pubs.acs.org/Biochemistry Published on Web 09/17/2010 r 2010 American Chemical Society 9140 Biochemistry 2010, 49, 9140–9151 DOI: 10.1021/bi1004427 Acetylation of GAGA Factor Modulates Its Interaction with DNA † Xavier Aran-Guiu, ‡ Miguel Ortiz-Lombardı´a, § Eliandre Oliveira, ) Carles Bonet Costa, ‡ Maria Antonia Odena, ) David Bellido, ^ and Jordi Bernu es* ,‡ ‡ Institut de Biologia Molecular de Barcelona-CSIC and Institute for Research in Biomedicine Barcelona, Parc Cientı´fic de Barcelona, Baldiri Reixac 10-12, 08028 Barcelona, Spain, § Architecture et Fonction des Macromol ecules Biologiques (UMR6098) CNRS, Universit es d’Aix-Marseille I & II, Marseille, France, ) Plataforma de Prote  omica, Parc Cientı´fic de Barcelona, Barcelona, Spain, and ^ Plataforma de Prote  omica, Parc Cientı´fic de Barcelona, Serveis Cientificot ecnics, Universitat de Barcelona, Barcelona, Spain Received March 24, 2010; Revised Manuscript Received September 16, 2010 ABSTRACT: GAGA is a Drosophila transcription factor that shows a high degree of post-translational modification. Here, we show that GAGA factor is acetylated in vivo. Lysine residues K325 and K373 on basic regions BR1 and BR3 of the DNA binding domain, respectively, are shown to be acetylated by PCAF. While BR1 is strictly required to stabilize DNA binding, BR3 is dispensable. However, acetylation of both lysine residues, either alone or in combination, weakens the binding to DNA. Despite the high degree of conserva- tion of K325 and K373 in flies, their mutation to glutamine does not affect DNA binding. Molecular dynamics simulations, using acetylated K325 and a K325Q mutant of GAGA DNA binding domain in complex with DNA, are fully consistent with these results and provide a thermodynamic explanation for this observation. We propose that while K325 and K373 are not essential for DNA binding they have been largely conserved for regulatory purposes, thus highlighting a key regulatory system for GAGA factor in flies. The regulation of transcription is a crucial process in gene expression. For this purpose, cells make use of diverse mecha- nisms. Among these, post-translational modification (PTM) 1 has been intensively studied in the chromatin research field. In par- ticular, since the proposal of the provocative histone code hypothesis (1), the PTM of histones has received much attention. Consequently, extensive knowledge of histone modifications has boosted interest in epigenetics. However, while some PTMs have been described for transcription factors, much less is known about their mapping and functions. A notable exception is p53, for which many PTMs have been shown to serve different functional roles (2, 3). Transcription factors are phosphorylated, acetylated, glyco- sylated, sumoylated, ubiquitinated, methylated, etc., in vivo, and in some cases, these modifications have been correlated with distinct functional states. While phosphorylation is the most studied PTM to date, acetylation and methylation are currently receiving much attention. In this respect, GAGA factor is remarkably modified in vivo (4). GAGA is a Drosophila tran- scription factor encoded in the single-copy gene Trithorax-like (5). GAGA factor is genetically classified in the Trithorax group of genes, which, in contrast to the action of the Polycomb group, maintain large regions of chromatin open to expression. GAGA factor is remarkably complex, acting as a transcrip- tional activator and repressor, and also in the insulator function, chromatin remodeling, and position effect variegation, among others (6-8). In addition to possibly being O-glycosylated (9), GAGA factor can be phosphorylated in the proximity of the zinc finger of its DNA binding domain. This phosphorylation weak- ens its specific interaction with DNA (4). These experiments also demonstrated the presence of additional modifications in GAGA factor to explain the complex isoform protein pattern observed in two-dimensional (2D) gel electrophoresis. Here, we show that GAGA factor is also acetylated in vivo, and we analyze the functional consequences of this modification. MATERIALS AND METHODS Recombinant Proteins, Mutants, and Peptides. GAGA 519 and GAGADBD (residues 310-392) (10) were expressed as six- His-tagged proteins using pET14b (Novagen). 6xHis-SirT2, in the pET30c expression plasmid, was kindly provided by A. Vaquero (11). His-tagged fusion proteins were expressed in Escherichia coli BL21-DE3, purified using a HisTrap column (GE), and dialyzed against 100 mM KCl, 20% glycerol, 50 mM Hepes (pH 7.8), 0.5 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF. The HAT domain of human PCAF (residues 352-658) (in pGEX-2TK) (12) was expressed in E. coli BL21-DE3, purified on glutathione-Sepharose beads, and resuspended in acetylation buffer. Flag-tagged proteins HDAC1 (in pcDNA3) and HDAC2 (in pME18S) were expressed in transiently transfected HeLa cells as described previously (13) and then extracted and purified by immunoprecipitation using the M2 anti-Flag antibody (Sigma). † X.A.-G. was supported in part by predoctoral fellowships FPI (from the CIRIT of the Generalitat de Catalunya) and I3P (from CSIC). The proteomics work was performed at the Proteomics Platform of Barcelona Science Park, University of Barcelona, a member of the ProteoRed network. This work was financed by Ministerio de Ciencia e Innovaci on of the Spanish Government Grants BFU2006-09761 and BFU2007- 64395/BMC to J.B., and it was performed in the frame of the “Centre de Refer encia en Biotecnologia” of the Generalitat de Catalunya. *To whom correspondence should be addressed: Institut de Biologia Molecular de Barcelona-CSIC and Institute for Research in Biomedicine Barcelona, Parc Cientı´fic de Barcelona, Baldiri Reixac 10-12, 08028 barcelona, Spain. E-mail: jordi.bernues@ibmb.csic.es. Phone: þ34 934034960. Fax: þ34 934034979. 1 Abbreviations: GAGADBD, GAGA factor DNA binding domain; BR, basic regions; PTM, post-translational modification; MD, molec- ular dynamics.