Heterozygous Disruption of the TATA-Binding Protein Gene in DT40 Cells Causes Reduced cdc25B Phosphatase Expression and Delayed Mitosis

Abstract

TATA-binding protein (TBP) is a key general transcription factor required for transcription by all three nuclear RNA polymerases. Although it has been intensively analyzed in vitro and in Saccharomyces cerevisiae, in vivo studies of vertebrate TBP have been limited. We applied gene-targeting techniques using chicken DT40 cells to generate heterozygous cells with one copy of the TBP gene disrupted. Such TBP-heterozygous (TBPHet) cells showed unexpected phenotypic abnormalities, resembling those of cells with delayed mitosis: a significantly lower growth rate, larger size, more G 2 /-M-than G 1 -phase cells, and a high proportion of sub-G 1 , presumably apoptotic, cells. Further evidence for delayed mitosis in TBP-Het cells was provided by the differential effects of several cell cycle-arresting drugs. To determine the cause of these defects, we first examined the status of cdc2 kinase, which regulates the G 2 /M transition, and unexpectedly observed more hyperphosphorylated, inactive cdc2 in TBP-Het cells. Providing an explanation for this, mRNA and protein levels of cdc25B, the trigger cdc2 phosphatase, were significantly and specifically reduced. These properties were all due to decreased TBP levels, as they could be rescued by expression of exogeneous TBP, including, in most but not all cases, a mutant form lacking the species-specific N-terminal domain. Our results indicate that small changes in TBP concentration can have profound effects on cell growth in vertebrate cells.TATA-binding protein (TBP) is a key general transcription factor involved in transcription by RNA polymerases (RNAPs) I, II, and III (23). In RNAP II-mediated transcription from TATA box-containing promoters, the direct interaction between the TATA box and TBP, as a component of TFIID, is the first and potentially rate-limiting step of transcription (11). Thus, this step has been shown to be a target for transcriptional activation and repression (21,40,68). Consistent with this, recruitment of TBP to a promoter by a heterologous DNA binding domain can bypass the need of transcriptional activators (8,33,49,80). Moreover, it was reported that the degree of TBP occupancy of promoters in yeast (Saccharomyces cerevisiae) is correlated with transcriptional activity (39, 43). Recently, Kuras et al. (38) and Li et al. (44) showed that TBP occupancy is constant at transcriptionally active promoters but that TBP-associated factor II (TAF II ) occupancies are variable, suggesting that binding of TBP to DNA is critical for transcription but that the TAF II requirement might be promoter dependent. Once TFIID interacts with the TATA box, either RNAP II holoenzyme is recruited or general transcription factors along with RNAP II are recruited stepwise to promoters and the preinitiation complex is formed (21, 57). For the class of promoters lacking TATA boxes, TBP is not sufficient for transcriptional initiation in vitro and TFIID is required (61, 75). The exact mechanism by which TBP functions on these promoters is not clear yet.TBP is divided into two d...

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In Vivo Functional Analysis of the Histone 3-like TAF9 and a TAF9-related Factor, TAF9L

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Increased Expression of TATA-Binding Protein, the Central Transcription Factor, Can Contribute to Oncogenesis

“…Decreases in the amount of TBP have also been shown to produce specific changes in gene expression. Expression of a cell cycle regulatory protein, cdc25B phosphatase, was found to be reduced in a chicken B-cell line when one copy of the TBP gene was disrupted (17). Furthermore, heterozygous disruption of the TBP gene in these cells caused abnormalities in cell growth and size and delayed mitosis (17).…”