The yeast histone acetyltransferase A2 complex, but not free Gcn5p, binds stably to nucleosomal arrays

We have investigated the structural basis for the differential catalytic function of the yeast Gcn5p-containing histone acetyltransferase (HAT) A2 complex and free recombinant yeast Gcn5p (rGcn5p). HAT A2 is shown to be a unique complex that contains Gcn5p, Ada2p, and Ada3p, but not proteins specific to other related HAT A complexes, e.g. ADA, SAGA. Nevertheless, HAT A2 produces the same unique polyacetylation pattern of nucleosomal substrates reported previously for ADA and SAGA, demonstrating that proteins specific to the ADA and SAGA complexes do not influence the enzymatic activity of Gcn5p within the HAT A2 complex. To investigate the role of substrate interactions in the differential behavior of free and complexed Gcn5p, sucrose density gradient centrifugation was used to characterize the binding of HAT A2 and free rGcn5p to intact and trypsinized nucleosomal arrays, H3/H4 tetramer arrays, and nucleosome core particles. We find that HAT A2 forms stable complexes with all nucleosomal substrates tested. In distinct contrast, rGcn5p does not interact stably with nucleosomal arrays, despite being able to specifically monoacetylate the H3 N terminus of nucleosomal substrates. Our data suggest that the ability of the HAT A2 complex to bind stably to nucleosomal arrays is functionally related to both local and global acetylation by the complexed and free forms of Gcn5p.     

Recruitment of chromatin remodelling factors during gene activation via the glucocorticoid receptor N-terminal domain

We have shown that yeast mutants with defects in the Ada adaptor proteins are defective in hormone-dependent gene activation by ectopically expressed human glucocorticoid receptor (GR). Others have shown that the Ada2 protein is required for physical interactions between some activation domains and TBP (TATA-binding protein), whereas the Gcn5 (Ada4) protein has a histone acetyltransferase (HAT) activity. Although all HAT enzymes are able to acetylate histone substrates, some also acetylate non-histone proteins. Taken together, these observations suggest that the Ada proteins have the ability to effect different steps in the process of gene activation. It has recently been shown that the Ada proteins are present in two distinct protein complexes, the Ada complex and a larger SAGA complex. Our recent work has focused on determining (1) which of the Ada-containing complexes mediates gene activation by GR, (2) whether the HAT activity encoded by GCN5 is required for GR-dependent gene activation, (3) whether the Ada proteins contribute to GR-mediated activation at the level of chromatin remodelling and (4) how the role of these HAT complexes is integrated with other chromatin remodelling activities during GR-mediated gene activation. Our results suggest a model in which GR recruits the SAGA complex and that this contributes to chromatin remodelling via a mechanism involving the acetylation of histones. Furthermore, recruitment of the SWI/SNF remodelling complex also has a role in GR-mediated activation that is independent of the role of SAGA. These complexes are similar to analogous mammalian complexes and therefore these results are likely to be relevant to the human system.     

A native peptide ligation strategy for deciphering nucleosomal histone modifications

Post-translational modifications of histones influence both chromatin structure and the binding and function of chromatin-associated proteins. A major limitation to understanding these effects has been the inability to construct nucleosomes in vitro that harbor homogeneous and site-specific histone modifications. Here, we describe a native peptide ligation strategy for generating nucleosomal arrays that can harbor a wide range of desired histone modifications. As a first test of this method, we engineered model nucleosomal arrays in which each histone H3 contains a phosphorylated serine at position 10 and performed kinetic analyses of Gcn5-dependent histone acetyltransferase activities. Recombinant Gcn5 shows increased histone acetyltransferase activity on nucleosomal arrays harboring phosphorylated H3 serine 10 and is consistent with peptide studies. However, in contrast to analyses using peptide substrates, we find that the histone acetyltransferase activity of the Gcn5-containing SAGA complex is not stimulated by H3 phosphorylation in the context of nucleosomal arrays. This difference between peptide and array substrates suggests that the ability to generate specifically modified nucleosomal arrays should provide a powerful tool for understanding the effects of post-translational histone modifications.     

Host cell factor and an uncharacterized SANT domain protein are stable components of ATAC, a novel dAda2A/dGcn5-containing histone acetyltransferase complex in Drosophila

Gcn5 is a conserved histone acetyltransferase (HAT) found in a number of multisubunit complexes from Saccharomyces cerevisiae, mammals, and flies. We previously identified Drosophila melanogaster homologues of the yeast proteins Ada2, Ada3, Spt3, and Tra1 and showed that they associate with dGcn5 to form at least two distinct HAT complexes. There are two different Ada2 homologues in Drosophila named dAda2A and dAda2B. dAda2B functions within the Drosophila version of the SAGA complex (dSAGA). To gain insight into dAda2A function, we sought to identify novel components of the complex containing this protein, ATAC (Ada two A containing) complex. Affinity purification and mass spectrometry revealed that, in addition to dAda3 and dGcn5, host cell factor (dHCF) and a novel SANT domain protein, named Atac1 (ATAC component 1), copurify with this complex. Coimmunoprecipitation experiments confirmed that these proteins associate with dGcn5 and dAda2A, but not with dSAGA-specific components such as dAda2B and dSpt3. Biochemical fractionation revealed that ATAC has an apparent molecular mass of 700 kDa and contains dAda2A, dGcn5, dAda3, dHCF, and Atac1 as stable subunits. Thus, ATAC represents a novel histone acetyltransferase complex that is distinct from previously purified Gcn5/Pcaf-containing complexes from yeast and mammalian cells.     

The ADA complex is a distinct histone acetyltransferase complex in Saccharomyces cerevisiae.

We have identified two Gcn5-dependent histone acetyltransferase (HAT) complexes from Saccharomyces cerevisiae, the 0.8-MDa ADA complex and the 1.8-MDa SAGA complex. The SAGA (Spt-Ada-Gcn5-acetyltransferase) complex contains several subunits which also function as part of other protein complexes, including a subset of TATA box binding protein-associated factors (TAFIIs) and Tra1. These observations raise the question of whether the 0.8-MDa ADA complex is a subcomplex of SAGA or whether it is a distinct HAT complex that also shares subunits with SAGA. To address this issue, we sought to determine if the ADA complex contained subunits that are not present in the SAGA complex. In this study, we report the purification of the ADA complex over 10 chromatographic steps. By a combination of mass spectrometry analysis and immunoblotting, we demonstrate that the adapter proteins Ada2, Ada3, and Gcn5 are indeed integral components of ADA. Furthermore, we identify the product of the S. cerevisiae gene YOR023C as a novel subunit of the ADA complex and name it Ahc1 for ADA HAT complex component 1. Biochemical functions of YOR023C have not been reported. However, AHC1 in high copy numbers suppresses the cold sensitivity caused by particular mutations in HTA1 (I. Pinto and F. Winston, personal communication), which encodes histone H2A (J. N. Hirschhorn et al., Mol. Cell. Biol. 15:1999-2009, 1995). Deletion of AHC1 disrupted the integrity of the ADA complex but did not affect SAGA or give rise to classic Ada(-) phenotypes. These results indicate that Gcn5, Ada2, and Ada3 function as part of a unique HAT complex (ADA) and represent shared subunits between this complex and SAGA.