Functional analysis of a transcriptional cofactor, TAF9, in vertebrate cells

This thesis focuses primarily on the functions of a transcriptional modulator, TBP-associated factor 9 (TAF9), in vertebrate cells. The first study, presented in Chapter 2, described the characterization of chicken DT40 cells (DT40-TAF9) containing a conditional TAF9 allele. Depletion of TAF9 resulted in apoptotic cell death, and codepletion of many other TAFs. Importantly, TAF9 functions in gene-specific, rather than general, transcription. Given that TAF9 is broadly required in yeast, this more limited role of TAF9 in transcription illustrate evolutionary divergence, likely due to a much higher degree of cofactor redundancy in vertebrates.; To gain more insights into the gene-specific TAF requirements, and to investigate the roles of core promoter elements in transcriptional activation, I examined expression and factor occupancy on representative promoters in DT40-TAF9 cells (Chapter 3). Core promoter elements were shown to play significant roles in promoter strength, response to activators, and factor recruitment and activation mechanisms. TAF9 requirement was highly promoter-specific, and TAF9 dependence and promoter occupancy were frequently but not always correlated. We also provide contrasting examples of activation mechanisms utilized by different promoters. Taken together, these results illustrate multiple and dynamic roles of core promoter elements and, in contrast to yeast, the unexpected degree of diversity and complexity of vertebrate transcriptional regulation.; In the study presented in Chapter 4, I investigated further the functions of TAF9 structural domains and a TAF9-related factor, TAF9L. TAF9 depletion was shown to disrupt TFIID, indicating a largely dispensable role of intact TFIID in maintaining transcription. In support of a critical role of TAF9 in cell growth, TAF9 levels are tightly regulated in DT40-TAF9 cells. Evidence is also provided for specific roles of TAF histone fold motifs (HFMs), highlighting the functional significance of HFM specificity and the TAF9-H3 HFM similarity. Despite partly redundant functions for TAF9 and TAF9L, RNAi experiments suggest that TAF9L is essential for cell growth. Strikingly, TAF9L plays a role in transcriptional repression.; Taken together, these studies constitute a detailed functional analysis of a transcriptional cofactor in vivo and illustrate the complexity of transcriptional regulation in vertebrates.