Robust mRNA transcription in chicken DT40 cells depleted of TAF(II)31 suggests both functional degeneracy and evolutionary divergence.

In eukaryotes, RNA polymerase II (Pol II)-mediated mRNA transcription requires several general transcription factors (GTFs). One important factor is TFIID, which recognizes core promoter elements to initiate preinitiation complex formation (45). TFIID is a multiprotein complex, consisting of the TATA-binding protein (TBP), which is primarily responsible for recognition of the TATA box, and approximately 10 to 12 phylogenetically conserved polypeptides called TBP-associated factors (TAFIIs) (6, 55, 59, 62). Early biochemical data suggested an important function of TAFIIs in transcriptional activation (6, 62). In reconstituted in vitro systems, TFIID, but not TBP alone, can stimulate transcription in response to activators in the presence of other GTFs and Pol II. Furthermore, interactions between different types of activators and distinct TAFIIs correlated well with the ability of the activators to enhance transcription. In light of these data, TAFIIs were considered to be essential coactivators, presumably functioning as the direct and specific targets of various activators. More recently, however, TAFII-independent activated transcription was found, under appropriate conditions, to occur in both yeast and mammalian reconstituted systems (12, 27, 43). Moreover, additional coactivators, including PC2 and PC4, were found to be required for TFIID-dependent transcriptional activation in cell-free systems (7), and in a highly purified system, PC4 can suffice for activation in the absence of TAFIIs (68). While the coactivator function of TAFIIs has thus not been fully established in vitro, another line of biochemical analysis provided strong evidence for core promoter functions of TAFIIs. Drosophila TAFII150 was first shown to be involved in recognition of the initiator element present in some promoters (62). A novel core promoter element, termed downstream promoter element (DPE), was identified in some TATA-less promoters, and this element might be recognized by dTAFII60 and dTAFII40 (5). These additional TAFII-promoter interactions might compensate for the absence of a TATA box to allow efficient initiation at a variety of promoters. The function of TAFIIs has also been extensively studied in vivo, mostly in yeast. While almost all TAFIIs studied are essential for cell viability, genetic depletion or inactivation of several TAFIIs appeared not to cause a significant decrease in general mRNA transcription (39, 63). Transcription of a subset of genes was, however, affected. Notably, depletion of yTAFII145 effected a reduction in transcription of several cell cycle genes, in agreement with the phenotypes of cell cycle arrest at the G1 phase observed in both yTAFII145 and homologous TAFII250 mutant cells (52, 57, 64, 66). In both yeast and mammals, the dependence on yTAFII145/TAFII250 was mapped, at least in part, to the core promoter region, suggesting a functional conservation of this TAFII. In Drosophila, a sensitized genetic assay was utilized to provide evidence suggesting roles for dTAFII110 and dTAFII60 in Dorsal-mediated activation of snail gene transcription, perhaps through direct interactions between TAFIIs and the Dorsal activator protein (70). Most recently, murine cells deprived of TAFII30 were shown to arrest at the G1/G0 phase, which correlated well with the impaired expression of cyclin E in these (37). The above in vivo data support a role for TAFIIs in transcription of specific genes, but they do not necessarily suggest a general requirement of TAFIIs for Pol II transcription. The possibility of a general role of TAFIIs in Pol II transcription in yeast was further explored in a recent set of studies focusing on a group of TAFIIs known as the histone-like TAFIIs (1, 38, 40, 42). Several lines of evidence strongly suggest the existence of canonical histone-fold motifs in three TAFIIs, yTAFII17/dTAFII40/42/hTAFII32/31, yTAFII60/dTAFII62/hTAFII80, and yTAFII61/68/dTAFII30α/hTAFII20, which have highest sequence similarity with histones H3, H4, and H2B, respectively (6). Inactivation of each of the histone-like TAFIIs was shown to cause an apparent decrease in general Pol II transcription as measured by reduced accumulation of total and specific mRNAs. Through similar assays, yTAFII40, a TFIID-specific TAFII, was also demonstrated to be essential for bulk Pol II transcription (28). Additionally, a genome-wide expression study showed that while a yTAFII145 mutation reduced expression of only 16% of genes tested, 67% required yTAFII17 for optimal expression (22). It is noteworthy that two earlier studies had suggested a limited role of yTAFII60, yTAFII68, and yTAFII40 in Pol II transcription (39, 63). Thus, while the essential role of yTAFII17 in transcription seems firmly established, a consensus regarding these other three yTAFIIs awaits additional studies. The histone-like TAFIIs are also components of a histone acetyl transferase (HAT)-containing complex, known as SAGA in yeast (14) and PCAF in humans (44). The discovery of TAFIIs in complexes other than TFIID (2) has added another level of complexity to the in vivo functions of TAFIIs: phenotypes observed after TAFII depletion could be due to TAFII function in the context of TFIID, SAGA, or both. Supporting an important role for SAGA in transcription, it has been found that SAGA can interact with TBP and some acidic activators, and mutational studies of SAGA revealed transcriptional defects (54). By contrast, only a small fraction of genes appear to be transcriptionally dependent on GCN5, the HAT activity of SAGA (22), and the non-TAFII components of SAGA, which are not required for viability. The exact role of PCAF/SAGA in transcriptional activation remains to be determined. Studies on the possible physiological functions of TAFIIs have been carried out mostly in yeast, and it remains unclear whether higher eukaryotic TAFIIs possess similar transcriptional and cellular functions as their yeast counterparts. To address this question, and to resolve the controversies regarding the general requirement for TAFIIs in activated transcription, we have employed gene targeting coupled with conditional expression (58, 65) to construct a chicken DT40 cell line in which the only source of cTAFII31, the homologue of yTAFII17 and dTAFII40, was from a tetracycline (Tet)-repressible promoter. Following the addition of Tet to the culture medium, cTAFII31 mRNA and protein were efficiently depleted, cell growth slowed, and the cells eventually died by an apoptotic pathway. Genetic depletion of cTAFII31 also significantly reduced the intracellular levels of several other TAFIIs, but there were only minimal or undetectable decreases of TBP and of several subunits of the PCAF complex. Surprisingly, 3H pulse-labeling experiments indicate that transcription of total poly(A)+ mRNA was not significantly compromised by cTAFII31 depletion and the abundance of several specific transcripts was either not affected or only minimally reduced. Taken together, these data suggest that cTAFII31 and perhaps other TAFIIs are not universally required for transcription in DT40 cells. We discuss these results in terms of the existence of extensive functional degeneracy in vertebrate cells.