The ts13 mutation in the TAF(II)250 subunit (CCG1) of TFIID directly affects transcription of D-type cyclin genes in cells arrested in G1 at the nonpermissive temperature.
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(II)250/CCG1 (TAF(II)250) gene shows temperature-sensitive expression of a subset of genes and arrests in late G1 at 39.5 degrees C. Here, we report the identification of cell cycle-specific (G1-specific) genes that appear to be regulated directly through TAF(II)250 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 degrees C) and nonpermissive (39.5 degrees 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 degrees 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(II)250 mutation is both core promoter and activator specific.
Suzuki-Yagawa
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Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021, USA.
Guermah
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Roeder
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D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D056741:Cyclin D; D019938:Cyclin D1; D056743:Cyclin D3; D016213:Cyclins / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D016193:G1 Phase; D051548:Histone Acetyltransferases; D006801:Humans; D008647:Mesocricetus; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000235:genetics*; D015513:Oncogene Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D016385:TATA Box / Q000235:genetics*; D035381:TATA-Binding Protein Associated Factors; D013696:Temperature; D015534:Trans-Activators / Q000235:genetics*; D035362:Transcription Factor TFIID; D019473:Transcription Factors, TFII / Q000235:genetics*; D014158:Transcription, Genetic
C534696:CCND3 protein, human; D056741:Cyclin D; D056743:Cyclin D3; D016213:Cyclins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D035381:TATA-Binding Protein Associated Factors; D015534:Trans-Activators; D035362:Transcription Factor TFIID; D019473:Transcription Factors, TFII; D019938:Cyclin D1; D051548:Histone Acetyltransferases; C069001:TATA-binding protein associated factor 250 kDa