The ts13 Mutation in the TAF_II250 Subunit (CCG1) of TFIID Directly Affects Transcription of D-Type Cyclin Genes in Cells Arrested in G₁ at the Nonpermissive Temperature

Abstract

The general transcription initiation factor TFIID contains the TATA-binding protein (TBP) and TBP-associated factors (TAFs) implicated in the function of gene-specific activators. Previous studies have indicated that a hamster cell line (ts13) with a point mutation in the TAF_II250/CCG1 (TAF_II250) gene shows temperature-sensitive expression of a subset of genes and arrests in late G₁ at 39.5°C. Here, we report the identification of cell cycle-specific (G₁-specific) genes that appear to be regulated directly through TAF_II250 both in vivo and in vitro. Transcription rates of several cell cycle-regulatory genes were determined by run-on assays in nuclei from ts13 cells grown at permissive (33°C) and nonpermissive (39.5°C) temperatures. Temperature-dependent differences in transcription rates were observed for cyclin A, D1, and D3 genes. In transient-transfection assays, the human cyclin D1 promoter fused to a luciferase reporter showed a temperature-dependent reduction in activity in ts13 cells but not in parental BHK cells. In in vitro assays, upstream sequence-dependent transcription from the human cyclin D1 promoter was significantly reduced in ts13 nuclear extracts preincubated at 30°C but not in similarly treated BHK nuclear extracts, and transcription in the ts13 extract was restored by addition of an affinity-purified human TFIID. Preincubation of the ts13 nuclear extracts did not affect the function of several GAL4-activation domain fusion proteins (GAL4-VP16, GAL4-p65, and GAL4-p53) on either the adenovirus major late or cyclin D1 core promoter bearing GAL4 sites, further indicating that the effect of the TAF_II250 mutation is both core promoter and activator specific.