Human Histone Acetyltransferase GCN5 Exists in a Stable Macromolecular Complex Lacking the Adapter ADA2 ² E. Camilla Forsberg, ‡ Lloyd T. Lam, ‡ Xiang-Jaio Yang, § Yoshihiro Nakatani, § and Emery H. Bresnick* ,‡ Department of Pharmacology, UniVersity of Wisconsin Medical School, 387 Medical Science Building, 1300 UniVersity AVenue, Madison, Wisconsin 53706, and National Institutes of Child Health and Human DeVelopment, National Institutes of Health, Bethesda, Maryland 20892-2753 ReceiVed July 9, 1997; ReVised Manuscript ReceiVed September 29, 1997 X ABSTRACT: Acetylation of core histones is an important regulatory step in transcriptional activation from chromatin templates. The yeast transcriptional coactivator protein GCN5 was recently shown to be a nuclear histone acetyltransferase (HAT). Genetic and biochemical studies in yeast suggest that GCN5 functions with the adapter proteins ADA1, ADA2, ADA3, and ADA5 in a heteromeric complex. We have established conditions for chromatographic fractionation of HATs and ADA2 from human K562 erythroleukemia cells. Gel-filtration chromatography revealed two populations of GCN5 with Stokes’ radii of 67 and 33 Å, consistent with a large macromolecular complex and a monomer, respectively. The GCN5-related HAT, PCAF, was resolved as a stable complex with a Stokes’ radius of 74 Å. The HAT complexes were resistant to 0.3 M NaCl and DNase I. ADA2 was characterized by a Stokes’ radius of 35 Å, consistent with a monomer. Thus, in contrast to the stable GCN5-adapter complex in yeast, human GCN5 and ADA2 are not stably associated with each other. The implications of this result are discussed Vis-a-Vis the mechanism of recruitment of GCN5 to regulatory regions of genes. The organization of DNA into chromatin plays an impor- tant role in transcriptional regulation. The wrapping of the DNA duplex around an octamer of histones to form the nucleosome can selectively occlude the binding of certain transcription factors to cognate DNA recognition sites (1). In contrast to the repressive effects of chromatin, nucleo- somes can also facilitate transcription by bringing together distant DNA sequences, allowing for protein-protein inter- actions between transcription factors (2, 3). On the basis of the impact of chromatin structure on protein-DNA interac- tions, it is not surprising that chromatin-modifying enzymes play a critical role in gene regulation. The binding of transcription factors to sites occluded by nucleosomes can be facilitated by the multiprotein complex SWI/SNF (4). The SWI/SNF complex consists of ap- proximately 12 proteins and appears to have a molecular mass of at least 2 megadaltons (5-7). Although the exact mechanism of SWI/SNF-induced chromatin remodeling is unknown, it has been proposed that SWI/SNF may enhance histone octamer mobility, thus exposing factor binding sites (8). In addition to SWI/SNF, nuclear type-A histone acetyl- transferases regulate chromatin structure and transcription. Acetylation of conserved lysine residues on the amino- terminal tails of core histones has long been correlated with gene activation (9). Genetic studies have provided strong evidence that the lysine acetylation sites are important in transcriptional activation (10). By neutralizing the positive charge of the lysine residue, acetylation may disrupt elec- trostatic interactions between the histone tail and the phos- phodiester backbone of DNA. Thus, a functional conse- quence of acetylation can be increased access of transcription factors to nucleosomal recognition sites (11, 12). Four type-A HATs 1 have recently been identified. Brownell et al. (13) discovered a Tetrahymena HAT with strong sequence homology to the yeast transcriptional coactivator GCN5. Yeast GCN5 physically interacts with the DNA binding protein GCN4 and is necessary for transcriptional enhancement by GCN4 (14). The mammalian coactivator, CBP, which is necessary for transcriptional activation by the cyclic AMP response element binding protein (CREB) (15), nuclear receptors (16), and certain other transactivators (17), was recently shown to be a HAT (18). CBP physically associates with a GCN5-related human protein, PCAF, which is also a HAT (19). Moreover, a component of the basal transcription machinery, TAF II 250, has intrinsic HAT activity (20). These studies have led to a model in which recruitment of HATs to genes is accomplished through protein-protein interactions with DNA-bound activator proteins (13, 18, 21). This model assumes that the HATs are recruited as part of large multimeric protein complexes. Genetic and biochemical studies have provided evidence that yeast GCN5 functions with the “adapter” proteins ADA1, ADA2, ADA3, and ADA5 (22-27). GCN5 physi- ² These studies were supported by a faculty development award from the Pharmaceutical Manufacturers of America Foundation, a Shaw Scholar Award from the Milwaukee Foundation, a Leukemia Society of America Scholar Award, and National Institutes of Health Grant DK50107. * Corresponding author: Tel 608-265-6446; FAX 608-262-1257; E-mail ehbresni@facstaff.wisc.edu. ‡ University of Wisconsin Medical School. § NIH. X Abstract published in AdVance ACS Abstracts, November 15, 1997. 1 Abbreviations: BSA, bovine serum albumin; CBP, CREB binding protein; CREB, cyclic AMP response element binding protein; DTT, dithiothreitol; f-GCN5, Flag epitope-tagged GCN5; f-PCAF, Flag epitope-tagged PCAF; FPLC, fast pressure liquid chromatography; HAT, histone acetyltransferase; LCR, locus control region; PCAF, p300/ CBP-associated factor; SDS-PAGE, sodium dodecyl sulfate-poly- acrylamide gel electrophoresis; Rs, Stokes’ radius. 15918 Biochemistry 1997, 36, 15918-15924 S0006-2960(97)01664-4 CCC: $14.00 © 1997 American Chemical Society