The Histone Chaperone TAF-I/SET/INHAT Is Required for Transcription In Vitro of Chromatin Templates

Abstract

To uncover factors required for transcription by RNA polymerase II on chromatin, we fractionated a mammalian cell nuclear extract. We identified the histone chaperone TAF-I (also known as INHAT [inhibitor of histone acetyltransferase]), which was previously proposed to repress transcription, as a potent activator of chromatin transcription responsive to the vitamin D₃ receptor or to Gal4-VP16. TAF-I associates with chromatin in vitro and can substitute for the related protein NAP-1 in assembling chromatin onto cloned DNA templates in cooperation with the remodeling enzyme ATP-dependent chromatin assembly factor (ACF). The chromatin assembly and transcriptional activation functions are distinct, however, and can be dissociated temporally. Efficient transcription of chromatin assembled with TAF-I still requires the presence of TAF-I during the polymerization reaction. Conversely, TAF-I cannot stimulate transcript elongation when added after the other factors necessary for assembly of a preinitiation complex on naked DNA. Thus, TAF-I is required to facilitate transcription at a step after chromatin assembly but before transcript elongation.

ACKNOWLEDGMENTS

We thank Stewart Shuman and Stéphane Larochelle for critical review of the manuscript. We are grateful to James Kadonaga, who provided the NAP-1, ISWI, and Acf1 baculoviruses; Kyosuke Nagata, who provided the anti-TAF-I antibodies; Jeffrey Parvin, who provided the TFIIA expression vector; and W. Lee Kraus, who provided the Gal4-VP16 expression vector and the pGEIO plasmid. HeLa cells were cultured by the National Cell Culture Center. We thank Brian Lemon for helpful discussion and Chao-Pei Chang for experimental support.

This work was supported by NIH grant DK45460 to L.P.F. and R.P.F.