Heterozygous disruption of the TATA-binding protein gene in DT40 cells causes reduced cdc25B phosphatase expression and delayed mitosis

TATA-binding protein (TBP) is a key general transcription factor required f or transcription by all three nuclear RNA polymerases. Although it has been intensively analyzed in vitro and in Saccharomyces cerevisiae, in vivo stu dies of vertebrate TBP have been limited. We applied gene-targeting techniq ues using chicken DT40 cells to generate heterozygous cells with one copy o f the TBP gene disrupted. Such TBP-heterozygous (TBP-Het) cells showed unex pected phenotypic abnormalities, resembling those of cells with delayed mit osis: 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, ce lls. Further evidence for delayed mitosis in TBP-Het cells was provided by the differential effects of several cell cycle-arresting drugs. To determin e the cause of these defects, we first examined the status of cdc2 kinase, which regulates the G(2)/M transition, and unexpectedly observed more hyper phosphorylated, inactive cdc2 in TBP-Het cells. Providing an explanation fo r this, mRNA and protein levels of cdc25B, the trigger cdc2 phosphatase, we re 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 speci es-specific N-terminal domain. Our results indicate that small changes in T BP concentration can have profound effects on cell growth in vertebrate cel ls.