The Nuclear Import of TAF10 Is Regulated by One of Its Three Histone Fold Domain-Containing Interaction Partners

Abstract

TFIID, comprising the TATA box binding protein (TBP) and 13 TBP-associated factors (TAFs), plays a role in nucleation in the assembly of the RNA polymerase II preinitiation complexes on protein-encoding genes. TAFs are shared among other transcription regulatory complexes (e.g., SAGA, TBP-free TAF-containing complex [TFTC], STAGA, and PCAF/GCN5). Human TAF10, a subunit of both TFIID and TFTC, has three histone fold-containing interaction partners: TAF3, TAF8, and SPT7Like (SPT7L). In human cells, exogenously expressed TAF10 remains rather cytoplasmic and leptomycin B does not affect this localization. By using fluorescent fusion proteins, we show that TAF10 does not have an intrinsic nuclear localization signal (NLS) and needs one of its three interaction partners to be transported into the nucleus. When the NLS sequences of either TAF8 or SPT7L are mutated, TAF10 remains cytoplasmic, but a heterologous NLS can drive TAF10 into the nucleus. Experiments using fluorescence recovery after photobleaching show that TAF10 does not associate with any cytoplasmic partner but that once transported into the nucleus it binds to nuclear structures. TAF10 binding to importin β in vitro is dependent on the coexpression of either TAF8 or TAF3, but not SPT7L. The cytoplasmic-nuclear transport of TAF10 is naturally observed during the differentiation of adult male germ cells. Thus, here we describe a novel role of the three mammalian interacting partners in the nuclear localization of TAF10, and our data suggest that a complex network of regulated cytoplasmic associations may exist among these factors and that this network is important for the composition of different TFIID and TFTC-type complexes in the nucleus.

ACKNOWLEDGMENTS

We are grateful to T. Misteli for helpful discussions, advice, and support; to the NCI and the IGBMC imaging facilities; to A. Jànoshàzi and T. Karpova for imaging training; to M. Oulad-Abdelghani for generating antibodies; to I. Davidson and G. Gangloff for TAF3 expression plasmids; to D. Gorlich, A. Kretsovali, and J. Papamatheakis for the importin plasmids; to S. Gorski and E. Meshorer for advice in FRAP experiments; and to M. Frontini, A. Magklara, E. Martinez, and S. John for critically reading the manuscript. We also thank the IGBMC cell culture facility for providing cells and I. Kolb-Cheynel for help with the recombinant baculoviruses.

E.S. was supported by an EMBO long-term fellowship, M.A.D. by a fellowship from the European Community (grant HPRN-CT-2000-00088), and G.F. by a fellowship of the Fondation de la Recherche Médicale. This work was supported by funds from INSERM, CNRS, Hôpital Universitaire de Strasbourg, Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, and the Front Nationale de la Science ACI and by European Community RTN (HPRN-CT-2000-00087, HPRN-CT-2000-00088, and HPRN-CT 00504228), STREP (LSHG-CT-2004-502950), and AICR (03-084) grants.