University of Washington     Department of Pharmacology

WANG LABORATORY

 

TAF1 Regulation of Cell Cycle Gene Transcription

The proper transcription of genes that control the cell division cycle is crucial for “normal” cell proliferation. Transcription is governed by the core machinery, which consists of the general transcription factors (TFIIA, B, D, E, F, and H) and the enzyme RNA polymerase II. The largest subunit of TFIID, TBP associated factor 1 (TAF1), possesses two serine/threonine kinase domains and intrinsic histone acetyltransferase (HAT) activity. Both TAF1 HAT and kinase activities are essential for transcription from a subset of genes and progression through G1 in mammalian cells.  TAF7, another TFIID subunit, directly binds TAF1 and inhibits TAF1 HAT activity.

Using synchronized HeLa cells, we have identified maximal TAF1 kinase activity and maximal TAF1 promoter binding during the G1 phase of the cell cycle.  In vitro kinase assays revealed that TAF1 phosphorylates TAF7 within the TFIID complex.  Pre-phosphorylation of purified TFIID increased TAF1 HAT activity, supporting a role for TAF1 kinase activity in activation of HAT activity.  In vivo and in vitro methods identified TAF7 serine-264 as a putative phosphosite for the kinase domains of TAF1.  Kinase assays and immunoprecipitation experiments revealed phosphorylated wild-type TAF7 and the phosphomimetic S264D mutant bound TAF1 less efficiently than a TAF7 S264A mutant.  These data suggest that TAF1 phosphorylation at TAF7 S-264 disrupts TAF1/7 binding, leading to activation of TAF1 HAT activity at target promoters during G1 and cell cycle progression.  In support of this model, we have observed a strong correlation between increased TAF1 recruitment and histone H3 acetylation, and decreased TAF7 promoter binding, at the cyclin D1 promoter.  The long-term objective of these studies is to elucidate the signaling pathways regulating the enzymatic activities of TAF1 and to determine if they represent potential therapeutic targets for cancer.