Proliferating Cell Nuclear Antigen (PCNA) Is Required for Cell Cycle-regulated Silent Chromatin on Replicated and Nonreplicated Genes

In Saccharomyces cerevisiae, silent chromatin is formed at HMR upon the passage through S phase, yet neither the initiation of DNA replication at silencers nor the passage of a replication fork through HMR is required for silencing. Paradoxically, mutations in the DNA replication processivity factor, POL30, disrupt silencing despite this lack of requirement for DNA replication in the establishment of silencing. We tested whether pol30 mutants could establish silencing at either replicated or non-replicated HMR loci during S phase and found that pol30 mutants were defective in establishing silencing at HMR regardless of its replication status. Although previous studies tie the silencing defect of pol30 mutants to the chromatin assembly factors Asf1p and CAF-1, we found pol30 mutants did not exhibit a gross defect in packaging HMR into chromatin. Rather, the pol30 mutants exhibited defects in histone modifications linked to ASF1 and CAF-1-dependent pathways, including SAS-I- and Rtt109p-dependent acetylation events at H4-K16 and H3-K9 (plus H3-K56; Miller, A., Yang, B., Foster, T., and Kirchmaier, A. L. (2008) Genetics 179, 793–809). Additional experiments using FLIM-FRET revealed that Pol30p interacted with SAS-I and Rtt109p in the nuclei of living cells. However, these interactions were disrupted in pol30 mutants with defects linked to ASF1- and CAF-1-dependent pathways. Together, these results imply that Pol30p affects epigenetic processes by influencing the composition of chromosomal histone modifications.     

The Snf2 Homolog Fun30 Acts as a Homodimeric ATP-dependent Chromatin-remodeling Enzyme

The Saccharomyces cerevisiae Fun30 (Function unknown now 30) protein shares homology with an extended family of Snf2-related ATPases. Here we report the purification of Fun30 principally as a homodimer with a molecular mass of about 250 kDa. Biochemical characterization of this complex reveals that it has ATPase activity stimulated by both DNA and chromatin. Consistent with this, it also binds to both DNA and chromatin. The Fun30 complex also exhibits activity in ATP-dependent chromatin remodeling assays. Interestingly, its activity in histone dimer exchange is high relative to the ability to reposition nucleosomes. Fun30 also possesses a weakly conserved CUE motif suggesting that it may interact specifically with ubiquitinylated proteins. However, in vitro Fun30 was found to have no specificity in its interaction with ubiquitinylated histones.     

The Saccharomyces cerevisiae Chromatin Remodeler Fun30 Regulates DNA End Resection and Checkpoint Deactivation

Fun30 is a Swi2/Snf2 homolog in budding yeast that has been shown to remodel chromatin both in vitro and in vivo. We report that Fun30 plays a key role in homologous recombination, by facilitating 5′-to-3′ resection of double-strand break (DSB) ends, apparently by facilitating exonuclease digestion of nucleosome-bound DNA adjacent to the DSB. Fun30 is recruited to an HO endonuclease-induced DSB and acts in both the Exo1-dependent and Sgs1-dependent resection pathways. Deletion of FUN30 slows the rate of 5′-to-3′ resection from 4 kb/h to about 1.2 kb/h. We also found that the resection rate is reduced by DNA damage-induced phosphorylation of histone H2A-S129 (γ-H2AX) and that Fun30 interacts preferentially with nucleosomes in which H2A-S129 is not phosphorylated. Fun30 is not required for later steps in homologous recombination. Like its homolog Rdh54/Tid1, Fun30 is required to allow the adaptation of DNA damage checkpoint-arrested cells with an unrepaired DSB to resume cell cycle progression.     

Replication-independent assembly of nucleosome arrays in a novel yeast chromatin reconstitution system involves antisilencing factor Asf1p and chromodomain protein Chd1p

Chromatin assembly in a crude DEAE (CD) fraction from budding yeast is ATP dependent and generates arrays of physiologically spaced nucleosomes which significantly protect constituent DNA from restriction endonuclease digestion. The CD fractions from mutants harboring deletions of the genes encoding histone-binding factors (NAP1, ASF1, and a subunit of CAF-I) and SNF2-like DEAD/H ATPases (SNF2, ISW1, ISW2, CHD1, SWR1, YFR038w, and SPT20) were screened for activity in this replication-independent system. ASF1 deletion substantially inhibits assembly, a finding consistent with published evidence that Asf1p is a chromatin assembly factor. Surprisingly, a strong assembly defect is also associated with deletion of CHD1, suggesting that like other SNF2-related groups of nucleic acid-stimulated ATPases, the chromodomain (CHD) group may contain a member involved in chromatin reconstitution. In contrast to the effects of disrupting ASF1 and CHD1, deletion of SNF2 is associated with increased resistance of chromatin to digestion by micrococcal nuclease. We discuss the possible implications of these findings for current understanding of the diversity of mechanisms by which chromatin reconstitution and remodeling can be achieved in vivo.     

A silencer promotes the assembly of silenced chromatin independently of recruitment

In Saccharomyces cerevisiae, silenced chromatin occurs at telomeres and the silent mating-type loci HMR and HML. At these sites, the Sir proteins are recruited to a silencer and then associate with adjacent chromatin. We used chromatin immunoprecipitation to compare the rates of Sir protein assembly at different genomic locations and discovered that establishment of silenced chromatin was much more rapid at HMR than at the telomere VI-R. Silenced chromatin also assembled more quickly on one side of HMR-E than on the other. Despite differences in spreading, the Sir proteins were recruited to HMR-E and telomeric silencers at equivalent rates. Additionally, insertion of HMR-E adjacent to the telomere VI-R increased the rate of Sir2p association with the telomere. These data suggest that HMR-E functions to both recruit Sir proteins and promote their assembly across several kilobases. Observations that association of Sir2p occurs simultaneously throughout HMR and that silencing at HMR is insensitive to coexpression of catalytically inactive Sir2p suggest that HMR-E acts by enabling assembly to occur in a nonlinear fashion. The ability of silencers to promote assembly of silenced chromatin over several kilobases is likely an important mechanism for maintaining what would otherwise be unstable chromatin at the correct genomic locations.     

Evolution and Functional Trajectory of Sir1 in Gene Silencing

We used the budding yeasts Saccharomyces cerevisiae and Torulaspora delbrueckii to examine the evolution of Sir-based silencing, focusing on Sir1, silencers, the molecular topography of silenced chromatin, and the roles of SIR and RNA interference (RNAi) genes in T. delbrueckii. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analysis of Sir proteins in T. delbrueckii revealed a different topography of chromatin at the HML and HMR loci than was observed in S. cerevisiae. S. cerevisiae Sir1, enriched at the silencers of HMLα and HMRa, was absent from telomeres and did not repress subtelomeric genes. In contrast to S. cerevisiae SIR1''s partially dispensable role in silencing, the T. delbrueckii SIR1 paralog KOS3 was essential for silencing. KOS3 was also found at telomeres with T. delbrueckii Sir2 (Td-Sir2) and Td-Sir4 and repressed subtelomeric genes. Silencer mapping in T. delbrueckii revealed single silencers at HML and HMR, bound by Td-Kos3, Td-Sir2, and Td-Sir4. The KOS3 gene mapped near HMR, and its expression was regulated by Sir-based silencing, providing feedback regulation of a silencing protein by silencing. In contrast to the prominent role of Sir proteins in silencing, T. delbrueckii RNAi genes AGO1 and DCR1 did not function in heterochromatin formation. These results highlighted the shifting role of silencing genes and the diverse chromatin architectures underlying heterochromatin.     

The BAH domain of Rsc2 is a histone H3 binding domain

Bromo-adjacent homology (BAH) domains are commonly found in chromatin-associated proteins and fall into two classes; Remodels the Structure of Chromatin (RSC)-like or Sir3-like. Although Sir3-like BAH domains bind nucleosomes, the binding partners of RSC-like BAH domains are currently unknown. The Rsc2 subunit of the RSC chromatin remodeling complex contains an RSC-like BAH domain and, like the Sir3-like BAH domains, we find Rsc2 BAH also interacts with nucleosomes. However, unlike Sir3-like BAH domains, we find that Rsc2 BAH can bind to recombinant purified H3 in vitro, suggesting that the mechanism of nucleosome binding is not conserved. To gain insight into the Rsc2 BAH domain, we determined its crystal structure at 2.4 Å resolution. We find that it differs substantially from Sir3-like BAH domains and lacks the motifs in these domains known to be critical for making contacts with histones. We then go on to identify a novel motif in Rsc2 BAH that is critical for efficient H3 binding in vitro and show that mutation of this motif results in defective Rsc2 function in vivo. Moreover, we find this interaction is conserved across Rsc2-related proteins. These data uncover a binding target of the Rsc2 family of BAH domains and identify a novel motif that mediates this interaction.     

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.     

Overcoming the chromatin barrier to end resection

Repair of double-strand breaks by homologous recombination requires Repair of double-strand breaks by homologous recombination requires 5′-3′ resection of the DNA ends to create 3′ single-stranded DNA tails. While much progress has been made in identifying the proteins that directly participate in end resection, how this process occurs in the context of chromatin is not well understood. Two papers in Nature report that Fun30, a poorly characterized member of the Swi2/Snf2 family of chromatin remodelers, plays a role in end processing by facilitating the Exo1 and Sgs1-Dna2 resection pathways.DNA double-strand breaks (DSBs) are highly cytotoxic lesions that must be repaired appropriately to prevent the formation of deleterious chromosome rearrangements associated with tumorigenesis. Cells use two major pathways to repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). Repair by HR requires a homologous donor duplex and is considered a high-fidelity process, whereas the homology-independent end joining pathway involves re-ligation of the broken ends and is more error prone. A critical determinant of repair pathway choice that commits cells to HR instead of NHEJ is the initiation of 5′-3′ resection of the DSB ends¹. Genetic studies in Saccharomyces cerevisiae identified the Mre11-Rad50-Xrs2 (Xrs2 is known as NBS1 or NBN in human) complex and Sae2 as key factors in the initiation of resection by removing oligonucleotides from the 5′ ends to form short 3′ single-stranded DNA (ssDNA) tails, while the Exo1 exonuclease or the Sgs1 helicase functioning with the Dna2 endonuclease promote extensive resection in a redundant manner^2,3,4. Resection has been reconstituted in vitro with these proteins^5,6,7; however, additional factors must be present in vivo to facilitate resection in the context of chromatin.Previous studies have suggested that cells use both histone modifying and remodeling complexes to relax chromatin and hence facilitate DNA repair. After sensing of a DSB by the MRX complex, the Tel1 kinase (ATM in human) is activated and phosphorylates histone H2A over a large region from the break site, followed by histone acetylation that unwinds chromatin and facilitates the recruitment of remodeling complexes⁸. ATP-dependent remodelers are large multi-subunit complexes that couple ATP hydrolysis to movement of histones or nucleosomes, including exchange or incorporation of core histones or histone variants, eviction of histones or nucleosomes, and repositioning or sliding of nucleosomes, thereby modifying chromatin structure⁹. Several chromatin remodeling complexes, including INO80, SWR1, SWI/SNF and RSC in budding yeast, have been reported to participate in the DNA damage response. It has been proposed that INO80 facilitates the eviction or sliding of nucleosomes in the immediate vicinity of the break site to allow 5′-3′ strand resection¹⁰. The SWR1 complex was suggested to exchange modified histones after repair, while SWI/SNF may facilitate clearing of nucleosomes surrounding the break site prior to Rad51-mediated strand invasion⁸. RSC is believed to affect resection initiation by facilitating Mre11 binding⁹.Two recent studies report that Fun30, a poorly characterized ATP-dependent chromatin remodeler, promotes DNA end resection in Saccharomyces cerevisiae. Both groups identified Fun30 by genome-wide screens for mutants with increased frequencies of recombination between a transformed linear DNA fragment and homologous chromosomal sequences. Chen et al.¹¹ found that deletion of FUN30 caused increased gene targeting, while Costelloe et al.¹² found higher break-induced replication and gap repair efficiencies in the fun30Δ mutant, properties shared by the resection mutants sgs1Δ and exo1Δ. Using several different assays to monitor the formation of ssDNA at endonuclease-induced DSBs, both groups demonstrated that Fun30 promotes extensive resection by Exo1-dependent and Sgs1-Dna2-dependent pathways (Figure 1). Indeed, both the fun30Δsgs1Δ and fun30Δexo1Δ double mutants exhibited a more severe resection defect than any of the three single mutants^11,12.Open in a separate windowFigure 1The involvement of chromatin remodelers in DSB end resection. Resection initiation is stimulated by RSC and to a lesser extent by INO80. Fun30 works with RPA, Dna2 and Exo1 to promote extensive resection, possibly through overcoming the resection barrier formed by Rad9-bound chromatin.The effect of Fun30 on end resection could be direct or indirect. Evidence in support of a direct role was provided by both studies showing that Fun30 localized to DSBs and along the DNA from the break site with similar kinetics as Sgs1, Dna2 and Exo1^11,12. Furthermore, Chen et al.¹¹ showed that Fun30 co-immunoprecipitates with RPA, Dna2 and Exo1, and enrichment of these resection factors at DSBs was reduced in the fun30Δ mutant. In addition, overexpression of Exo1 in the fun30Δ strain was able to rescue both resection and resistance to the topoisomerase I inhibitor camptothecin (CPT)¹². These data suggest a direct involvement of Fun30 in long-range end resection, possibly through its interaction with extensive resection factors. It remains to be determined whether Fun30 directly recruits the resection machinery, or Fun30-mediated chromatin remodeling facilitates access of resection proteins to ssDNA.Importantly, the ATPase activity of Fun30, which is essential for its chromatin remodeling activity¹³, was found to be required for efficient resection and resistance to CPT^11,12, indicating a correlation between the two processes. Chen et al.¹¹ reported impaired recruitment of Fun30 to DSBs in the resection-defective mre11Δ and sgs1Δexo1Δ mutants, suggesting that Fun30-mediated chromatin remodeling is coupled with resection. Consistently, ChIP analysis of histone H3 and H2B occupancy around an endonuclease-induced DSB showed the same trend as resection in wild-type, fun30Δ and sgs1Δexo1Δ cells^11,12. Further studies are needed to investigate which one is the causal process, histone eviction or resection. Although histone loss appeared to be slower in fun30Δ and sgs1Δexo1Δ than in wild-type cells, it could be due to impaired long-range resection. Thus Fun30 does not seem to function via evicting histones. It remains to be determined how Fun30 remodels chromatin structure to facilitate resection.Costelloe et al.¹² extended their findings to human cells by showing that SMARCAD1, the potential human counterpart of Fun30, participates in end resection. SMARCAD1 co-localizes with γH2AX to DSBs and the pattern of its accumulation at DSBs is similar to that of Exo1. Knockdown of SMARCAD1 caused a dramatic reduction in ionizing radiation-induced ssDNA formation and RPA loading, indicating impaired resection. Accordingly, cells depleted of SMARCAD1 displayed hypersensitivity to genotoxic drugs and reduced HR.Previous studies suggested that the ATP-dependent nucleosome remodeling complexes, INO80, RSC and SWR, affect resection. Chen et al.¹¹ sought to characterize the genetic interaction of these remodelers with Fun30 in promoting resection. Of all the single mutants, fun30Δ showed the strongest phenotype. Deleting components of the INO80 or RSC complexes together with Fun30 further delayed resection and elimination of all three remodeling factors resulted in a severe resection defect, indicating that Fun30 is the primary activity with RSC and INO80 playing redundant roles (Figure 1).A further clue to the mechanism by which Fun30 promotes resection was revealed by its genetic interaction with Rad9, a histone-bound checkpoint mediator known to inhibit resection. Surprisingly, rad9Δ was able to suppress the resection defect of fun30Δ¹¹, suggesting that Fun30 is able to overcome the barrier to resection by Rad9-bound chromatin. Consistent with this hypothesis, elimination of Fun30 led to more Rad9 accumulation at DSBs. Understanding how Fun30 is recruited to DSBs and how it recruits other factors is likely to shed some light on its role in resection. γH2A is required for the recruitment of INO80 and SWR⁸, while recruitment of RSC absolutely requires Mre11 and partially depends on yKu70¹⁴. It will be interesting to know whether Fun30 directly interacts with γH2A and Rad9, since Rad9 is partly recruited by γH2A. According to Chen et al., recruitment of Fun30 and extensive resection factors to DSBs occurs in a mutually dependent manner. One possible explanation for this paradox is that some initial binding of extensive resection factors facilitates Fun30 localization, which in turn remodels the chromatin and makes it more accessible for more resection factors, forming a positive feedback loop.As more ATP-dependent chromatin remodelers with roles in DNA DSB repair are identified, more questions regarding their apparent functional redundancy are raised. Why do cells need so many complexes to remodel chromatin during DSB repair? How is their sequential recruitment to DSBs regulated and does the apparent redundancy reflect the ordered recruitment? Is recruitment of the early- and late-acting chromatin remodelers coordinated? Do the chromatin remodelers that facilitate end resection participate in later steps of repair, such as invasion of the donor locus by the Rad51-ssDNA complex and resolution of recombination intermediates?     

CAF-1 and Rtt101p function within the replication-coupled chromatin assembly network to promote H4 K16ac,preventing ectopic silencing

Replication-coupled chromatin assembly is achieved by a network of alternate pathways containing different chromatin assembly factors and histone-modifying enzymes that coordinate deposition of nucleosomes at the replication fork. Here we describe the organization of a CAF-1-dependent pathway in Saccharomyces cerevisiae that regulates acetylation of histone H4 K16. We demonstrate factors that function in this CAF-1-dependent pathway are important for preventing establishment of silenced states at inappropriate genomic sites using a crippled HMR locus as a model, while factors specific to other assembly pathways do not. This CAF-1-dependent pathway required the cullin Rtt101p, but was functionally distinct from an alternate pathway involving Rtt101p-dependent ubiquitination of histone H3 and the chromatin assembly factor Rtt106p. A major implication from this work is that cells have the inherent ability to create different chromatin modification patterns during DNA replication via differential processing and deposition of histones by distinct chromatin assembly pathways within the network.