Activation of a DNA damage checkpoint response in a TAF1-defective cell line.

The ts13 and tsBN462 cell lines have been used as model systems to study the relationship between transcription and cell cycle control. These lines were originally isolated in screens to identify cells that underwent a cell cycle arrest upon a shift in temperature from 33 to 39°C (28, 45). It was subsequently discovered that both lines contain the same point substitution mutation (G690D) in TBP-associated factor 1 (TAF1) (TAFII250/CCG1), a key member of the TFIID complex (32, 35, 36). TAF1 is the largest of several TAFs, forming a scaffold between TBP and other TAFs and contributing to activated transcription (9). The ability of TAF1 to bind to TBP and other TAFs appears to be unaffected in ts13 cells shifted to the restrictive temperature (14, 37). However, TAF1 may lose the ability to bind to TBP at the cyclin D1 promoter in cells shifted to the restrictive temperature (18). TAF1 is associated with at least three enzymatic activities. The N- and C-terminal ends of TAF1 contain a kinase activity that phosphorylates RAP74, a subunit of TFIIF (12). This kinase activity appears to be unaffected by the ts13 mutation (30). The central domain of TAF1 contains a histone acetyltransferase (HAT) activity that can acetylate TFIIEβ and histones H3 and H4 (21, 26). The ts13 and tsBN462 point mutation in TAF1 is located within the HAT domain, and a mutation of the corresponding residue in human TAF1 resulted in temperature-sensitive elimination of the HAT activity in vitro (14). TAF1 also contains an E1 and E2 ubiquitin activating and conjugating activity that participates in the monoubiquitination of histone H1 (23). It is not known whether this activity of TAF1 is affected by the ts13 and tsBN462 mutation. Since TAF1 contributes to activated transcription, loss-of-function mutations could be expected to have significant effects on the transcription profile. Surprisingly, the ts13 and tsBN462 point mutation affected the transcription of a limited set of genes, including those that encode the cyclin-dependent kinase Cdk1/Cdc2 and cyclins D1, D3, and A (44, 49, 50). The transcription of several other genes, including c-fos and c-myc, was not reduced at the restrictive temperature (49). Since several cell cycle-related genes were found to be downregulated upon a shift to the restrictive temperature, it had been proposed that the decreased expression of cell cycle-related genes accounted for the cell cycle arrest, linking a defect in a general transcription factor to cell cycle regulation. In contrast to the decreased transcription of cell cycle-related genes, several reports have suggested that the transcription of p53-dependent genes increased in ts13 cells after a shift to the restrictive temperature. For example, expression of the cyclin kinase inhibitor p21Cip1 increased in ts13 cells shifted to the restrictive temperature (33, 37). A microarray analysis of gene expression revealed that although 18% of the more than 4,000 genes examined had decreased expression, the mRNA levels of several p53-dependent genes, including those that encode Gadd45 and Pai-1, increased dramatically at the restrictive temperature (29). Finally, it has been reported that temperature restriction of tsBN462 cells led to increased expression of p53 itself (53). These reports suggested the possibility that the cell cycle arrest of ts13 cells might be mediated by a p53-dependent pathway. p53 can be activated in response to a variety of cellular stresses, including hypoxia, heat shock, and DNA damage (17, 43). DNA damage, in particular, leads to the stabilization and activation of p53 by phosphorylation and acetylation of specific residues (8, 40). For example, two mediators of the DNA damage response, the ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3 related) kinases, phosphorylate p53 on the serine 15 residue, inhibiting the ability of p53 to bind to and become degraded by Mdm2 (40, 46). ATR and ATM are members of a family of large phosphatidylinositol 3-kinase-related protein kinases. Both ATR and ATM are activated in the presence of abnormal DNA structures, including stalled replication forks and double-strand breaks. In response to activation, these kinases phosphorylate a number of checkpoint and DNA repair proteins, including p53, the kinases Chk1 and Chk2, Rad17, Rad9, and BRCA1 (1, 6). Phosphorylation of these substrates contributes to cell cycle arrest and repair of DNA damage. Here we provide evidence that the cell cycle arrest of ts13 cells is dependent on the rapid activation of p53 in response to a DNA damage checkpoint. Furthermore, ATR is specifically activated in response to the TAF1 defect and contributes to the phosphorylation of p53 and the G1 phase arrest. These results indicate that a defect in TAF1 can trigger the activation of a DNA damage pathway.