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 (TBP-Het) cells showed unexpected phenotypic abnormalities, resembling those of cells with delayed mitosis: a significantly lower growth rate, larger size, more G₂/-M- than G₁-phase cells, and a high proportion of sub-G₁, 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 G₂/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.

ACKNOWLEDGMENTS

We thank W. Moon for valuable advice and help; Z. Chen, J. Wang, T. Kashima, and Y. Takagaki for providing plasmids, cells, and advice; L. Yamasaki, F. McKeon, X. Jacq, E. Abali, and W. Zhao for helpful discussion and advice; L. Tora for providing anti-TBP MAb 3G3; K. Okamoto for providing anti-cyclin D1 and anti-cdk2 antibodies; and M. K. Kim for technical assistance.

This work was supported by National Institutes of Health grant R01 GM 37971.