Interaction of Histone Acetylases and Deacetylases In Vivo

Satoshi Yamagoe

Tomohiko Kanno

Yuka Kanno

Shigakazu Sasaki

Yoshihiro Nakatani+2 authors

Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development,^{Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892,}^{Dana Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115}³

^{Corresponding author. Mailing address: Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, Bldg. 6, Rm. 2A01, National Institutes of Health, Bethesda, MD 20892-2753. Phone: (301) 496-9184. Fax: (301) 480-9354. E-mail:}vog.hin@kotazo.

^{Present address: Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo, Japan.}

^{Present address: Second Division of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan.}

Received 2002 Jun 12; Revised 2002 Aug 1; Accepted 2002 Oct 29.

Abstract

Having opposing enzymatic activities, histone acetylases (HATs) and deacetylases affect chromatin and regulate transcription. The activities of the two enzymes are thought to be balanced in the cell by an unknown mechanism that may involve their direct interaction. Using fluorescence resonance energy transfer analysis, we demonstrated that the acetylase PCAF and histone deacetylase 1 (HDAC1) are in close spatial proximity in living cells, compatible with their physical interaction. In agreement, coimmunoprecipitation assays demonstrated that endogenous HDACs are associated with PCAF and another acetylase, GCN5, in HeLa cells. We found by glycerol gradient sedimentation analysis that HATs are integrated into a large multiprotein HDAC complex that is distinct from the previously described HDAC complexes containing mSin3A, Mi-2/NRD, or CoREST. This HDAC-HAT association is partly accounted for by a direct protein-protein interaction observed in vitro. The HDAC-HAT complex may play a role in establishing a dynamic equilibrium of the two enzymes in vivo.

Abstract

Specific lysines on the core histones are acetylated by a series of histone acetylases (HATs). The status of acetylation constitutes one basis for the histone code, an important basis of chromatin-mediated regulatory processes, including transcription, replication, and chromosome dynamics (12, 23, 29). Acetylation of histone tails can be reversed by a diverse series of histone deacetylases (5, 15).

Both HATs and histone deacetylases are classified into several distinct groups. The GNAT family of HATs, one of the best-studied families, is conserved throughout eukaryotes (23, 28). In mammalian species there are two GNAT members, GCN5 and PCAF. They are structurally similar to each other and are expressed in overlapping sets of cells and tissues. They predominantly regulate acetylation of histone H3 and are generally involved in transcriptional activation (12, 25, 39). Both GCN5 and PCAF form large multiprotein complexes whose compositions are also conserved (9, 21, 23, 28). Among histone deacetylases, class I histone deacetylases (HDACs) were the first to be identified and are conserved from yeasts such as Saccharomyces cerevisiae to humans. Four HDACs are currently known in humans: HDAC1, HDAC2, HDAC3, and HDAC8 (15). These histone deacetylases are generally associated with transcriptional repression mediated by various DNA binding transcription factors (5, 15, 20, 24). HDAC1 and HDAC2 form at least three distinct complexes that contain representative factors, mSin3A, Mi-2/NRD, and CoREST/kiaa0071 (13, 32, 40, 44, 45, 46). HDAC3 also forms a complex that contains N-CoR and SMRT, among other components (10, 16, 36).

Although rapid progress has been made on understanding the structure and function of individual HATs and HDACs, a remaining question is how the activities of these two enzymes, which exert opposite functions, are mutually balanced in the cell (23). A series of genetic studies and promoter analyses suggest that the two enzymes may not act independently and that their activities in some cases may be linked to one another (22, 34, 37). Other lines of evidence indicate that some HATs and HDACs occupy the common space in the nucleus and coordinately regulate the same set of target genes. For example, transcriptional activation mediated by nuclear receptors such as retinoic acid receptor and retinoid X receptor involves ligand-dependent association and dissociation of HAT and HDAC, respectively, on a given promoter (2, 38). Coordinated activity of the two enzymes may also be inferred for cell growth-regulated genes controlled by E2F, whose promoter activity is repressed by the HDAC-associated retinoblastoma protein but is activated by subsequent HAT recruitment (5). Furthermore, YY1 and Sp1 interact with both HATs and HDACs, thereby acquiring an activator or repressor function depending on promoter context and other factors (5). A close interrelationship between the two enzymes may also be presumed, based on the earlier observation that histones are rapidly acetylated and deacetylated with a half-life of less than 10 min in some regions of a nucleus while in other regions histone acetylation is turned over more slowly (4, 6). More-recent studies demonstrate that HATs and HDACs are engaged in a rapid cycle of global histone acetylation and deacetylation that affects the whole yeast genome (1, 14, 34). The global, untargeted alteration of histone acetylation is likely to be critical for rapid reversal of targeted chromatin modification in a specific promoter associated with transcriptional activation and/or repression (14, 34).

In this work we wished to address the mechanisms that may balance the activities of the two enzymes. We surmised that among the mechanisms that help coordinate their activities, one might involve a physical interaction between the two enzymes. To search for evidence indicating the presence of HDAC-HAT interaction in vivo, we first employed a novel flow cytometry technique based on fluorescence resonance energy transfer (FRET) (26, 30). This technique allows an assessment of molecular interactions between two proteins in the living cell. Although not heretofore applied extensively to the analysis of nuclear events, this approach provides a powerful new tool to investigate the molecular behavior of transcription factors and chromatin modifiers in the nucleus. By introducing PCAF and HDAC1 labeled with distinct fluorochromes into HeLa cells, we observed clear FRET signals ascribable to their physical proximity. Using coimmunoprecipitation assays, it was shown that HDAC1, HDAC2, and HDAC3 are all associated with GCN5 and PCAF in HeLa cells. Glycerol gradient sedimentation analysis of HDAC1 complexes revealed that GCN5 is contained in a large multiprotein complex(es) distinct from the three HDAC complexes reported before. In vitro binding analysis indicated that the HATs are incorporated into the HDAC complex(es) at least in part by directly binding to HDACs. Finally, we present evidence suggesting that HDAC-HAT interactions occur in a dynamic fashion depending on the physiological state of cells. Taken together, these results point to a mechanism that internally maintains HDAC-HAT equilibrium in the cell.

Acknowledgments

We thank S. Schreiber and E. Seto for HDAC plasmids, T. Howard for viral transduction of HeLa cells, and R. Swofford and K. Holmes for flow cytometry analysis.

Acknowledgments

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