Condensin binding at distinct and specific chromosomal sites in the Saccharomyces cerevisiae genome

Mitotic chromosome condensation is chiefly driven by the condensin complex. The specific recognition (targeting) of chromosomal sites by condensin is an important component of its in vivo activity. We previously identified the rRNA gene cluster in Saccharomyces cerevisiae as an important condensin-binding site, but both genetic and cell biology data suggested that condensin also acts elsewhere. In order to characterize the genomic distribution of condensin-binding sites and to assess the specificity of condensin targeting, we analyzed condensin-bound sites using chromatin immunoprecipitation and hybridization to whole-genome microarrays. The genomic condensin-binding map shows preferential binding sites over the length of every chromosome. This analysis and quantitative PCR validation confirmed condensin-occupied sites across the genome and in the specialized chromatin regions: near centromeres and telomeres and in heterochromatic regions. Condensin sites were also enriched in the zones of converging DNA replication. Comparison of condensin binding in cells arrested in G(1) and mitosis revealed a cell cycle dependence of condensin binding at some sites. In mitotic cells, condensin was depleted at some sites while enriched at rRNA gene cluster, subtelomeric, and pericentromeric regions.     

Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing

BACKGROUND: Position-dependent gene silencing in yeast involves many factors, including the four HIR genes and nucleosome assembly proteins Asf1p and chromatin assembly factor I (CAF-I, encoded by the CAC1-3 genes). Both cac Delta asfl Delta and cac Delta hir Delta double mutants display synergistic reductions in heterochromatic gene silencing. However, the relationship between the contributions of HIR genes and ASF1 to silencing has not previously been explored. RESULTS: Our biochemical and genetic studies of yeast Asf1p revealed links to Hir protein function. In vitro, an active histone deposition complex was formed from recombinant yeast Asf1p and histones H3 and H4 that lack a newly synthesized acetylation pattern. This Asf1p/H3/H4 complex generated micrococcal nuclease--resistant DNA in the absence of DNA replication and stimulated nucleosome assembly activity by recombinant yeast CAF-I during DNA synthesis. Also, Asf1p bound to the Hir1p and Hir2p proteins in vitro and in cell extracts. In vivo, the HIR1 and ASF1 genes contributed to silencing the heterochromatic HML locus via the same genetic pathway. Deletion of either HIR1 or ASF1 eliminated telomeric gene silencing in combination with pol30--8, encoding an altered form of the DNA polymerase processivity factor PCNA that prevents CAF-I from contributing to silencing. Conversely, other pol30 alleles prevented Asf1/Hir proteins from contributing to silencing. CONCLUSIONS: Yeast CAF-I and Asf1p cooperate to form nucleosomes in vitro. In vivo, Asf1p and Hir proteins physically interact and together promote heterochromatic gene silencing in a manner requiring PCNA. This Asf1/Hir silencing pathway functionally overlaps with CAF-I activity.     

The selfish yeast plasmid utilizes the condensin complex and condensed chromatin for faithful partitioning

Equipartitioning by chromosome association and copy number correction by DNA amplification are at the heart of the evolutionary success of the selfish yeast 2-micron plasmid. The present analysis reveals frequent plasmid presence near telomeres (TELs) and centromeres (CENs) in mitotic cells, with a preference towards the former. Inactivation of Cdc14 causes plasmid missegregation, which is correlated to the non-disjunction of TELs (and of rDNA) under this condition. Induced missegregation of chromosome XII, one of the largest yeast chromosomes which harbors the rDNA array and is highly dependent on the condensin complex for proper disjunction, increases 2-micron plasmid missegregation. This is not the case when chromosome III, one of the smallest chromosomes, is forced to missegregate. Plasmid stability decreases when the condensin subunit Brn1 is inactivated. Brn1 is recruited to the plasmid partitioning locus (STB) with the assistance of the plasmid-coded partitioning proteins Rep1 and Rep2. Furthermore, in a dihybrid assay, Brn1 interacts with Rep1-Rep2. Taken together, these findings support a role for condensin and/or condensed chromatin in 2-micron plasmid propagation. They suggest that condensed chromosome loci are among favored sites utilized by the plasmid for its chromosome-associated segregation. By homing to condensed/quiescent chromosome locales, and not over-perturbing genome homeostasis, the plasmid may minimize fitness conflicts with its host. Analogous persistence strategies may be utilized by other extrachromosomal selfish genomes, for example, episomes of mammalian viruses that hitchhike on host chromosomes for their stable maintenance.     

Repeat expansion in the budding yeast ribosomal DNA can occur independently of the canonical homologous recombination machinery

Major eukaryotic genomic elements, including the ribosomal DNA (rDNA), are composed of repeated sequences with well-defined copy numbers that must be maintained by regulated recombination. Although mechanisms that instigate rDNA recombination have been identified, none are directional and they therefore cannot explain precise repeat number control. Here, we show that yeast lacking histone chaperone Asf1 undergo reproducible rDNA repeat expansions. These expansions do not require the replication fork blocking protein Fob1 and are therefore independent of known rDNA expansion mechanisms. We propose the existence of a regulated rDNA repeat gain pathway that becomes constitutively active in asf1Δ mutants. Cells lacking ASF1 accumulate rDNA repeats with high fidelity in a processive manner across multiple cell divisions. The mechanism of repeat gain is dependent on highly repetitive sequence but, surprisingly, is independent of the homologous recombination proteins Rad52, Rad51 and Rad59. The expansion mechanism is compromised by mutations that decrease the processivity of DNA replication, which leads to progressive loss of rDNA repeats. Our data suggest that a novel mode of break-induced replication occurs in repetitive DNA that is dependent on high homology but does not require the canonical homologous recombination machinery.     

Activation of the DNA damage checkpoint in yeast lacking the histone chaperone anti-silencing function 1

The packaging of the eukaryotic genome into chromatin is likely to be important for the maintenance of genomic integrity. Chromatin structures are assembled onto newly synthesized DNA by the action of chromatin assembly factors, including anti-silencing function 1 (ASF1). To investigate the role of chromatin structure in the maintenance of genomic integrity, we examined budding yeast lacking the histone chaperone Asf1p. We found that yeast lacking Asf1p accumulate in metaphase of the cell cycle due to activation of the DNA damage checkpoint. Furthermore, yeast lacking Asf1p are highly sensitive to mutations in DNA polymerase alpha and to DNA replicational stresses. Although yeast lacking Asf1p do complete DNA replication, they have greatly elevated rates of DNA damage occurring during DNA replication, as indicated by spontaneous Ddc2p-green fluorescent protein foci. The presence of elevated levels of spontaneous DNA damage in asf1 mutants is due to increased DNA damage, rather than the failure to repair double-strand DNA breaks, because asf1 mutants are fully functional for double-strand DNA repair. Our data indicate that the altered chromatin structure in asf1 mutants leads to elevated rates of spontaneous recombination, mutation, and DNA damage foci formation arising during DNA replication, which in turn activates cell cycle checkpoints that respond to DNA damage.     

At Short Telomeres Tel1 Directs Early Replication and Phosphorylates Rif1

The replication time of Saccharomyces cerevisiae telomeres responds to TG_1–3 repeat length, with telomeres of normal length replicating late during S phase and short telomeres replicating early. Here we show that Tel1 kinase, which is recruited to short telomeres, specifies their early replication, because we find a tel1Δ mutant has short telomeres that nonetheless replicate late. Consistent with a role for Tel1 in driving early telomere replication, initiation at a replication origin close to an induced short telomere was reduced in tel1Δ cells, in an S phase blocked by hydroxyurea. The telomeric chromatin component Rif1 mediates late replication of normal telomeres and is a potential substrate of Tel1 phosphorylation, so we tested whether Tel1 directs early replication of short telomeres by inactivating Rif1. A strain lacking both Rif1 and Tel1 behaves like a rif1Δ mutant by replicating its telomeres early, implying that Tel1 can counteract the delaying effect of Rif1 to control telomere replication time. Proteomic analyses reveals that in yku70Δ cells that have short telomeres, Rif1 is phosphorylated at Tel1 consensus sequences (S/TQ sites), with phosphorylation of Serine-1308 being completely dependent on Tel1. Replication timing analysis of a strain mutated at these phosphorylation sites, however, suggested that Tel1-mediated phosphorylation of Rif1 is not the sole mechanism of replication timing control at telomeres. Overall, our results reveal two new functions of Tel1 at shortened telomeres: phosphorylation of Rif1, and specification of early replication by counteracting the Rif1-mediated delay in initiation at nearby replication origins.     

Hir Proteins Are Required for Position-Dependent Gene Silencing in Saccharomyces cerevisiae in the Absence of Chromatin Assembly Factor I

Chromatin assembly factor I (CAF-I) is a three-subunit histone-binding complex conserved from the yeast Saccharomyces cerevisiae to humans. Yeast cells lacking CAF-I (cacΔ mutants) have defects in heterochromatic gene silencing. In this study, we showed that deletion of HIR genes, which regulate histone gene expression, synergistically reduced gene silencing at telomeres and at the HM loci in cacΔ mutants, although hirΔ mutants had no silencing defects when CAF-I was intact. Therefore, Hir proteins are required for an alternative silencing pathway that becomes important in the absence of CAF-I. Because Hir proteins regulate expression of histone genes, we tested the effects of histone gene deletion and overexpression on telomeric silencing and found that alterations in histone H3 and H4 levels or in core histone stoichiometry reduced silencing in cacΔ mutants but not in wild-type cells. We therefore propose that Hir proteins contribute to silencing indirectly via regulation of histone synthesis. However, deletion of combinations of CAC and HIR genes also affected the growth rate and in some cases caused partial temperature sensitivity, suggesting that global aspects of chromosome function may be affected by the loss of members of both gene families.     

The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange

Histone chaperone Asf1 participates in heterochromatin silencing, DNA repair and regulation of gene expression, and promotes the assembly of DNA into chromatin in vitro. To determine the influence of Asf1 on genetic stability, we have analysed the effect of asf1Delta on homologous recombination. In accordance with a defect in nucleosome assembly, asf1Delta leads to a loss of negative supercoiling in plasmids. Importantly, asf1Delta increases spontaneous recombination between inverted DNA sequences. This increase correlates with an accumulation of double-strand breaks (DSBs) as determined by immunodetection of phosphorylated histone H2A and fluorescent detection of Rad52-YFP foci during S and G2/M phases. In addition, asf1Delta shows high levels of sister chromatid exchange (SCE) and is proficient in DSB-induced SCE as determined by physical analysis. Our results suggest that defective chromatin assembly caused by asf1Delta leads to DSBs that can be repaired by SCE, affecting genetic stability.     

Actin-Related Protein Arp6 Influences H2A.Z-Dependent and -Independent Gene Expression and Links Ribosomal Protein Genes to Nuclear Pores

Actin-related proteins are ubiquitous components of chromatin remodelers and are conserved from yeast to man. We have examined the role of the budding yeast actin-related protein Arp6 in gene expression, both as a component of the SWR1 complex (SWR-C) and in its absence. We mapped Arp6 binding sites along four yeast chromosomes using chromatin immunoprecipitation from wild-type and swr1 deleted (swr1Δ) cells. We find that a majority of Arp6 binding sites coincide with binding sites of Swr1, the catalytic subunit of SWR-C, and with the histone H2A variant Htz1 (H2A.Z) deposited by SWR-C. However, Arp6 binding detected at centromeres, the promoters of ribosomal protein (RP) genes, and some telomeres is independent of Swr1 and Htz1 deposition. Given that RP genes and telomeres both show association with the nuclear periphery, we monitored the ability of Arp6 to mediate the localization of chromatin to nuclear pores. Arp6 binding is sufficient to shift a randomly positioned locus to nuclear periphery, even in a swr1Δ strain. Arp6 is also necessary for the pore association of its targeted RP promoters possibly through cell cycle-dependent factors. Loss of Arp6, but not Htz1, leads to an up-regulation of these RP genes. In contrast, the pore-association of GAL1 correlates with Htz1 deposition, and loss of Arp6 reduces both GAL1 activation and peripheral localization. We conclude that Arp6 functions both together with the nucleosome remodeler Swr1 and also without it, to mediate Htz1-dependent and Htz1-independent binding of chromatin domains to nuclear pores. This association is shown to have modulating effects on gene expression.     

Yeast Silent Mating Type Loci Form Heterochromatic Clusters through Silencer Protein-Dependent Long-Range Interactions

The organization of eukaryotic genomes is characterized by the presence of distinct euchromatic and heterochromatic sub-nuclear compartments. In Saccharomyces cerevisiae heterochromatic loci, including telomeres and silent mating type loci, form clusters at the nuclear periphery. We have employed live cell 3-D imaging and chromosome conformation capture (3C) to determine the contribution of nuclear positioning and heterochromatic factors in mediating associations of the silent mating type loci. We identify specific long-range interactions between HML and HMR that are dependent upon silencing proteins Sir2p, Sir3p, and Sir4p as well as Sir1p and Esc2p, two proteins involved in establishment of silencing. Although clustering of these loci frequently occurs near the nuclear periphery, colocalization can occur equally at more internal positions and is not affected in strains deleted for membrane anchoring proteins yKu70p and Esc1p. In addition, appropriate nucleosome assembly plays a role, as deletion of ASF1 or combined disruption of the CAF-1 and HIR complexes abolishes the HML-HMR interaction. Further, silencer proteins are required for clustering, but complete loss of clustering in asf1 and esc2 mutants had only minor effects on silencing. Our results indicate that formation of heterochromatic clusters depends on correctly assembled heterochromatin at the silent loci and, in addition, identify an Asf1p-, Esc2p-, and Sir1p-dependent step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions.     

Functional characterization of individual genomic condensin-binding sites in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using minichromosome mitotic segregation stability assay

Proper chromatin compaction in mitosis (condensation) is required for equal chromosome distribution and the precise inheritance of genetic information. A protein complex called condensin is responsible for mitotic chromosome condensation, chromosome individualization, the timely separation of sister chromatids in mitosis, and proper tension in the mitotic spindle. The mitotic function of condensin depends on the recognition of specific binding sites in the chromosome. The mechanism for binding condensin to individual sites of mitotic chromosomes, as well as the molecular anatomy of these sites, remains to be elucidated. Even less is known about the process that translates condensin binding to individual sites into the segregation of chromosomes in anaphase. In the present work, by using minichromosome assay, we analyze seven individual condensin-binding sites in S. cerevisiae identified in the whole-genome ChIP-on-chip screening. This approach allowed us to estimate the individual contribution of condensin-binding sites to the segregation fidelity of minichromosomes.     

Genome stability is guarded by yeast Rtt105 through multiple mechanisms

Ty1 mobile DNA element is the most abundant and mutagenic retrotransposon present in the genome of the budding yeast Saccharomyces cerevisiae. Protein regulator of Ty1 transposition 105 (Rtt105) associates with large subunit of RPA and facilitates its loading onto a single-stranded DNA at replication forks. Here, we dissect the role of RTT105 in the maintenance of genome stability under normal conditions and upon various replication stresses through multiple genetic analyses. RTT105 is essential for viability in cells experiencing replication problems and in cells lacking functional S-phase checkpoints and DNA repair pathways involving homologous recombination. Our genetic analyses also indicate that RTT105 is crucial when cohesion is affected and is required for the establishment of normal heterochromatic structures. Moreover, RTT105 plays a role in telomere maintenance as its function is important for the telomere elongation phenotype resulting from the Est1 tethering to telomeres. Genetic analyses indicate that rtt105Δ affects the growth of several rfa1 mutants but does not aggravate their telomere length defects. Analysis of the phenotypes of rtt105Δ cells expressing NLS-Rfa1 fusion protein reveals that RTT105 safeguards genome stability through its role in RPA nuclear import but also by directly affecting RPA function in genome stability maintenance during replication.     

Analysis of the epigenetic status of telomeres by using ChIP-seq data

The chromatin structure of eukaryotic telomeres plays an essential role in telomere functions. However, their study might be impaired by the presence of interstitial telomeric sequences (ITSs), which have a widespread distribution in different model systems. We have developed a simple approach to study the chromatin structure of Arabidopsis telomeres independently of ITSs by analyzing ChIP-seq data. This approach could be used to study the chromatin structure of telomeres in some other eukaryotes. The analysis of ChIP-seq experiments revealed that Arabidopsis telomeres have higher density of histone H3 than centromeres, which might reflects their short nucleosomal organization. These experiments also revealed that Arabidopsis telomeres have lower levels of heterochromatic marks than centromeres (H3K9^Me2 and H3K27^Me), higher levels of some euchromatic marks (H3K4^Me2 and H3K9Ac) and similar or lower levels of other euchromatic marks (H3K4^Me3, H3K36^Me2, H3K36^Me3 and H3K18Ac). Interestingly, the ChIP-seq experiments also revealed that Arabidopsis telomeres exhibit high levels of H3K27^Me3, a repressive mark that associates with many euchromatic genes. The epigenetic profile of Arabidopsis telomeres is closely related to the previously defined chromatin state 2. This chromatin state is found in 23% of Arabidopsis genes, many of which are repressed or lowly expressed. At least, in part, this scenario is similar in rice.