Yaf9, a novel NuA4 histone acetyltransferase subunit,is required for the cellular response to spindle stress in yeast

Yaf9 is one of three proteins in budding yeast containing a YEATS domain. We show that Yaf9 is part of a large complex and that it coprecipitates with three known subunits of the NuA4 histone acetyltransferase. Although Esa1, the catalytic subunit of NuA4, is essential for viability, we found that yaf9 Delta mutants are viable but hypersensitive to microtubule depolymerizing agents and synthetically lethal with two different mutants of the mitotic apparatus. Microtubules depolymerized more readily in the yaf9Delta mutant compared to the wild type in the presence of nocodazole, and recovery of microtubule polymerization and cell division from limiting concentrations of nocodazole was inhibited. Two other NuA4 mutants (esa1-1851 and yng2 Delta) and nonacetylatable histone H4 mutants were also sensitive to benomyl. Furthermore, wild-type budding yeast were more resistant to benomyl when grown in the presence of trichostatin A, a histone deacetylase inhibitor. These results strongly suggest that acetylation of histone H4 by NuA4 is required for the cellular resistance to spindle stress.     

Elucidation of binding preferences of YEATS domains to site-specific acetylated nucleosome core particles

Acetylated lysine residues (Kac) in histones are recognized by epigenetic reader proteins, such as Yaf9, ENL, AF9, Taf14, and Sas5 (YEATS) domain-containing proteins. Human YEATS domains bind to the acetylated N-terminal tail of histone H3; however, their Kac-binding preferences at the level of the nucleosome are unknown. Through genetic code reprogramming, here, we established a nucleosome core particle (NCP) array containing histones that were acetylated at specific residues and used it to compare the Kac-binding preferences of human YEATS domains. We found that AF9-YEATS showed basal binding to the unmodified NCP and that it bound stronger to the NCP containing a single acetylation at one of K4, K9, K14, or K27 of H3, or to histone H4 multi-acetylated between K5 and K16. Crystal structures of AF9-YEATS in complex with an H4 peptide diacetylated either at K5/K8 or K8/K12 revealed that the aromatic cage of the YEATS domain recognized the acetylated K8 residue. Interestingly, E57 and D103 of AF9, both located outside of the aromatic cage, were shown to interact with acetylated K5 and K12 of H4, respectively, consistent with the increase in AF9-YEATS binding to the H4K8-acetylated NCP upon additional acetylation at K5 or K12. Finally, we show that a mutation of E57 to alanine in AF9-YEATS reduced the binding affinity for H4 multiacetylated NCPs containing H4K5ac. Our data suggest that the Kac-binding affinity of AF9-YEATS increases additively with the number of Kac in the histone tail.     

Evaluation of acyllysine isostere interactions with the aromatic pocket of the AF9 YEATS domain

Amide−π interactions, in which an amide interacts with an aromatic group, are ubiquitous in biology, yet remain understudied relative to other noncovalent interactions. Recently, we demonstrated that an electrostatically tunable amide−π interaction is key to recognition of histone acyllysine by the AF9 YEATS domain, a reader protein which has emerged as a therapeutic target due to its dysregulation in cancer. Amide isosteres are commonly employed in drug discovery, often to prevent degradation by proteases, and have proven valuable in achieving selectivity when targeting epigenetic proteins. However, like amide−π interactions, interactions of amide isosteres with aromatic rings have not been thoroughly studied despite widespread use. Herein, we evaluate the recognition of a series of amide isosteres by the AF9 YEATS domain using genetic code expansion to evaluate the amide isostere−π interaction. We show that compared to the amide−π interaction with the native ligand, each isostere exhibits similar electrostatic tunability with an aromatic residue in the binding pocket, demonstrating that the isosteres maintain similar interactions with the aromatic residue. We identify a urea-containing ligand that binds with enhanced affinity for the AF9 YEATS domain, offering a promising starting point for inhibitor development. Furthermore, we demonstrate that carbamate and urea isosteres of crotonyllysine are resistant to enzymatic removal by SIRT1, a protein that cleaves acyl post-translational modifications, further indicating the potential of amide isosteres in YEATS domain inhibitor development. These results also provide experimental precedent for interactions of these common drug discovery moieties with aromatic rings that can inform computational methods.     

Exploring the mechanism how AF9 recognizes and binds H3K9ac by molecular dynamics simulations and free energy calculations

Histone acetylation is a very important regulatory mechanism in gene expression in the chromatin context. A new protein family‐YEATS domains have been found as a novel histone acetylation reader, which could specific recognize the histone lysine acetylation. AF9 is an important one in the YEATS family. Focused on the AF9‐H3K9ac (K9 acetylation) complex (ALY) (PDB code: 4TMP) and a serials of mutants, MUT (the acetyllsine of H3K9ac was mutated to lysine), F59A, G77A, and D103A, we applied molecular dynamics simulation and molecular mechanics Poisson?Boltzmann (MM‐PBSA) free energy calculations to examine the role of AF9 protein in recognition interaction. The simulation results and analysis indicate that some residues of the protein have significant influence on recognition and binding to H3K9ac peptides and hydrophobic surface show the hydrophobic interactions play an important role in the binding. Our work can give important information to understand how the protein AF9 recognizes the peptides H3K9ac. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 779–786, 2016.     

Comparative analysis of SET domain proteins in maize and Arabidopsis reveals multiple duplications preceding the divergence of monocots and dicots

Histone proteins play a central role in chromatin packaging, and modification of histones is associated with chromatin accessibility. SET domain [Su(var)3-9, Enhancer-of-zeste, Trithorax] proteins are one class of proteins that have been implicated in regulating gene expression through histone methylation. The relationships of 22 SET domain proteins from maize (Zea mays) and 32 SET domain proteins from Arabidopsis were evaluated by phylogenetic analysis and domain organization. Our analysis reveals five classes of SET domain proteins in plants that can be further divided into 19 orthology groups. In some cases, such as the Enhancer of zeste-like and trithorax-like proteins, plants and animals contain homologous proteins with a similar organization of domains outside of the SET domain. However, a majority of plant SET domain proteins do not have an animal homolog with similar domain organization, suggesting that plants have unique mechanisms to establish and maintain chromatin states. Although the domains present in plant and animal SET domain proteins often differ, the domains found in the plant proteins have been generally implicated in protein-protein interactions, indicating that most SET domain proteins operate in complexes. Combined analysis of the maize and Arabidopsis SET domain proteins reveals that duplication of SET domain proteins in plants is extensive and has occurred via multiple mechanisms that preceded the divergence of monocots and dicots.     

Swc2 is a widely conserved H2AZ-binding module essential for ATP-dependent histone exchange

The histone variant H2AZ is incorporated preferentially at specific locations in chromatin to modulate chromosome functions. In Saccharomyces cerevisiae, deposition of histone H2AZ is mediated by the multiprotein SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Here, we define interactions between SWR1 components and H2AZ, revealing a link between the ATPase domain of Swr1 and three subunits required for the binding of H2AZ. We discovered that Swc2 binds directly to and is essential for transfer of H2AZ. Swc6 and Arp6 are necessary for the association of Swc2 and for nucleosome binding, whereas other subunits, Swc5 and Yaf9, are required for H2AZ transfer but neither H2AZ nor nucleosome binding. Finally, the C-terminal alpha-helix of H2AZ is crucial for its recognition by SWR1. These findings provide insight on the initial events of histone exchange.     

The Saccharomyces cerevisiae Set1 complex includes an Ash2 homologue and methylates histone 3 lysine 4.

The SET domain proteins, SUV39 and G9a have recently been shown to be histone methyltransferases specific for lysines 9 and 27 (G9a only) of histone 3 (H3). The SET domains of the Saccharomyces cerevisiae Set1 and Drosophila trithorax proteins are closely related to each other but distinct from SUV39 and G9a. We characterized the complex associated with Set1 and Set1C and found that it is comprised of eight members, one of which, Bre2, is homologous to the trithorax-group (trxG) protein, Ash2. Set1C requires Set1 for complex integrity and mutation of Set1 and Set1C components shortens telomeres. One Set1C member, Swd2/Cpf10 is also present in cleavage polyadenylation factor (CPF). Set1C methylates lysine 4 of H3, thus adding a new specificity and a new subclass of SET domain proteins known to methyltransferases. Since methylation of H3 lysine 4 is widespread in eukaryotes, we screened the databases and found other Set1 homologues. We propose that eukaryotic Set1Cs are H3 lysine 4 methyltransferases and are related to trxG action through association with Ash2 homologues.     

The topology of chromatin-binding domains in the NuRD deacetylase complex

Class I histone deacetylase complexes play essential roles in many nuclear processes. Whilst they contain a common catalytic subunit, they have diverse modes of action determined by associated factors in the distinct complexes. The deacetylase module from the NuRD complex contains three protein domains that control the recruitment of chromatin to the deacetylase enzyme, HDAC1/2. Using biochemical approaches and cryo-electron microscopy, we have determined how three chromatin-binding domains (MTA1-BAH, MBD2/3 and RBBP4/7) are assembled in relation to the core complex so as to facilitate interaction of the complex with the genome. We observe a striking arrangement of the BAH domains suggesting a potential mechanism for binding to di-nucleosomes. We also find that the WD40 domains from RBBP4 are linked to the core with surprising flexibility that is likely important for chromatin engagement. A single MBD2 protein binds asymmetrically to the dimerisation interface of the complex. This symmetry mismatch explains the stoichiometry of the complex. Finally, our structures suggest how the holo-NuRD might assemble on a di-nucleosome substrate.     

A flagellar A-kinase anchoring protein with two amphipathic helices forms a structural scaffold in the radial spoke complex

A-kinase anchoring proteins (AKAPs) contain an amphipathic helix (AH) that binds the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase A (PKA). Many AKAPs were discovered solely based on the AH–RIIa interaction in vitro. An RIIa or a similar Dpy-30 domain is also present in numerous diverged molecules that are implicated in critical processes as diverse as flagellar beating, membrane trafficking, histone methylation, and stem cell differentiation, yet these molecules remain poorly characterized. Here we demonstrate that an AKAP, RSP3, forms a dimeric structural scaffold in the flagellar radial spoke complex, anchoring through two distinct AHs, the RIIa and Dpy-30 domains, in four non-PKA spoke proteins involved in the assembly and modulation of the complex. Interestingly, one AH can bind both RIIa and Dpy-30 domains in vitro. Thus, AHs and D/D domains constitute a versatile yet potentially promiscuous system for localizing various effector mechanisms. These results greatly expand the current concept about anchoring mechanisms and AKAPs.     

Domain cross-talk in regulation of histone modifications: Molecular mechanisms and targeting opportunities

Functional cross-talk between the catalytic and reader domains in chromatin-modifying enzymes and protein complexes enable coordinated regulation of chromatin modification status, and consequently impacts chromatin-associated processes. ZZ domains are a recently identified class of chromatin readers that recognize the N-terminal region of histone H3 to direct and regulate acetylation activity of several histone acetylation complexes. Cross-talk between chromatin readers sensitive to methylation, and catalytic domains of methyltransferases and demethylases impacts substrate specificity, catalytic activity, and propagation of chromatin marks. Recently described allosteric ligands that target domain communication highlight the potential of domain cross-talk in the development of the next-generation of chromatin-directed therapeutics.