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

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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 de- acetylation, 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
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