Two Different Drosophila ADA2 Homologues Are Present in Distinct GCN5 Histone Acetyltransferase-Containing Complexes

Abstract

We have isolated a novel Drosophila (d) gene coding for two distinct proteins via alternative splicing: a homologue of the yeast adaptor protein ADA2, dADA2a, and a subunit of RNA polymerase II (Pol II), dRPB4. Moreover, we have identified another gene in the Drosophila genome encoding a second ADA2 homologue (dADA2b). The two dADA2 homologues, as well as many putative ADA2 homologues from different species, all contain, in addition to the ZZ and SANT domains, several evolutionarily conserved domains. The dada2a/rpb4 and dada2b genes are differentially expressed at various stages of Drosophila development. Both dADA2a and dADA2b interacted with the GCN5 histone acetyltransferase (HAT) in a yeast two-hybrid assay, and dADA2b, but not dADA2a, also interacted with Drosophila ADA3. Both dADA2s further potentiate transcriptional activation in insect and mammalian cells. Antibodies raised either against dADA2a or dADA2b both immunoprecipitated GCN5 as well as several Drosophila TATA binding protein-associated factors (TAFs). Moreover, following glycerol gradient sedimentation or chromatographic purification combined with gel filtration of Drosophila nuclear extracts, dADA2a and dGCN5 were detected in fractions with an apparent molecular mass of about 0.8 MDa whereas dADA2b was found in fractions corresponding to masses of at least 2 MDa, together with GCN5 and several Drosophila TAFs. Furthermore, in vivo the two dADA2 proteins showed different localizations on polytene X chromosomes. These results, taken together, suggest that the two Drosophila ADA2 homologues are present in distinct GCN5-containing HAT complexes.

ACKNOWLEDGMENTS

We are grateful to D. Allis, E. Smith, S. Berger, Y. Nakatani, A. Imhof, P. Becker, E. vom Baur, C. Gaudon, and R. Losson for reagents and advice; to O. Poch for help in the sequence alignments; and to Katalin ükrüsné for technical help. We also thank P. Eberling for peptide synthesis and G. Duval for polyclonal antibody production.

S.M. was supported by an EMBO short-term fellowship (ASTF 9604). This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the CNRS, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer, the FRM, European Community RTN grants (RTN1-1999-00401 and RTN-1999-00171), and the Human Frontier Science Program (RG 196/98) (to L.T.) and by Hungarian Ministry of Education OTKA grants (T29939 and T29207 to I.B. and to A.U.) and FKFP grant (0060/2000 to I.B.).