Structure of the NuA4 acetyltransferase complex bound to the nucleosome.

Deoxyribonucleic acid in eukaryotes wraps around the histone octamer to form nucleosomes1, the fundamental unit of chromatin. The N termini of histone H4 interact with nearby nucleosomes and play an important role in the formation of high-order chromatin structure and heterochromatin silencing2-4. NuA4 in yeast and its homologue Tip60 complex in mammalian cells are the key enzymes that catalyse H4 acetylation, which in turn regulates chromatin packaging and function in transcription activation and DNA repair5-10. Here we report the cryo-electron microscopy structure of NuA4 from Saccharomyces cerevisiae bound to the nucleosome. NuA4 comprises two major modules: the catalytic histone acetyltransferase (HAT) module and the transcription activator-binding (TRA) module. The nucleosome is mainly bound by the HAT module and is positioned close to a polybasic surface of the TRA module, which is important for the optimal activity of NuA4. The nucleosomal linker DNA carrying the upstream activation sequence is oriented towards the conserved, transcription activator-binding surface of the Tra1 subunit, which suggests a potential mechanism of NuA4 to act as a transcription co-activator. The HAT module recognizes the disk face of the nucleosome through the H2A-H2B acidic patch and nucleosomal DNA, projecting the catalytic pocket of Esa1 to the N-terminal tail of H4 and supporting its function in selective acetylation of H4. Together, our findings illustrate how NuA4 is assembled and provide mechanistic insights into nucleosome recognition and transcription co-activation by a HAT.

Catalysis by protein acetyltransferase Gcn5.

Gcn5 serves as the defining member of the Gcn5-related N-acetyltransferase (GNAT) superfamily of proteins that display a common structural fold and catalytic mechanism involving the transfer of the acyl-group, primarily acetyl-, from CoA to an acceptor nucleophile. In the case of Gcn5, the target is the ε-amino group of lysine primarily on histones. Over the years, studies on Gcn5 structure-function have often formed the basis by which we understand the complex activities and regulation of the entire protein acetyltransferase family. It is now appreciated that protein acetylation occurs on thousands of proteins and can reversibly regulate the function of many cellular processes. In this review, we provide an overview of our fundamental understanding of catalysis, regulation of activity and substrate selection, and inhibitor development for this archetypal acetyltransferase.

The Ada2/Ada3/Gcn5/Sgf29 histone acetyltransferase module.

Histone post-translational modifications are essential for the regulation of gene expression in eukaryotes. Gcn5 (KAT2A) is a histone acetyltransferase that catalyzes the post-translational modification at multiple positions of histone H3 through the transfer of acetyl groups to the free amino group of lysine residues. Gcn5 catalyzes histone acetylation in the context of a HAT module containing the Ada2, Ada3 and Sgf29 subunits of the parent megadalton SAGA transcriptional coactivator complex. Biochemical and structural studies have elucidated mechanisms for Gcn5's acetyl- and other acyltransferase activities on histone substrates, for histone H3 phosphorylation and histone H3 methylation crosstalks with histone H3 acetylation, and for how Ada2 increases Gcn5's histone acetyltransferase activity. Other studies have identified Ada2 isoforms in SAGA-related complexes and characterized variant Gcn5 HAT modules containing these Ada2 isoforms. In this review, we highlight biochemical and structural studies of Gcn5 and its functional interactions with Ada2, Ada3 and Sgf29.

Structure of the transcription coactivator SAGA.

Gene transcription by RNA polymerase II is regulated by activator proteins that recruit the coactivator complexes SAGA (Spt-Ada-Gcn5-acetyltransferase)1,2 and transcription factor IID (TFIID)2-4. SAGA is required for all regulated transcription5 and is conserved among eukaryotes6. SAGA contains four modules7-9: the activator-binding Tra1 module, the core module, the histone acetyltransferase (HAT) module and the histone deubiquitination (DUB) module. Previous studies provided partial structures10-14, but the structure of the central core module is unknown. Here we present the cryo-electron microscopy structure of SAGA from the yeast Saccharomyces cerevisiae and resolve the core module at 3.3 Å resolution. The core module consists of subunits Taf5, Sgf73 and Spt20, and a histone octamer-like fold. The octamer-like fold comprises the heterodimers Taf6-Taf9, Taf10-Spt7 and Taf12-Ada1, and two histone-fold domains in Spt3. Spt3 and the adjacent subunit Spt8 interact with the TATA box-binding protein (TBP)2,7,15-17. The octamer-like fold and its TBP-interacting region are similar in TFIID, whereas Taf5 and the Taf6 HEAT domain adopt distinct conformations. Taf12 and Spt20 form flexible connections to the Tra1 module, whereas Sgf73 tethers the DUB module. Binding of a nucleosome to SAGA displaces the HAT and DUB modules from the core-module surface, allowing the DUB module to bind one face of an ubiquitinated nucleosome.

Crystal structure of TbEsa1 presumed Tudor domain from Trypanosoma brucei.

The essential SAS2-related acetyltransferase 1 (Esa1), as a acetyltransferase of MYST family, is indispensable for the cell cycle and transcriptional regulation. The Tudor domain consists of 60 amino acids and belongs to the Royal family, which serves as a module interacting with methylated histone and/or DNA. Although Tudor domain has been widely studied in higher eukaryotes, its structure and function remain unclear in Trypanosoma brucei (T. brucei), a protozoan unicellular parasite causing sleeping sickness in human and nagana in cattle in sub-Saharan Africa. Here, we determined a high-resolution structure of TbEsa1 presumed Tudor domain from T. brucei by X-ray crystallography. TbEsa1 Tudor domain adopts a conserved Tudor-like fold, which is comprised of a five-stranded ß-barrel surrounded by two short α-helices. Furthermore, we revealed a non-specific DNA binding pattern of TbEsa1 Tudor domain. However, TbEsa1 Tudor domain showed no methyl-histone binding ability, due to the absence of key aromatic residues forming a conserved aromatic cage.

Molecular Architecture of the Essential Yeast Histone Acetyltransferase Complex NuA4 Redefines Its Multimodularity.

Conserved from yeast to humans, the NuA4 histone acetyltransferase is a large multisubunit complex essential for cell viability through the regulation of gene expression, genome maintenance, metabolism, and cell fate during development and stress. How the different NuA4 subunits work in concert with one another to perform these diverse functions remains unclear, and addressing this central question requires a comprehensive understanding of NuA4's molecular architecture and subunit organization. We have determined the structure of fully assembled native yeast NuA4 by single-particle electron microscopy. Our data revealed that NuA4 adopts a trilobal overall architecture, with each of the three lobes constituted by one or two functional modules. By performing cross-linking coupled to mass spectrometry analysis and in vitro protein interaction studies, we further mapped novel intermolecular interfaces within NuA4. Finally, we combined these new data with other known structural information of NuA4 subunits and subassemblies to construct a multiscale model to illustrate how the different NuA4 subunits and modules are spatially arranged. This model shows that the multiple chromatin reader domains are clustered together around the catalytic core, suggesting that NuA4's multimodular architecture enables it to engage in multivalent interactions with its nucleosome substrate.

Structure of the transcription activator target Tra1 within the chromatin modifying complex SAGA.

The transcription co-activator complex SAGA is recruited to gene promoters by sequence-specific transcriptional activators and by chromatin modifications to promote pre-initiation complex formation. The yeast Tra1 subunit is the major target of acidic activators such as Gal4, VP16, or Gcn4 but little is known about its structural organization. The 430 kDa Tra1 subunit and its human homolog the transformation/transcription domain-associated protein TRRAP are members of the phosphatidyl 3-kinase-related kinase (PIKK) family. Here, we present the cryo-EM structure of the entire SAGA complex where the major target of activator binding, the 430 kDa Tra1 protein, is resolved with an average resolution of 5.7 Å. The high content of alpha-helices in Tra1 enabled tracing of the majority of its main chain. Our results highlight the integration of Tra1 within the major epigenetic regulator SAGA.

Structures and Activities of the Elongator Complex and Its Cofactors.

Elongator is a highly conserved eukaryotic protein complex consisting of two sets of six Elp proteins, while homologues of its catalytic subunit Elp3 are found in all the kingdoms of life. Although it was originally described as a transcription elongation factor, cumulating evidence suggests that its primary function is catalyzing tRNA modifications. In humans, defects in Elongator subunits are associated with neurological disorders and cancer. Although further studies are still required, a clearer picture of the molecular mechanism of action of Elongator and its cofactors has started to emerge within recent years that have witnessed significant development in the field. In this review we summarize recent Elongator-related findings provided largely by crystal structures of several subunits of the complex, the electron microscopy structure of the entire yeast holoenzyme, as well as the structure of the Elongator cofactor complex Kti11/Kti13.

Live imaging of H3K9 acetylation in plant cells.

Proper regulation of histone acetylation is important in development and cellular responses to environmental stimuli. However, the dynamics of histone acetylation at the single-cell level remains poorly understood. Here we established a transgenic plant cell line to track histone H3 lysine 9 acetylation (H3K9ac) with a modification-specific intracellular antibody (mintbody). The H3K9ac-specific mintbody fused to the enhanced green fluorescent protein (H3K9ac-mintbody-GFP) was introduced into tobacco BY-2 cells. We successfully demonstrated that H3K9ac-mintbody-GFP interacted with H3K9ac in vivo. The ratio of nuclear/cytoplasmic H3K9ac-mintbody-GFP detected in quantitative analysis reflected the endogenous H3K9ac levels. Under chemically induced hyperacetylation conditions with histone deacetylase inhibitors including trichostatin A, Ky-2 and Ky-14, significant enhancement of H3K9ac was detected by H3K9ac-mintbody-GFP dependent on the strength of inhibitors. Conversely, treatment with a histone acetyltransferase inhibitor, C646 caused a reduction in the nuclear to cytoplasmic ratio of H3K9ac-mintbody-GFP. Using this system, we assessed the environmental responses of H3K9ac and found that cold and salt stresses enhanced H3K9ac in tobacco BY-2 cells. In addition, a combination of H3K9ac-mintbody-GFP with 5-ethynyl-2'-deoxyuridine labelling confirmed that H3K9ac level is constant during interphase.

Regulation of SRC-3 intercompartmental dynamics by estrogen receptor and phosphorylation.

The steroid receptor coactivator 3 gene (SRC-3) (AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160 family transcription coactivator and a known oncogene. Despite its importance, the functional regulation of SRC-3 remains poorly understood within a cellular context. Using a novel combination of live-cell, high-throughput, and fluorescent microscopy, we report SRC-3 to be a nucleocytoplasmic shuttling protein whose intracellular mobility, solubility, and cellular localization are regulated by phosphorylation and estrogen receptor alpha (ERalpha) interactions. We show that both chemical inhibition and small interfering RNA reduction of the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MEK1/2) pathway induce a cytoplasmic shift in SRC-3 localization, whereas stimulation by epidermal growth factor signaling enhances its nuclear localization by inducing phosphorylation at T24, S857, and S860, known participants in the phosphocode that regulates SRC-3 activity. Accordingly, the cytoplasmic localization of a nonphosphorylatable SRC-3 mutant further supported these results. In the presence of ERalpha, U0126 also dramatically reduces (i) ligand-dependent colocalization of SRC-3 and ERalpha, (ii) the formation of ER-SRC-3 complexes in cell lysates, and (iii) SRC-3 targeting to a visible, ERalpha-occupied and -regulated prolactin promoter array. Taken together, these results indicate that phosphorylation coordinates SRC-3 coactivator function by linking the probabilistic formation of transient nuclear receptor-coactivator complexes with its molecular dynamics and cellular compartmentalization. Technically and conceptually, these findings have a new and broad impact upon evaluating mechanisms of action of gene regulators at a cellular system level.