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 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.
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
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Um
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Yamauchi
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Kato
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Manley
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics*; D002478:Cells, Cultured; D002645:Chickens; D004268:DNA-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D008938:Mitosis / Q000235:genetics*; D012441:Saccharomyces cerevisiae; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000235:genetics*; D020687:cdc25 Phosphatases / Q000235:genetics*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D020687:cdc25 Phosphatases