heterochromatin are still unclear. The fission yeast proteins Abp1, Cbh1, and Cbh2 are homologs of the human protein CENP-B that bind to centromeric α-satellite DNA and associate with centromeric heterochromatin. We show that the CENP-B homologs are functionally redundant at centromeres, and that Abp1 binds specifically to centromeric heterochromatin. In the absence of Abp1 or Cbh1, the centromeric association of Swi6 is diminished, resulting in a decrease in silencing of the region. CENP-B-homolog double disruptants show a synergistic reduction of Swi6 at centromeric heterochromatin, indicating that the three proteins are functionally redundant in the recruitment of Swi6. Furthermore, using chromatin immunoprecipitation assays, we show that disruption of CENP-B homologs causes a decrease in heterochromatin-specific modifications of histone H3. These results indicate that the CENP-B homologs act as site-specific nucleation factors for the formation of centromeric heterochromatin by heterochromatin-specific modifications of histone tails.

Although telomerase activity in hepatocellular carcinoma (HCC) increases in accordance with degree of histological undifferentiation, it is unknown whether the level of telomerase activity in HCC reflects of the degree of activity in individual cells or the frequency of telomerase-positive HCC cells. Non-cancerous liver tissues exhibit low but significant levels of telomerase activity, but the nature of telomerase-positive cells in these tissues is unclear. In this study, we performed immunohistochemical staining using specific antibody against telomerase reverse transcriptase (hTERT) protein in 15 HCC samples and 13 adjacent non-cancerous liver tissues. There were hTERT-positive hepatocytes, though very low frequency, in non-cancerous liver tissues. The frequencies in hTERT positive hepatocytes were very well correlated with clinicopathological parameters and telomerase activity levels: the average frequencies of chronic hepatitis was 0.2%, liver cirrhosis 0.2%, well-differentiated HCC 3.0%, moderately differentiated HCC 28%, and poorly differentiated HCC 95%. The intensity of staining varied among cells within a given specimen, and correlation with degree of histological undifferentiation was less obvious. Portions of migrating lymphocytes and biliary epithelial cells were also hTERT-positive. These findings indicate that the up-regulation of telomerase activity with degree of undifferentiation of HCC is mainly due to the increase in frequency of hTERT positive HCC cells.

Background: Telomerase consists of two essential subunits, the template RNA (TR; telomerase RNA) and the catalytic subunit TERT (telomerase reverse transcriptase). Knockout mice with a mTR (mouse TR) deletion have been described and well characterized. However, mice with a mTERT (mouse TERT) deletion have not been reported. Results: mTERT-knockout mice have been constructed. The first generation mTERT ˇ/ˇ mice were fertile, and did not show any noticeable macroscopic or microscopic phenotypic change. All tissue cells derived from mTERT ˇ/ˇ mice that were examined lacked telomerase activity, indicating that mTERT is the only gene encoding the telomerase catalytic subunit. Pulse field gel electrophoresis (PFGE) and nondenaturing in-gel hybridization analyses showed that mouse telomeric DNA has G-strand 5 0 -overhangs, as demonstrated for human and yeast cells. This telomeric single-stranded G-tail was also observed in MEF (mouse embryonic fibroblast) and liver cells derived from mTERT ˇ/ˇ mice. Conclusions: mTERT-knockout mice show phenotypes that are apparently normal at least during the early generations. This observation is similar to that obtained with the mTR-knockout mice. The presence of the telomeric G-strand tails in mTERT ˇ/ˇ mice suggests that these telomeric 5 0 overhangs are produced by telomerase-independent mechanisms, as has been proposed for yeast and human.

Human telomerase is expressed in germ tissues and in the majority of primary tumors. Cell renewal tissues and some pre-cancerous tissues also have weak telomerase activity. Yet, neither the exact location and frequency of telomerase-positive cells nor the changes in telomerase expression during differentiation or carcinogenesis of individual cells are known. This paper reports on the expression of hTERT (telomerase reverse transcriptase) protein in tumor and non-tumor colorectal tissues by Western blotting and tissue sections by immuno-histochemistry using antibodies raised against partial peptides of hTERT. Though telomerase activity and hTERT expression at both mRNA and protein levels were generally higher in tumor part than in non-tumor part, these two were not always correlated: expression of hTERT did not always give rise to high telomerase activity. Colonic carcinoma cell nuclei were stained with anti-hTERT antibodies but not with antigen-preabsorbed antibodies. In normal mucosa, hTERT protein was expressed, though weaker than in carcinoma, in all colonic crypt epithelial cells except those at the tip; the expressing-cell distribution was much wider than that of Ki-67 positive cells which were located at the bottom of the crypt. Isolated crypt contained a significant level of hTERT protein revealed by Western blotting, while having very weak telomerase activity. Telomerase activity was detected in epithelial cells only at the bottom half of the crypt. Specific hTERT-staining was positive in tissue lymphocytes but negative in almost all other stromal cells. It is of interest to see whether a significant level of hTERT expression with low telomerase activity is characteristic of physiologically regenerating tissues containing stem cells. In situ detection of the hTERT protein will permit further analysis of cancer diagnosis and stem cell differentiation.

A single-cell method allows the assessment of relationships between the dynamic epigenetic behavior of yeast heterochromatin boundaries over multiple generations., Differences in gene expression between individual cells can be mediated by epigenetic regulation; thus, methods that enable detailed analyses of single cells are crucial to understanding this phenomenon. In this study, genomic silencing regions of Saccharomyces cerevisiae that are subject to epigenetic regulation, including the HMR, HML, and telomere regions, were investigated using a newly developed single cell analysis method. This method uses fluorescently labeled proteins to track changes in gene expression over multiple generations of a single cell. Epigenetic control of gene expression differed depending on the specific silencing region at which the reporter gene was inserted. Correlations between gene expression at the HMR-left and HMR-right regions, as well as the HMR-right and HML-right regions, were observed in the single-cell level; however, no such correlations involving the telomere region were observed. Deletion of the histone acetyltransferase GCN5 gene from a yeast strain carrying a fluorescent reporter gene at the HMR-left region reduced the frequency of changes in gene expression over a generation. The results presented here suggest that epigenetic control within an individual cell is reversible and can be achieved via regulation of histone acetyltransferase activity., Author Summary Although eukaryotic gene repression usually acts on individual genes, cells can also repress larger chromosomal regions via the establishment of a high order chromatin structure called heterochromatin. Once initiated, heterochromatin spreads until halted by a boundary, and in this study we focus on how this boundary is formed. Because the mechanism is epigenetic and can differ from cell to cell, we wanted to assess the dynamics of the process by tracking individual cells over multiple generations. Here we develop a novel method employing protein fluorescence to monitor gene expression at the boundaries of several yeast heterochromatic regions simultaneously. This allows us to assess whether different boundaries within a single cell fluctuate in concert or independently of each other. In addition, we use histone modification mutants to probe the specific types of epigenetic regulation responsible for fluctuations in heterochromatin boundary positioning. Using this method, we show that epigenetic gene expression within individual cells is reversible and that this process is regulated by histone acetylation state. Future work will identify connections between variation in boundary positioning and novel transcription control systems.

The budding yeast Saccharomyces cerevisiae contains active and inactive chromatin separated by boundary domains. Previously, we used genome-wide screening to identify 55 boundary-related genes. Here, we focus on Sgf73, a boundary protein that is a component of the Spt-Ada-Gcn5 acetyltransferase (SAGA) and SLIK (SAGA-like) complexes. These complexes have histone acetyltransferase (HAT) and histone deubiquitinase activity, and Sgf73 is one of the factors necessary to anchor the deubiquitination module. Domain analysis of Sgf73 was carried out, and the minimum region (373-402 aa) essential for boundary function was identified. This minimum region does not include the domain involved in anchoring the deubiquitination module, suggesting that the histone deubiquitinase activity of Sgf73 is not important for its boundary function. Next, Sgf73-mediated boundary function was analyzed in disruption strains in which different protein subunits of the SAGA/SLIK/ADA complexes were deleted. Deletion of ada2, ada3 or gcn5 (a HAT module component) caused complete loss of the boundary function of Sgf73. The importance of SAGA or SLIK complex binding to the boundary function of Sgf73 was also analyzed. Western blot analysis detected both the full-length and truncated forms of Spt7, suggesting that SAGA and SLIK complex formation is important for the boundary function of Sgf73.

In fission yeast, siRNA is generated from pericentromeric noncoding RNA by the RNAi machinery. siRNA synthesis and heterochromatin formation are interdependent, forming a self-reinforcing loop on chromatin. In this system, siRNA is amplified by the RNA-dependent RNA polymerase complex (RDRC) and the endoribonuclease Dcr1, which synthesizes dsRNA and processes the dsRNA, respectively. The amplification is essential for stable heterochromatin formation. Here, a novel gene, dsh1+ (defect of the gene silencing at centromeric heterochromatin), is identified as an essential component of RNAi-directed heterochromatin assembly. Loss of dsh1+ abolishes normal RNAi function and heterochromatic gene silencing at pericentromeres. Dsh1 interacts with Dcr1 and RDRC and couples the reactions of both proteins to the effective production of siRNA in vivo. Dsh1 binds to heterochromatin in the absence of RDRC, while RDRC requires Dsh1 for its chromatin-binding activity, suggesting that Dsh1 recruits RDRC to chromatin. Immunofluorescence analysis shows that Dsh1 forms foci at the nuclear periphery, and some Dsh1 foci colocalize with Dcr1 and RDRC. Dsh1 is required for the colocalization of Dcr1 and RDRC. Moreover, loss of the nuclear periphery localization of Dsh1 abolishes Dsh1 function. Taken together, these results suggest that Dsh1 assembles the RNAi machinery on heterochromatin and forms a perinuclear compartment for amplification of heterochromatic siRNA.

Grc3 is an evolutionarily conserved protein. Genome-wide budding yeast studies suggest that Grc3 is involved in rRNA processing. In the fission yeast Schizosaccharomyces pombe, Grc3 was identified as a factor exhibiting distinct nuclear dot localization, yet its exact physiological function remains unknown. Here, we show that S. pombe Grc3 is required for both rRNA processing and heterochromatic gene silencing. Cytological analysis revealed that Grc3 nuclear dots correspond to heterochromatic regions and that some Grc3 is also present in the nucleolar peripheral region. Depleting the heterochromatic proteins Swi6 or Clr4 abolished heterochromatic localization of Grc3 and resulted in its preferential accumulation in the perinucleolar region, suggesting its dynamic association with these nuclear compartments. Cells expressing mutant grc3 showed defects in 25 S rRNA maturation and in heterochromatic gene silencing. Protein analysis of Grc3-containing complexes led to the identification of Las1 and components of the IPI complex (Rix1, Ipi1, and Crb3). All of these Grc3-interacting proteins showed a dynamic nuclear localization similar to that observed for Grc3, and those conditional mutants showed defects in both rRNA processing and silencing of centromeric transcripts. Our data suggest that Grc3 functions cooperatively with Las1 and the IPI complex in both ribosome biogenesis and heterochromatin assembly.

Epigenetic gene silencing is one of the fundamental mechanisms for ensuring proper gene expression patterns during cellular differentiation and development. Histone deacetylases (HDACs) are evolutionally conserved enzymes that remove acetyl modifications from histones and play a central role in epigenetic gene silencing. In cells, HDAC forms a multiprotein complex (HDAC complex) in which the associated proteins are believed to help HDAC carry out its cellular functions. Though each HDAC complex contains distinct components, the presence of isoforms for some of the components expands the variety of complexes and the diversity of their cellular roles. Recent studies have also revealed a functional link between HDAC complexes and specific histone demethylases. In this paper, we summarize the distinct and cooperative roles of four class I HDAC complexes, Sin3, NuRD, CoREST, and NCoR/SMRT, with respect to their component diversity and their relationship with specific histone demethylases.

Heterochromatin protein 1 (HP1) is a conserved chromosomal protein with important roles in chromatin packaging and gene silencing. In fission yeast, two HP1 family proteins, Swi6 and Chp2, are involved in transcriptional silencing at heterochromatic regions, but how they function and whether they act cooperatively or differentially in heterochromatin assembly remain elusive. Here, we show that both Swi6 and Chp2 are required for the assembly of fully repressive heterochromatin, in which they play distinct, nonoverlapping roles. Swi6 is expressed abundantly and plays a dose-dependent role in forming a repressive structure through its self-association property. In contrast, Chp2, expressed at a lower level, does not show a simple dose-dependent repressive activity. However, it contributes to the recruitment of chromatin-modulating factors Clr3 and Epe1 and possesses a novel ability to bind the chromatin-enriched nuclear subfraction that is closely linked with its silencing function. Finally, we demonstrate that a proper balance between Swi6 and Chp2 is critical for heterochromatin assembly. Our findings provide novel insight into the distinct and cooperative functions of multiple HP1 family proteins in the formation of higher-order chromatin structure. In eukaryotic cells, heterochromatin is a distinct chromatin structure that plays pivotal roles in chromosomal function and epigenetic gene regulation. Heterochromatin is generally transcriptionally silent, and its higher-order chromatin structure can spread, causing the heritable inactivation of euchromatic genes adjacent to heterochromatin and leading to varied patterns of gene expression (12, 13, 37). The establishment of heterochromatin is intimately correlated with changes in the posttranslational modifications of histone tails. The methylation of lysine 9 of histone H3 (H3K9me) is a hallmark of

Besides serving to package nuclear DNA, histones carry information in the form of a diverse array of post-translational modifications. Methylation of histones H3 and H4 has been implicated in long-term epigenetic 'memory'. Dimethylation or trimethylation of Lys4 of histone H3 (H3 Lys4) has been found in expressible euchromatin of yeasts and mammals. In contrast, methylation of Lys9 of histone H3 (H3 Lys9) has been implicated in establishing and maintaining the largely quiescent heterochromatin of mammals, yeasts, Drosophila melanogaster and plants. We have previously shown that a DNA methylation mutant of Neurospora crassa, dim-5 (defective in methylation), has a nonsense mutation in the SET domain of an H3-specific histone methyltransferase and that substitutions of H3 Lys9 cause gross hypomethylation of DNA. Similarly, the KRYPTONITE histone methyltransferase is required for full DNA methylation in Arabidopsis thaliana. We used biochemical, genetic and immunological methods to investigate the specific mark for DNA methylation in N. crassa. Here we show that trimethylated H3 Lys9, but not dimethylated H3 Lys9, marks chromatin regions for cytosine methylation and that DIM-5 specifically creates this mark.

The fission yeast Schizosaccharomyces pombe has been widely used as a model eukaryote to study a diverse range of biological processes. However, population genetic studies of this species have been limited to date, and we know very little about the evolutionary processes and selective pressures that are shaping its genome. Here, we sequenced the genomes of 32 worldwide S. pombe strains and examined the pattern of polymorphisms across their genomes. In addition to introns and untranslated regions (UTRs), intergenic regions also exhibited lower levels of nucleotide diversity than synonymous sites, suggesting that a considerable amount of noncoding DNA is under selective constraint and thus likely to be functional. A number of genomic regions showed a reduction of nucleotide diversity probably caused by selective sweeps. We also identified a region close to the end of chromosome 3 where an extremely high level of divergence was observed between 5 of the 32 strains and the remain 27, possibly due to introgression, strong positive selection, or that region being responsible for reproductive isolation. Our study should serve as an important starting point in using a population genomics approach to further elucidate the biology of this important model organism.

The assembly of higher order chromatin structures has been linked to the covalent modifications of histone tails. We provide in vivo evidence that lysine 9 of histone H3 (H3 Lys 9 ) is preferentially methylated by the Clr4 protein at heterochromatin-associated regions in fission yeast. Both the conserved chromo- and SET domains of Clr4 are required for H3 Lys 9 methylation in vivo. Localization of Swi6, a homolog of Drosophila HP1, to heterochomatic regions is dependent on H3 Lys 9 methylation. Moreover, an H3-specific deacetylase Clr3 and a β-propeller domain protein Rik1 are required for H3 Lys 9 methylation by Clr4 and Swi6 localization. These data define a conserved pathway wherein sequential histone modifications establish a “histone code” essential for the epigenetic inheritance of heterochromatin assembly.

Inheritance of stable states of gene expression is essential for cellular differentiation. In fission yeast, an epigenetic imprint marking the mating-type (mat2/3) region contributes to inheritance of the silenced state, but the nature of the imprint is not known. We show that a chromodomain-containing Swi6 protein is a dosage-critical component involved in imprinting the mat locus. Transient overexpression of Swi6 alters the epigenetic imprint at the mat2/3 region and heritably converts the expressed state to the silenced state. The establishment and maintenance of the imprint are tightly coupled to the recruitment and the persistence of Swi6 at the mat2/3 region during mitosis as well as meiosis. Remarkably, Swi6 remains bound to the mat2/3 interval throughout the cell cycle and itself seems to be a component of the imprint. Our analyses suggest that the unit of inheritance at the mat2/3 locus comprises the DNA plus the associated Swi6 protein complex.

Telomerase is a specialized type of reverse transcriptase which catalyzes the synthesis and extension of telomeric DNA (for review, see ref.1). This enzyme is highly active in most cancer cells, but is inactive in most somatic cells2. This striking observation led to the suggestion that telomerase might be important for the continued growth3 or progression4 of cancer cells. However, little is known about the molecular mechanism of telomerase activation in cancer cells. Human telomerase reverse transcriptase (hTRT) has recently been identified as a putative human telomerase catalytic subunit5,6. We transfected the gene encoding hTRT into telomerase-negative human normal fibroblast cells and demonstrated that expression of wild-type hTRT induces telomerase activity, whereas hTRT mutants containing mutations in regions conserved among other reverse transcriptases did not. Hepatocellular carcinoma (2O samples) and non-cancerous liver tissues (19 samples) were examined for telomerase activity and expression of hTRT, the human telomerase RNA component (hTR; encoded by TER)7 and the human telomerase-associated protein (HTLP1; encoded by 7EP7)8,9. A significant correlation between hTRT expression and telomerase activity was observed. These results indicate that the hTRT protein is the catalytic subunit of human telomerase, and that it plays a key role in the activation of telomerase in cancer cells.

The chromosomal locations of the human TEP1 (telomerase protein component 1) and mouse Tep1 genes, which were originally named TLP1 (telomerase protein 1) or TP1 (telomerase-associated protein 1), were determined by direct R-banding FISH and a molecular linkage analysis with interspecific backcross mice. The human TEP1 and mouse Tep1 genes were mapped by FISH to human chromosome 14q11.2 and to the C2-D1 band of mouse chromosome 14, respectively. By means of genetic linkage mapping, the mouse gene was further localized as being 2.7 cM distal toD14Mit18andD14Mit134and 2.0 cM proximal toD14Mit5on mouse chromosome 14, where conserved linkage homology with human chromosome 14q11–q12 has been identified.

MRG15 is a conserved chromodomain protein that associates with histone deacetylases (HDACs) and Tip60-containing histone acetyltransferase (HAT) complexes. Here we further characterize MRG15-containing complexes and show a functional link between MRG15 and histone H3K4 demethylase activity in mammalian cells. MRG15 was predominantly localized to discrete nuclear subdomains enriched for Ser(2)-phosphorylated RNA polymerase II, suggesting it is involved specifically with active transcription. Protein analysis of the MRG15-containing complexes led to the identification of RBP2, a JmjC domain-containing protein. Remarkably, over-expression of RBP2 greatly reduced the H3K4 methylation in culture human cells in vivo, and recombinant RBP2 efficiently removed H3K4 methylation of histone tails in vitro. Knockdown of RBP2 resulted in increased H3K4 methylation levels within transcribed regions of active genes. Our findings demonstrate that RBP2 associated with MRG15 complex to maintain reduced H3K4 methylation at transcribed regions, which may ensure the transcriptional elongation state.