Two Surfaces on the Histone Chaperone Rtt106 Mediate PNAS PLUS Histone Binding, Replication, and Silencing

Two surfaces on the histone chaperone Rtt106 mediate PNAS PLUS histone binding, replication, and silencing

Rachel M. Zunder, Andrew J. Antczak, James M. Berger, and Jasper Rine1

Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720

Contributed by Jasper Rine, November 22, 2011 (sent for review September 22, 2011) The histone chaperone Rtt106 binds histone H3 acetylated at lysine proteins are acetylated at K56, incorporated into chromatin 56 (H3K56ac) and facilitates nucleosome assembly during several during replication-dependent and -independent nucleosome as- fi molecular processes. Both the structural basis of this modi cation- sembly, and then deacetylated as the cell passes through G2 (10, specific recognition and how this recognition informs Rtt106 11). Therefore, the H3K56ac-binding specificity of Rtt106 may function are presently unclear. Guided by our crystal structure of act as a sorting mechanism to distinguish newly synthesized Rtt106, we identified two regions on its double-pleckstrin homol- histones from recycled histones bearing other marks. ogy domain architecture that mediated histone binding. When The Rtt106-mediated incorporation of H3K56ac into chro- histone binding was compromised, Rtt106 localized properly to matin is important for several processes. In replication-coupled chromatin but failed to deliver H3K56ac, leading to replication nucleosome assembly, Rtt106 is thought to deliver H3K56ac to and silencing defects. By mutating analogous regions in the struc- sites of DNA synthesis through a direct physical interaction with turally homologous chromatin-reorganizer Pob3, we revealed a the CAF-1 histone chaperone complex (Cac1, Cac2, and Msi1) conserved histone-binding function for a basic patch found on both (7, 8). CAF-1 is targeted to replication forks by directly binding proteins. In contrast, a loop connecting two β-strands was required to proliferating cell nuclear antigen (PCNA) (12). Like Rtt106, for histone binding by Rtt106 but was dispensable for Pob3 func- CAF-1 binds H3 in a K56ac-specific manner (7). The rtt106Δ tion. Unlike Rtt106, Pob3 histone binding was modification-in- cac1Δ strains have synergistic sensitivities to S-phase DNA dependent, implicating the loop of Rtt106 in H3K56ac-specific damaging agents, suggesting that Rtt106 and CAF-1 perform recognition in vivo. Our studies described the structural origins of overlapping functions during replication-coupled nucleosome GENETICS Rtt106 function, identified a conserved histone-binding surface, turnover (7). During silencing, Rtt106 interacts physically with and defined a critical role for Rtt106:H3K56ac-binding specificity Sir4, a member of the silent information regulator (Sir) complex, in silencing and replication-coupled nucleosome turnover. which forms a repressive domain at silent regions (9, 13). Si- lencing is defective in rtt106Δ cac1Δ strains (8, 9); however, the histone acetylation | yFACT | CAF-1 | Sir | Saccharomyces cerevisiae role of H3K56ac in silencing is currently undefined. Although H3K56ac is important for replication and silencing, the mecha- ackaging DNA into chromatin is dynamic, reversible, and nism by which Rtt106 specifically recognizes H3K56ac has not Pessential for eukaryotic cell viability. The principal packaging been elucidated. unit of chromatin is the nucleosome, consisting of an octamer of The affinity of Rtt106 for acetylated histones is unexpected two copies each of the four canonical histones (H2A, H2B, H3, because it lacks either of the two known acetyl-lysine–binding and H4) wrapped in 146 bp of DNA (1). Histone proteins are domains: a bromodomain or a plant homeodomain (14, 15). decorated with posttranslational modifications, including lysine While our structural studies were in progress, two groups deacetylation, which influence chromatin architecture by altering termined three crystal structures of Rtt106, revealing a double nucleosome contacts or by affecting interactions with nonhistone pleckstrin homology (PH) domain architecture [PDB ID codes proteins (2). During DNA-dependent processes, nucleosomes 3GYP and 3GYO (16) and PDB ID code 3FSS]. One group disassemble to grant access to specific regions of DNA and assigned a DNA-binding function to the N-terminal domain and reassemble in a way that preserves the local chromatin land- a histone-binding function to the C-terminal domain (16). Here, scape. By virtue of their highly basic charge, histones are prone we have determined a structure of Rtt106 from a distinct crystal to both aggregation and promiscuous interactions when they are lattice and performed extensive and precise mutational analyses fi not associated with DNA, such as when they are newly synthe- of the protein. We de ned fully the critical positions on its his- sized or during nucleosome turnover. To prevent these delete- tone-binding surface, providing unique interpretations and in- rious effects, a network of histone chaperones regulates each sights into function. In contrast to earlier studies, which broadly fi step of chromatin assembly and disassembly. Although individual de ned a loop in the C-terminal domain as important (16), we fi chaperones have been implicated in specific DNA-dependent identi ed single substitutions that blocked histone binding. In processes, the principles governing which chaperone operates on addition, we discovered that a conserved basic patch within the which histone and when are largely unknown. N-terminal PH domain, previously implicated solely for DNA Histone chaperones are molecular escorts that bind histones binding, was necessary for interactions with histones. These point and stimulate their transfer without ATP hydrolysis, either to mutations, in turn, were used to dissect the role of Rtt106 lo- another chromatin-associated protein or directly on or off DNA. calization and H3K56ac delivery during replication and silenc- Chaperone:histone interactions are influenced by histone type and oligomeric status (3, 4). However, much less is known about how histone modifications regulate chaperone binding. One of fi fi fi Author contributions: R.M.Z., A.J.A., J.M.B., and J.R. designed research; R.M.Z. and A.J.A. the rst chaperones found to have modi cation-speci c histone- performed research; R.M.Z. and A.J.A. contributed new reagents/analytic tools; R.M.Z., binding activity is Rtt106, a fungal-specific histone chaperone A.J.A., J.M.B., and J.R. analyzed data; and R.M.Z. and J.R. wrote the paper. that escorts newly synthesized H3 and H4 histones into chro- The authors declare no conflict of interest. – matin during replication and transcription (5 7). Rtt106 also Data deposition: The atomic coordinates and structure factors have been deposited in the plays a poorly defined role in silent chromatin (heterochromatin) Protein Data Bank, www.pdb.org (PDB ID code 3TO1). formation in Saccharomyces cerevisiae (8, 9). The histone-binding 1To whom correspondence should be addressed. E-mail: [email protected]. fi af nity of Rtt106 is enhanced by the acetylation of H3 at lysine This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 56 (H3K56ac) (7). During S-phase, all newly translated H3 1073/pnas.1119095109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1119095109 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 ing. Additionally, our comparative analysis of the structurally in binding their ligands, examining the unbound Rtt106 structure homologous chromatin-reorganizer Pob3 (17) suggested an ori- could not predict the residues that directly interact with histones gin for Rtt106’s modification specificity and established a pre- (22). To establish the importance of the relative orientation of viously undescribed conserved mechanism for PH domain- the two PH domains, we compared our structure with the pre- histone binding. viously deposited structures of Rtt106 (PDB ID codes 3FSS, 3GYO, and 3GYP) (16). Although each structure was solved Results from a distinct crystal lattice and environment, the Cα rmsds Rtt106 Contained Two Rigidly Opposed PH Domains. During nucle- between pairs of structures were very close, ranging from 0.62 to osome assembly, Rtt106 binds and chaperones newly synthesized 1.05 Å (Fig. S1B). Additionally, hydrophobic contacts between H3/H4 histones into chromatin. Before incorporation, newly the N- and C-terminal PH domains were superimposable be- translated H3 proteins are acetylated at lysine 56 (H3K56ac), tween structures, indicating a fixed orientation despite the dis- and the histone-binding function of Rtt106 is H3K56ac-specific ordered connecting loop (Fig. S1C). Because all four structures (7, 11), presumably in the context of an H3/H4 dimer or tetra- displayed a conserved relative orientation between PH domains, mer. Until recently, bromodomains and plant homeodomains a rigid attachment between domains appeared necessary for were the only structural motifs known to bind specifically to Rtt106 function. acetyl-lysine (15, 18). However, Pfam analysis of the Rtt106 protein sequence revealed a single PH domain (19), suggesting Two Surfaces on Rtt106 Mediated Replication and Silencing Functions. that PH domains may represent a previously undescribed struc- To define regions of Rtt106 necessary for histone binding, we tural mechanism for binding acetylated histones. screened targeted mutations for defects in two distinct Rtt106- To resolve the structural basis for Rtt106:H3K56ac binding, mediated processes: silencing and replication-coupled nucleo- we determined the crystal structure of a truncated form of some assembly. Because Rtt106:H3 binding likely precedes nu- Rtt106 (Rtt1062PH, residues 69–300 out of a total length of 455 cleosome assembly, we reasoned that Rtt106:H3 binding mutants residues) at a resolution of 2.6 Å using multiwavelength anom- might disrupt multiple processes (7, 9). In contrast, mutants that alous dispersion. This construct excluded N- and C-terminal disrupted an interaction between Rtt106 and a replication factor residues, which were predicted to be disordered by Phyre (20) or a Sir protein might disrupt only replication-coupled assembly and were variable in a fungal sequence alignment. The structure or silencing, respectively. We generated 72 single- or double- revealed that Rtt106 contained two tandem PH domains con- amino- acid substitutions of Rtt106, covering 106 residues and nected by a disordered loop (Fig. 1A and Fig. S1A). Although 78% of the surface area of the two PH domains (Fig. 1B and PH domains share a common fold, the size and composition of Table S1). All mutations were generated on full-length RTT106 2PH the loops connecting the individual β-strands vary. This variation because the truncated rtt106 construct had no detectable renders some PH domains, including the N-terminal PH domain function in vivo (Fig. S2). Mutants were screened for replication of Rtt106, impossible to detect based on primary sequence (21). and silencing phenotypes by growth on selective media (Fig. 1C). Because PH domains use different surfaces, loops, and pockets Because the CAF-1 histone chaperone has overlapping functions

Fig. 1. Two functionally important surfaces on Rtt106. (A) The X-ray crystal structure of Rtt106 revealed a double-PH domain architecture. Link- ers between the N- (dark green) and C- (light green) PH domains and a loop in the N-terminal PH domain were disordered (represented as dashed green lines). (B) Surface-exposed residues on Rtt106 were mutated and screened for function. The 106 mutated residues, colored green, covered 78% of the surface area. (C) rtt106 mutantswerescreenedfordefectsofgrowthon CPT (3.5 μg/mL) and defects in silencing of an hmr- a1Δ::URA3 reporter gene (shown in schematic, described in Materials and Methods). Complete synthetic medium (CSM)-HIS media here and elsewhere maintained selection for RTT106 plasmids. Shown are all mutants that yielded phenotypes distinct from WT. R86A, K88A, T265E, and T268E single mutants were generated to decon- volute the phenotypes associated with the double mutants RK86,88AA and TT265,268EE tested in the original screen. (D) Mutations that altered Rtt106’s in vivo functions formed two distinct clusters on the N- and C-terminal PH domain surfaces. Residues that, when mutated, impaired Rtt106 function were qualitatively categorized as severe (red), medium (orange), or mild (yellow) (severe: S80E, R86A, and T265E; medium: RL266, 267AA and SM284,285AA; and mild: I264A, K88A, and T268E). Mild mutants included those with phenotypes in combination with additional mutated residues but not alone. A close-up of each region shows that the N-terminal cluster occurred on β-strands, whereas the C-terminal cluster localized to an extended loop. Side chains that were mutated are shown in stick representation.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1119095109 Zunder et al. Downloaded by guest on September 26, 2021 with Rtt106 (7–9), we expanded the dynamic range of the assays PNAS PLUS by monitoring each process in a cac1Δ mutant. We screened Rtt106 mutants for replication defects by growth on the DNA-damaging agent camptothecin (CPT). CPT impairs topoisomerase I and has synergistic sensitivities with many DNA replication factors, including chromatin assembly proteins (7, 23, 24). Of the 106 targeted residues, mutations of only 10 residues produced observable CPT growth phenotypes (Fig. 1C). These mutations segregated into two spatially distinct clusters on the surface of Rtt106: a region within the β-strands on the N-terminal PH domain and a nine-residue loop connecting two β-strands within the C-terminal PH domain (Fig. 1D). These two surface clusters were inferred to mediate Rtt106 binding to either histones or replication-specific factors. We also screened each RTT106 mutant for silencing defects using an HMR-a1 reporter stain (hmr-a1Δ::URA3) (Fig. 1C). Strains with WT silencing were able to grow on medium containing 5-fluoroortic acid (+FOA), a counterselection for URA3 expression. Conversely, mutants with silencing defects failed to grow on +FOA medium and grew on medium lacking uracil (−URA). As in the CPT-sensitivity screen, mutations of only 10 residues led to silencing defects. Surprisingly, these residues were identical to those uncovered by the replication screen, highlighting the broad functional importance of these two spatially distinct clusters (Fig. 1D). These results were consistent with a recently published structure-function analysis of Rtt106 GENETICS that used multiple alanine substitutions within the N-terminal PH domain and 3–5 residue alanine substitutions within the C- terminal loop, broadly defining functional areas (16). Below, we used our single-substitution mutants to examine Rtt106-histone binding and to investigate further the importance of Rtt106- mediated H3K56ac delivery during replication and silencing. To determine precisely which biochemical properties were important for Rtt106 function, we generated mutations that pro- bed the electrostatic and conformational features of the two distinct surfaces (Table S1). Two mutants isolated from our screen, S80E and R86A, disrupted the charge of a basic region on the N- terminal PH domain (Fig. 2A). To test whether the change in Fig. 2. C-terminal loop and the charge of an N-terminal basic patch were electrostatics caused the deleterious phenotypes, we generated critical for Rtt106 function. (A) Electrostatic surface representation of Rtt106 conservative mutations that maintained the charge of the basic revealed that critical residues within the N-terminal PH domain clustered patch (S80T, S80A, and R86K). Cells with these conservative within a basic patch. Arrows indicate critical residues. (B) Rtt106 S80, R86, T265, and T268 residues were mutated to conservative and nonconservative mutations behaved like WT, suggesting that the basic charge of B amino acids and assayed for function as in Fig. 1C.(C) Mapping sequence this N-terminal surface was essential for Rtt106 function (Fig. 2 ). conservation from a fungal multiple sequence alignment onto the surface of In contrast to the importance of charge in the N-terminal patch, Rtt106 showed that functionally critical residues map to the highly con- within the C-terminal loop, TT265,268EE was phenocopied by a served face (purple) but not to the variable face (white). double-alanine mutation, TT265,268AA. Thus, the deleterious phenotypes were not solely attributable to a change in loop electrostatics (Fig. 2B). Examination of the corresponding single Two Structurally Distinct Regions of Rtt106 Were Important for mutants revealed T265E produced the strongest effect, indicating Histone Binding. Because Rtt106:H3 binding was likely a pre- its importance in maintaining loop function (Fig. 2B). These single requisite for both silencing and replication-coupled chromatin point mutations revealed how Rtt106 function depended critically assembly, we tested our mutated proteins for the ability to bind on the electrostatic surface of the N-terminal PH domain. In histones using a coimmunoprecipitation (CoIP) assay. This assay addition, these studies highlighted the importance of threonine showed that the Rtt106 S80E, R86A, and T265E mutations residues, particularly T265, within the C-terminal loop. substantially reduced H3 binding in whole-cell lysates, and pre- All point mutations within these two structurally distinct clus- sumably in vivo, compared with WT Rtt106 (Fig. 3A). Because ters had highly correlated replication and silencing phenotypes Rtt106 interacts with the CAF-1 complex and the HIR (Hir1, (Figs. 1C and 2B). Thus, despite the ∼30-Å distance between Hir2, Hir3, and Hpc2) complex, which are H3/H4 chaperones, clusters, the two sites appeared to define a common interaction reduced histone binding by mutant Rtt106 proteins could result surface. Furthermore, both clusters fell along a highly conserved either from direct effects on binding H3 or from indirect effects face of the rigid double-PH domain structure (Fig. 2C). Because from a failure to bind CAF-1 or HIR. However, neither cac1Δ the amino acid sequence of histone H3 is among the most con- nor hir1Δ mutations altered Rtt106:H3 copurification (Fig. 3B). served of all proteins, we reasoned that its interaction surface was Therefore, the Rtt106 mutants likely disrupted a direct in- likely to involve conserved positions on Rtt106. Both the se- teraction. The requirement of H3K56ac for detectable Rtt106: quence conservation patterns and the overlapping residues im- H3 binding in yeast whole-cell lysate suggests that the physical plicated in silencing and replication suggested that these clusters interaction is dominated by direct contacts at or around acetyl- defined a functionally important histone-binding surface. modified K56 (7). However, the distance between the N-terminal

Zunder et al. PNAS Early Edition | 3of10 Downloaded by guest on September 26, 2021 replication in each mutant background. The localization of these three mutant Rtt106 proteins at early (ARS305 and ARS607) and late (ARS501) origins of replication in asynchronously dividing cells was indistinguishable from the localization of WT Rtt106 (Fig. 4B). However, in a cac1Δ strain, the rtt106 mutants had signiﬁcantly reduced H3K56ac enrichment compared with WT (Fig. 4C). Additionally, reduced H3K56ac enrichment at a sequence downstream of origins (ARS305 + 1 kb) suggested that Rtt106:H3 binding and CAF-1 were required for H3K56ac incorporation during replication elongation as well as initiation. In rtt106Δ cac1Δ mutants, the total cellular level of H3K56ac was similar to that of WT (Fig. 4D). Therefore, the CPT, MMS, and HU sensitivities were attributable to Rtt106 mutants’ decreased afﬁnity for H3 rather than a shortage of H3K56ac.

Rtt106 Chaperoned H3K56ac Within Silent Chromatin. To understand the molecular basis of the rtt106 mutant silencing phenotypes, we examined the interdependence between Rtt106:H3 binding, Rtt106 localization, and H3K56ac deposition at HMR. The rtt106 mutants with compromised H3 binding, in combination with the cac1Δ mutation, were initially characterized as silencing-defective by monitoring growth of a URA3 reporter strain (hmr- a1Δ::URA3)on−URA medium (Fig. 1C). In agreement with the growth assay, quantitative RT-PCR of URA3 mRNA verified that rtt106(Δ, S80E, R86A, and T265E) cac1Δ mutants were all significantly derepressed compared with both WT and the cac1Δ single-mutant strains (P < 0.01; Fig. 5A). At HMR, Rtt106 mutant proteins were recruited to the locus (Fig. 5B); however, as at Fig. 3. Rtt106 S80, R86, and T265 defined a histone interaction surface. (A) origins of replication, the inability of each mutant to bind H3, in rtt106 S80E, R86A, and T265E mutants disrupted Rtt106:H3 binding in vivo. combination with the cac1Δ mutation, resulted in H3K56 hypo- WT and mutant Rtt106-FLAG proteins were immunoprecipitated (IP) from acetylation throughout the region (Fig. 5C). yeast whole-cell extract (WCE) with anti-FLAG resin. Copurifying proteins Intriguingly, unlike in cells with defects in replication-coupled were detected by immunoblotting with antibodies against the indicated nucleosome assembly, HML and HMR remain silent in rtt109Δ proteins. (B) Neither CAF-1 nor HIR chaperone complexes were required for fi fi strains, indicating that the H3K56ac modi cation is not required Rtt106:H3 binding. Rtt106 was puri ed as in A in the indicated chaperone for silencing (27) (Fig. 5A). Therefore, the silencing phenotypes mutant backgrounds. (C) rtt106 S80E, R86A, and T265E mutants disrupted rtt106Δ cac1Δ Rtt106:H3 binding in vitro. Although H3K56ac was required for Rtt106:H3 associated with strains did not result simply from binding, and H3K56 was not acetylated in E. coli, Rtt106 still binds reduced H3K56ac within silent chromatin. We hypothesized that recombinant H3 at a lower affinity compared with histones isolated from in the absence of H3K56ac-specific chaperones, either the ac-

yeast (7). His6-RTT106 and yeast H3 and H4 were coexpressed in E. coli. cumulation of unincorporated H3K56ac or the inappropriate Binding was monitored by nickel affinity purification from bacteria lysate assembly of H3K56ac within chromatin could be antagonistic to and immunoblotting with antibodies against the indicated proteins. silencing. This model predicted that the absence of this modification might alleviate the rtt106Δ cac1Δ silencing defect. Indeed, the HMR silencing defects observed in rtt106(Δ, S80E, R86A, basic patch and the C-terminal loop indicated that there were and T265E) cac1Δ mutants were all partially suppressed by an multiple energetically important contacts between Rtt106 and rtt109Δ null mutation (Fig. 5A). Suppression of the silencing H3. Consistent with this idea, Rtt106 S80E, R86A, and T265E defect was also observed at endogenous HMR-a1, indicating that A mutant proteins had reduced binding to both H3K56ac (Fig. 3 ) the phenotype was not specific to the URA3 reporter (Fig. 5D). fi C as well as recombinant, unmodi ed histones (Fig. 3 ). Although These results suggested that although H3K56ac was not required these experiments did not discriminate whether the N- or C- for silencing, the weakened silencing in the absence of the terminal interface directly interacted with H3K56ac, our com- Rtt106 and CAF-1 chaperones suggested that H3K56ac must be parative studies with Pob3 (below) suggested that functional properly chaperoned within HMR to maintain the silent state. differences, related to modification-specific binding, exist between the two surfaces. Pob3 and Rtt106 Were Similar in Structure but Differed in Histone- Binding Specificity. Our findings suggested that the histone-bind- Rtt106:H3 Binding Was Required for the Delivery of H3K56ac During ing mechanism of Rtt106 relied on two interaction surfaces, Replication. During S-phase, the histone chaperones Rtt106 and one within each PH domain. Strikingly, Pob3, a member of the CAF-1 are thought to promote incorporation of H3K56ac at the chromatin-reorganizing complex facilitates chromatin transcrip- replication fork (7, 25). An rtt109Δ strain, which cannot acetylate tion (yFACT), contains a double-PH domain with architecture H3K56, was sensitive to CPT, methyl methanesulfonate (MMS), similar to Rtt106 (16, 17). The yFACT complex destabilizes and hydroxyurea (HU), suggesting that this acetylation plays an nucleosomes and is thought to promote the recycling of parental important role during replication-coupled chromatin assembly histones during replication and transcription (28). Intriguingly, (Fig. 4A) (26). In a cac1Δ strain, the rtt106(S80E, R86A, and the tandem PH domains of Pob3 are rigidly opposed in an T265E) mutants, which had reduced H3-binding activity (Fig. 3 analogous orientation to that found in Rtt106 (Fig. 6A). Rtt106 A and C), were also CPT-, MMS-, and HU-sensitive, suggesting and Pob3 both interact with histones H3/H4, and a mutant allele that Rtt106:H3 binding was necessary for Rtt106’s contribution of pob3 interacts genetically with mutations on H3 at K56 (7, 29). to replication-coupled chromatin assembly (Fig. 4A). Therefore, we determined whether the histone-binding mecha- To analyze the nature of this defect, we used ChIP to monitor nism and acetyl-lysine specificity were conserved between these Rtt106 localization and H3K56ac incorporation at origins of structurally similar chromatin-associated proteins.

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Fig. 4. Rtt106 mediated H3K56ac delivery to origins of replication. (A) rtt106 mutants were sensitive to DNA damaging agents. Growth on CPT (3.5 μg/mL), MMS (0.0075%), and HU (150 mM) was monitored as in Fig. 1C.(B) Rtt106 mutant proteins localized to origins of replication. ChIP analysis of WT and mutant Rtt106-FLAG at early and late origins of replication was performed. Immunoprecipitation (IP)/input ratios of amplified DNA for ARS305, ARS305 + 1 kb, ARS607, and ARS501 primer sets are shown for each strain. WT ratios were normalized to 1. Error bars here and elsewhere represent SDs (n ≥ 3). (C) H3K56ac occupancy was reduced at early and late origins of replication in rtt106 mutant backgrounds. ChIP analysis used antibodies against H3K56ac and total H3. Amplified DNA from ARS305, ARS305 + 1 kb, ARS607, and ARS501 was normalized to a previously described control gene, SSC1 (27). WT ratios were normalized to 1. rtt106(Δ, S80E, R86A, and T265E) cac1Δ mutants each had significantly reduced H3K56ac occupancy compared with WT (all ARSs, P < 0.01) and the cac1Δ single mutant (ARS305 and ARS305 + 1 kb, P < 0.01; ARS607 and ARS501, P < 0.05). (D) Total cellular H3K56ac was not altered in rtt106Δ cac1Δ backgrounds. H3K56ac levels were detected by immunoblotting whole-cell extract (WCE) with antibodies against either H3K56ac or total H3, normalized to an anti-Pgk1 loading control. WT values were normalized to 1.

To compare the histone-binding specificities of Rtt106 and would be functionally conserved between Pob3 and Rtt106, Pob3, we performed CoIP experiments in strains that abolished or whereas the H3K56ac-specific recognition surface would be im- increased acetylation on H3K56. Consistent with previous results portant only for Rtt106-histone binding. (7), although Rtt106 was able to bind unmodified, recombinant To test whether the functional importance of the histone- H3 at high concentrations (Fig. 3C), Rtt106:H3 binding was un- binding clusters in Rtt106 was conserved in Pob3, we generated detectable in vivo in strains that lacked the H3K56-specific his- mutations in POB3 that were structurally analogous to those af- tone acetyl-transferase (rtt109Δ) (Fig. 6B). Conversely, Rtt106 fecting H3 binding in Rtt106 and monitored Pob3:H3 binding showed increased H3 binding in strains that lacked the H3K56- (Table S2). The Pob3 structure has a basic patch on the N-ter- fi hst3Δ hst4Δ B speci c deacetylases ( , ) (Fig. 6 ). Unlike Rtt106, minal PH domain in a position analogous to the basic region Pob3 still bound H3 in the absence of H3K56ac, albeit at a re- fi C identi ed as critical for Rtt106-mediated nucleosome assembly duced level (Fig. 6 ). Additionally, rather than increased binding, (Fig. 7A, Upper). Notably, Q308, a well-studied residue that dis- elevated levels of H3K56ac led to decreased Pob3:H3 binding rupts the role of Pob3 in transcription initiation and replication (Fig. 6C). Together, these results suggested mechanistic differ- when mutated to R or K (17), is located at the edge of the N- ences between Pob3:H3 and Rtt106:H3 recognition. Our finding terminal basic patch (Fig. 7A, Upper). To test whether the positive that Pob3:H3 binding did not require H3K56ac was consistent with a role of the yFACT complex in recycling parental histones, charge of this surface was necessary for Pob3 function, we mutated most of which would have been deacetylated at H3K56 by Hst3 two arginines that were in a similar position to Rtt106 R86 in the and Hst4 following chromatin deposition (10). Additionally, al- aligned structures to alanines (Pob3 RR254,256AA). Three in- though H3K56ac was not necessary for Pob3:H3 binding, at least dividual glutamic acid substitutions in Pob3 were generated at a portion of the H3 that copurified with Pob3 was acetylated at positions near Rtt106 S80 (Pob3 F249E, T251E, and T252E). The K56 (Fig. 6C). This physical interaction was consistent with the loop within the C-terminal PH domain of Pob3 was longer (11 recently reported genetic interactions that suggest a role for the residues) than the similar region of Rtt106 (9 residues), and unlike yFACT complex in guiding newly synthesized histones into Rtt106’s structure, the loop was disordered and not modeled in chromatin (29). The unique H3K56ac specificity of Rtt106 sug- the Pob3 structure (Fig. 6A). To test whether the Pob3 loop was gested that the modification-independent recognition surface critical for function, all 11 residues were mutated to glycine [Pob3

Zunder et al. PNAS Early Edition | 5of10 Downloaded by guest on September 26, 2021 Fig. 5. Unchaperoned H3K56ac was antagonistic to silencing. (A) The rtt109Δ mutation partially suppressed rtt106Δ cac1Δ silencing defects at HMR. Quantitative RT-PCR (qRT-PCR) analysis of hmr-a1Δ::URA3 mRNA was normalized to ACT1. Expression of sir3Δ was normalized to 100. hmr-a1Δ::URA3 was significantly derepressed in each rtt106(Δ, S80E, R86A, and T265E) cac1Δ mutant compared with both WT and the cac1Δ single mutant (P < 0.01). Expression for each rtt106 mutant was significantly suppressed in the cac1Δ rtt109Δ background compared with each mutant in the cac1Δ background, respectively (P < 0.01). (B) Rtt106 mutant proteins localized to HMR. ChIP analysis of Rtt106-FLAG was performed as in Fig. 4B with a primer set amplifying HMR-a1.(C) H3K56ac was significantly reduced at HMR-a1 in each rtt106(Δ, S80E, R86A, and T265E) cac1Δ mutant background compared with WT (P < 0.01) and the cac1Δ single mutant (P < 0.05). ChIP analysis of H3K56ac was performed as in Fig. 4C with a primer set amplifying HMR-a1.(D) Silencing of endogenous HMR-a1. Quantitative RT-PCR analysis of HMR-a1 mRNA was performed as in A.

423–433(G)11] and Pob3 TT428,430EE was constructed specifi- lication. Transcription initiation phenotypes were analyzed using − cally to test the importance of threonine residues (Fig. 7A, Lower). the Spt reporter lys2-128δ (17). Mutants with transcription As with Rtt106, mutations within the N-terminal basic patch led initiation defects bypass a Ty1 insertion within the LYS2 pro- to reduced Pob3:H3 binding in CoIP experiments (Fig. 7B). Pob3 moter, allowing growth on medium lacking lysine. Replication T252E had a strong H3-binding defect, F249E and T251E had was assayed by growth on medium containing HU. Consistent slightly reduced binding, and RR254,256AA bound the same with the severity of H3-binding results, pob3 T252E had a strong − amount of H3 as WT (Fig. 7B). The reduced binding in F249E and Spt phenotype and was the only mutant in our set of site-di- T251E was more predominant when a lower concentration of rected alleles to cause any detectable HU sensitivity. Addition- antibody was used to detect copurifying H3, further suggesting ally, mutations within the C-terminal loop did not affect Pob3 that the N-terminal basic patch was important for binding (Fig. function (Fig. 7C). Although F249E and T251E mutants had S3). Pob3 RR254,256AA lacked a strong binding phenotype (Fig. slightly reduced Pob3:H3 binding (Fig. 7B and Fig. S3), neither − 7B). Therefore, in the N-terminal basic patches of Rtt106 and mutation caused substantial Spt or HU sensitivity phenotypes. Pob3, insertion of a negative charge resulted in a stronger phe- Thus, the function of Pob3 appeared to be sensitive to large notype than removal of a positive charge. Surprisingly, unlike changes in H3-binding levels but tolerant of smaller changes. In Rtt106, mutations within the C-terminal loop [Pob3 423–433(G)11 summary, the N-terminal basic patch was a functionally impor- and TT428,430EE] did not affect Pob3:H3 binding (Fig. 7B). tant histone-binding interface conserved across chromatin as- Therefore, in contrast to Rtt106, where single point mutations sembly proteins containing a double-PH domain architecture. within the N-terminal basic patch or the C-terminal loop abol- The importance of the N-terminal basic patch suggested a ished the interaction with H3 (Fig. 3 A and C), the H3-binding mechanistic explanation for the transcription and replication function of Pob3 was sensitive only to mutation of the N-terminal defects associated with the previously isolated pob3 Q308K mu- basic patch and not to perturbations of the analogous C-terminal tant: The change in electrostatics associated with Pob3 Q308K loop. These results suggested that the N-terminal basic patch affected histone binding. Indeed, CoIP experiments revealed that represented a conserved histone-binding surface, for either H3 or Pob3 Q308K protein, which inserts an additional basic charge at H4, within the double-PH domain architecture. In contrast, the C- the edge of the N-terminal patch, had an increased affinity for H3 terminal loop was important only for Rtt106 and specific residues compared with WT (Fig. 7D). This increase was in contrast to the were not required for the histone-binding function of Pob3. reduction observed when a negative charge was inserted into the Next, we tested whether Pob3 mutations within the N-terminal N-terminal basic patch (Fig. 7 B and D). Although Pob3:H3 basic patch or the C-terminal loop affected two yFACT-medi- binding was increased in the Q308K mutant and decreased in − ated DNA-dependent processes: transcription initiation and rep- the T252E mutant, either mutation led to similarly severe Spt

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1119095109 Zunder et al. Downloaded by guest on September 26, 2021 directly. Although our experiments monitored H3 copurification, PNAS PLUS reduced H3 could potentially result from disruption of an H4 interaction surface. Indeed, the histone chaperone Asf1 uses distinct histone-binding surfaces for H3 and H4 (30). Consistent with this idea, the distance between the critical binding surfaces of Rtt106 (∼30 Å) is conducive to one site interacting with H3K56ac and the other site interacting with either H3 or H4. Future experiments should identify the histone surface targeted by the basic patches on Rtt106 and Pob3. Interestingly, either reduced Pob3 binding to H3 or H4 through addition of a negative charge (T252E) or increased Pob3:H3 binding through addition of a positive charge (Q308K) was deleterious to Pob3 function (Fig. 7 C and E). Combining these mutations in a T252E Q308K double mutant resulted in cosuppression and restored near-WT levels of Pob3:H3 binding and in vivo functions (Fig. 7 D and E). This set of mutations may be useful to determine the importance of the relative amount of histones bound by distinct interacting proteins as nucleosomes are assembled and disassembled. At face value, these data would be consistent with suppression resulting from a restoration of electrostatic interactions. Although there are no gross structural changes in the Q308K mutant of Pob3, the loop adjacent to the mutation adopts a distinct conformation from WT (17). There- fore, T252E may stabilize the WT conformation in the Q308K Fig. 6. Rtt106 and Pob3 shared a double-PH domain architecture but dif- mutant background. Further structural studies are needed to fered in binding specificity for acetylated histones. (A) Double-PH domain of determine if electrostatic changes or conformational epistasis Rtt106 (green) was similar to that of Pob3 (blue) (PDB ID code 2GCL). The C- underlies these mutant phenotypes. GENETICS terminal loop between residues 425 and 432 on Pob3 is disordered in the Although it is counterintuitive that a basic surface on Rtt106 crystal and is represented as a dashed blue line. (B) H3K56ac was required for Rtt106:H3 binding in vivo. Rtt106-FLAG was immunoprecipitated from yeast would mediate binding with the highly basic histone proteins, whole-cell extract (WCE) as in Fig. 3A.(C) Pob3:H3 binding did not correlate histones do contain acidic patches that have previously been with H3K56ac. Pob3-MYC was immunoprecipitated from yeast whole-cell implicated in histone-histone interactions (1). Therefore, histone extract with anti-MYC resin. Copurifying proteins were visualized by immu- chaperones may exploit a similar binding mechanism to regulate noblotting with antibodies against the indicated proteins. nucleosome assembly by occluding these regions before histone octamer formation. Alternatively, the basic patch could co- pob3 T252E,Q308K ordinate histone binding via an intramolecular interaction with and HU-sensitive phenotypes. Strikingly, the Rtt106’s acidic C-terminal tail. This idea is consistent with our double mutant, which preserved the overall charge of the patch, findings that full-length Rtt106 was able to bind H3 in vivo, restored histone binding, transcription initiation, and replication whereas Rtt1062PH could not (Fig. S2). Unstructured acidic tails D E to near-WT levels (Fig. 7 and ). This analysis indicated that are a common feature of histone chaperones, including Pob3, as a precise level of H3 binding was required for WT Pob3 function are basic surfaces within structured domains (Fig. S4). There- fi and further implicated this speci c region on the N-terminal PH fore, intramolecular interactions between basic surfaces and domain as a functionally conserved histone-binding surface. acidic tails may represent a conserved feature of histone binding during chaperone-mediated nucleosome assembly. Discussion In addition to the histone-binding surface described here, the To mediate nucleosome assembly during replication and silenc- basic surface within the N-terminal PH domain is necessary for ing, Rtt106 must recognize and deposit H3K56ac-containing Rtt106’s weak DNA-binding activity (∼22 μM Kd) (16). DNA histones into chromatin. To understand the structural basis of binding has been reported for only one other chaperone, the fi this interaction, we identi ed two surfaces on Rtt106, one within human NAP-1 family member SET (31). Interestingly, similar to each PH domain, as important for histone binding and in vivo our findings, SET uses overlapping surfaces to bind histones and function. Comparative studies with Pob3 revealed a conserved DNA. Like Rtt106, mutations within the binding surface disrupt histone-binding motif in the N-terminal PH domain that, coun- interactions with both histones and DNA, suggesting that bind- terintuitively, was dependent on basic residues within the motif. ing each target might be mutually exclusive. These findings A feature that distinguishes Rtt106 from other histone chaper- suggest that a competition between DNA and histones for this ones is its modification-specific recognition of H3 that is acety- binding surface may function as a conserved ATP-independent lated on K56 (7). Our comparative studies implicated a loop mechanism for unloading histones onto DNA. within the C-terminal PH domain as the origin of H3K56ac specificity. We confirmed the importance of both histone-binding Loop Within the C-Terminal PH Domain Allowed Rtt106 to Target sites in Rtt106’s replication and silencing functions. These H3K56ac. In addition to the basic patch within the N-terminal PH studies expanded our understanding of the molecular recogni- domain of Rtt106, we defined specific residues on a loop con- tion of posttranslational modifications beyond the bromodomain necting two β-sheets within the C-terminal PH domain that were and plant homeodomain and of the regulation of nucleosome necessary for histone binding and Rtt106 function (Figs. 1 C and assembly by histone chaperones. D and 3 A and C). In contrast to the conserved histone-binding residues in the N-terminal PH domains of Rtt106 and Pob3, the Rtt106 and Pob3 Bound Histones Using a Conserved Basic Patch on the C-terminal loop of Pob3 could be mutated entirely without a loss N-Terminal PH Domain. The structural, electrostatic, and func- of function (Fig. 7 B and C). Because Pob3:H3 binding was not tional overlap of the N-terminal PH domains suggested that H3K56ac-dependent (Fig. 6C), this divergence suggested that the Rtt106 and Pob3 shared a common mechanism to bind histones C-terminal loop of Rtt106 contributed to its H3K56ac specificity.

Zunder et al. PNAS Early Edition | 7of10 Downloaded by guest on September 26, 2021 Fig. 7. A basic patch within the N-terminal PH domain of Pob3 was necessary for histone binding and chromatin assembly function. (A)(Upper) Mutations were generated to test the function of an analogous basic patch on the N-terminal PH domain of Pob3. The electrostatic surface representations for Rtt106 and Pob3 are shown in the aligned orientations from Fig. 6A.TheCα atoms of residues targeted on Rtt106 and Pob3 are shown as spheres (Rtt106 S80 and R86; Pob3 F249, T251, T252, R254, and R256). The Cα atom of Pob3 Q308K, a previously described mutation (17), is also shown as a sphere. (Lower) Residues from the C-terminal loop are shown for Rtt106 and Pob3. Mutated threonines are highlighted in red. (B) Pob3-MYC was immunoprecipitated as in Fig. 6C. Immunoblotting with antibodies against the indicated proteins monitored H3 copurification. (C)(Left) Expression of the Spt− reporter, lys2-128∂, was monitored by fivefold serial dilutions of each strain onto media lacking lysine (-LYS). (Right) DNA damage phenotypes were monitored by fivefold serial − dilutions onto HU (150 mM). Spt and HU phenotypes were monitored in S288c and W303 backgrounds, respectively. Strains are described in Materials and − Methods. The pob3 T252E Q308K double mutants had suppressed H3-binding defects (D), Spt phenotypes (E, Upper), and HU sensitivity (E, Lower). Pob3- MYC immunopurifications (D) and serial-dilution-based growth assays (E) were performed as in Fig. 6C and C, respectively.

However, the dramatic loss of Rtt106:H3 binding observed in conformation (37). Alternatively, rather than directly binding rtt109Δ cells is difficult to reconcile with the observation that H3K56ac, the loop may recognize a change in H3 conformation Rtt106 can bind recombinantly produced, unmodified H3/H4 or modification profile that is correlated with the acetylation (Figs. 3C and 6B). Therefore, in addition to the direct loss of of H3K56. affinity attributable to the absence of the H3K56 acetyl moiety in the rtt109Δ background, indirect factors, such as altered histone Rtt106 Mutants Localized but Did Not Deliver H3K56ac During DNA binding by other chaperones (7), changes to the dynamics of Replication and Silencing. Rtt106 played a key role in delivering histone incorporation into chromatin (25, 32), or variations to newly synthesized H3K56ac molecules into chromatin during the modification profile of H3 (33), likely contribute to the ab- DNA replication and silencing. Although previous studies relied sence of detectable Rtt106:H3 binding. Thus, although the on null alleles to probe Rtt106 function, we generated point Rtt106:H3 association in vivo appears to be highly modification- mutations that specifically disrupted Rtt106:H3 binding. These dependent (Fig. 6B), the association in vitro is only modestly mutations allowed us to untangle the requirements for Rtt106 specific for the acetylated form of H3 (7). Quantitative measures recruitment and H3K56ac delivery. Our results demonstrated of binding affinities, perhaps enabled by recent technologies that Rtt106 could localize to replication origins and silent that allow site-specific modifications of recombinant histones chromatin independent of its H3-binding activity and indepen- (34, 35), will reveal the direct contributions of modifications to dent of CAF-1 (Figs. 4B and 5B). The WT level of H3K56ac Rtt106:H3-binding specificity. enrichment in chromatin in cac1Δ strains further supported Modification-specific recognition represents an additional the idea that Rtt106 localization, and subsequent H3K56ac mechanism for chaperones to distinguish among different types of deposition, was independent of CAF-1 (Figs. 4C and 5C). At histone cargo. Curiously, structural analyses of distinct chaperone replication origins, Rtt106 localization was possibly achieved family members have not revealed any of the known modification- through a proposed interaction with PCNA (5) or through a di- recognition motifs (e.g., bromodomain, plant homeodomain, rect interaction with DNA (discussed above) (16). Because the chromodomain) (3). The importance of the C-terminal loop on localization of mutant Rtt106 proteins to HMRa1 was equivalent Rtt106 suggested a previously undescribed structural basis for in WT (silent) and cac1Δ (partially derepressed) backgrounds acetyl-lysine recognition and was consistent with earlier reports (Fig. 5B), Rtt106 recruitment was likely not Sir-dependent. that the loops connecting β-strands in PH domains are the source Therefore, our data are compatible with Rtt106 localizing to of ligand specificity (36). The loop may mediate H3K56ac binding HMR by the same mechanism that is used to localize elsewhere directly via an antibody-like–binding mechanism, where antigen in the genome. Alternatively, the phenotypes associated with the specificity is largely determined by antibody loop sequence and RTT106 mutants may have resulted from a kinetic delay in the

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1119095109 Zunder et al. Downloaded by guest on September 26, 2021 recruitment of the mutant Rtt106 proteins to chromatin that resolution native data using molecular replacement (Table S3; PDB ID code PNAS PLUS would not have been revealed by our assays. 3TO1). Further details of purification procedures and analysis are provided in Although H3K56ac weakens contacts between histones and SI Materials and Methods. DNA, creating a looser nucleosome structure (35), its role in replication is less well established. Our results are consistent with Yeast Strains, Plasmids, and Culture. All yeast strains were generated in the S. cerevisiae W303 background unless otherwise indicated (Table S4). Genes a model in which loosely assembled acetylated nucleosomes are were deleted by one-step integration of KO cassettes (42), followed by PCR thought to reduce the energetic costs of ATP-dependent remod- verification of the 5′ and 3′ ends of the targeted gene. The hmr-a1Δ::URA3 eling, which slides nucleosomes along newly replicated DNA. In reporter strain was previously described (43). The lys2-128∂ reporter strain the absence of incorporated H3K56ac, because of defects in was previously described (17). Mutant rtt106 plasmids were introduced into Rtt106-histone binding, nucleosomes may be less accessible to each strain by transformation (44). For the initial screen, JRY9243 was remodelers, leading to stalled replication and subsequent dam- transformed with each of the rtt106 mutant plasmids. The pob3 plasmids age, which we observed as CPT sensitivity. harboring mutations of interest were introduced into strains containing WT The role of H3K56ac in silencing is paradoxical. We and versions of the relevant gene (JRY9282 and JRY9285) by plasmid swap using others have shown that rtt109Δ cells, which lack H3K56ac, have FOA counterselection. Plasmids are described in Table S2. fi RTT106 was cloned into pRS313 using gap repair to generate pJR2877. fully functional silent chromatin, suggesting that the modi cation fi is not critical for silencing (27, 38) (Fig. 5A). Therefore, the small RTT106 was ampli ed from WT genomic DNA (JRY3009) using Phusion polymerase (New England Biolabs). The PCR fragment and EcoRI SalI-diges- yet significant silencing defects observed in rtt106Δ cac1Δ strains HMR ted pRS313 were cotransformed into JRY3009. Plasmids were rescued from at could not simply result from the reduction of H3K56ac His+ transformants and sequenced. Site-directed mutagenesis generated the within chromatin. Intriguingly, cells with elevated levels of rtt106(69-300) truncation (pJR2878) as described (45). A C-terminal 3× FLAG H3K56ac (hst3Δ hst4Δ) have telomeric silencing defects, sug- tag (46) was integrated on the plasmid in frame with RTT106 and rtt106(69- gesting that excess H3K56ac is antagonistic to silencing (27). 300) using homologous recombination, resulting in pJR2879 and pJR2880, Therefore, if a cell makes H3K56ac yet does not incorporate it respectively. The 72 rtt106 point mutations and additional follow-up mu- (as in rtt106Δ cac1Δ mutants) or allows it to accumulate (as in tations were generated on pJR2879 by site-directed mutagenesis with Pfu hst3Δ hst4Δ mutants), silencing is disrupted. Consistent with Ultra polymerase (Stratagene) (Table S1). these results, rtt106Δ cac1Δ silencing defects at HMR and hst3Δ Full-length RTT106 was inserted into pET3a-Tr by ligation-independent – hst4Δ silencing defects at telomeres are both suppressed by an cloning to generate His6-RTT106 expressing plasmids (pJR2883) (47). Point mutations were generated as described above (pJR2884–2886). The poly- rtt109Δ mutation (27) (Fig. 5A). Because H3K56ac is not re- GENETICS cistronic expression plasmid, containing full-length untagged HHT1 and quired for silencing, these data suggested that the silencing de- rtt106Δ cac1Δ fl HHF1, was previously described (pJR2881) (48, 49). fect in double mutants re ected an inhibitory role POB3 and POB3-13×MYC were cloned into pRS313 by gap repair as de- of unincorporated H3K56ac on silencing. The role of Rtt106 and scribed above creating pJR2887 and pJR2888, respectively. POB3 and POB3- CAF-1 in silencing may be either to remove excess H3K56ac 13×MYC::KanMX were amplified from JRY3009 and JRY9281 genomic DNA, from the free histone pool through nucleosome assembly and/or respectively, using Phusion polymerase. POB3 point mutations were created to create an arrangement of H3K56ac-containing nucleosomes as above (Table S2). POB3 mutants had increased chemical sensitivities in the that is compatible with Sir protein association. W303 background (JRY9285) compared with S288c (JRY9282). Therefore, HU sensitivity was monitored in the W303 background. All other POB3 experi- Potential for Promiscuous Binding Domains in Histone Recognition. ments were performed in the S288c background. This work expands the study of modification-specific histone To screen rtt106 and pob3 mutants for silencing, transcription initiation, recognition by demonstrating that the PH domain architecture and/or chemical sensitivity phenotypes, fivefold serial dilutions of midlog- can recognize and regulate acetylated histones. Because PH phase cultures were spotted onto selective media, as described (17, 43). All selective media lacked histidine (−HIS) to maintain selection of rtt106 or domains can fold from nearly unrecognizable sequences, it is pob3 plasmids. likely that additional PH domain-containing proteins with fi fi modi cation-speci c histone-binding activity have yet to be dis- Protein Analysis and Copurification. Yeast whole-cell extract analysis. Yeast covered in yeast and other species. However, as we observed for whole-cell extracts were precipitated using 20% (wt/vol) trichloroacetic acid Pob3, the modification dependence of PH domain-histone and solubilized in SDS loading buffer. SDS/PAGE and immunoblotting were binding is variable, and therefore must be determined empiri- performed using standard procedures and evaluated with a LI-COR Odyssey cally. Here, H3K56ac recognition by Rtt106 regulates replication imaging system. Anti-H3 (Ab1791) or anti-H3K56ac (07-677; Millipore) was and silencing. In other eukaryotes, H3K56ac has been implicated used to monitor bulk or modified H3. Anti-Pgk1 (Invitrogen) was used as in stem cell differentiation and the DNA damage response (39, a loading control. × × 40); however, the proteins that specifically recognize this modi- Yeast CoIP. Rtt106-3 FLAG and Pob3-13 MYC CoIPs were performed as de- fi scribed (50). Solubilized yeast lysate was incubated with 50 μL of anti-FLAG cation remain unknown. Thus, although Rtt106 is a fungal- × μ fi M2 agarose (Sigma) (for Rtt106-3 FLAG) or 30 L of anti-c-Myc agarose speci c chaperone, a similar architecture may have evolved (Sigma) (for Pob3-13×MYC). SDS/PAGE, immunoblotting, and imaging were convergently to recognize this mark in other species. performed as described above. Anti-Flag M2 antibody (F3165; Sigma), anti-c- In addition to PH domains, other binding domains with pro- Myc antibody (M4439; Sigma), and anti-H3 (Ab1791) or anti-H3K56ac (07- miscuous ligand specificity (e.g., Ig-like domains) may interact 677; Millipore) were used to detect Rtt106-3×FLAG, Pob3-13×MYC, or his- with histones in a modification-specific manner. For example, tones, respectively. Further details are provided in SI Materials and Methods. although Asf1, which contains an Ig-like domain (41), is cur- Escherichia coli extract histone-binding assays. Bacterial coexpression of HHT1 – fi fi rently thought to be a modification-independent binder, mod- HHF1 (pJR2881) and His6-RTT106 (pJR2883 2886) and nickel af nity puri - ifications to histones H3 and H4 may modulate its affinity for cations were performed as described (48). SDS/PAGE, immunoblotting, and histones. Even small modification-dependent changes in binding imaging were performed as described above. Anti-His (34670; Qiagen) was fi fl used to detect His6-Rtt106, and copurifying histones were detected with af nity could in uence nucleosome function. The discovery of anti-H3 (Ab1791). Further details are provided in SI Materials and Methods. additional promiscuous binding domains in histone-interacting proteins will aid in understanding the network of interactions RNA Preparation and Analysis. RNA analysis was performed exactly as de- that regulate nucleosome assembly. scribed (51). cDNA was analyzed by quantitative PCR as described in SI Materials and Methods. Amplification values for all primer sets were nor- Materials and Methods malized to ACT1 cDNA amplification values. Samples were analyzed in tripli- Rtt106 Structural Analysis. A truncated Rtt106 construct (Rtt1062PH) was pu- cate for at least three independent RNA preparations. Statistical comparisons rified, crystallized, and phased by multiwavelength anomalous dispersion. were performed using a two-tailed unpaired t test. Primer sequences are listed An initial model built into experimental maps was used to phase higher in Table S5.

Zunder et al. PNAS Early Edition | 9of10 Downloaded by guest on September 26, 2021 Rtt106-FLAG ChIP. ChIP analyses were performed as described (51) with minor Sepharose (17-5280-01; GE Healthcare). The sonicated sample was split in

modifications. Fifty OD600 units of log-phase cells were cross-linked with 1% half and incubated with either H3- or H3K56ac-coupled beads. Precipitated formaldehyde for 30 min at room temperature. Chromatin was sonicated to DNA fragments were analyzed by quantitative PCR as described above. an average size of 500 bp. Rtt106-FLAG was immunoprecipitated using anti- Amplification values for all primer sets were normalized to a previously FLAG M2 agarose (Sigma). Precipitated DNA fragments were analyzed by described reference locus, SSC1 (27). Samples were analyzed as described for fi quantitative PCR as above. Ampli cation values for all primer sets were Rtt106-FLAG ChIP. normalized to percent input. Samples were analyzed in triplicate for at least three independent chromatin preps. Statistical comparisons were performed ACKNOWLEDGMENTS. We thank James Fraser and Tim Formosa for advice, using a two-tailed unpaired t test. Primer sequences are listed in Table S5. comments, and strains. We thank Paul Kaufman, Ann Kirchmaier, and David Stillman for thoughtful critiques and insights. We acknowledge technical H3K56ac ChIP. ChIP analyses were performed as previously described (52) with support from Caitlin Schartner, the QB3 MacroLab, and the staff of the fi minor modi cations. Fifty OD600 units of log-phase cells were cross-linked Advanced Light Source Beamline 8.3.1. This research was supported by a Na- with 1% formaldehyde for 20 min at room temperature. Chromatin was tional Science Foundation Graduate Research Fellowship (to R.M.Z.), Na- sheared as described above. H3 antibody (1.2 μg, Ab1791) or H3K56ac an- tional Cancer Institute Research Grant CA0777373 (to A.J.A. and J.M.B.), tibody (1 μL, 07-677; Millipore) was coupled to a 30-μL slurry of Protein A and National Institute of Health Research Grant GM31105 (to J.R.).

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