Coactivator cross-talk specifies transcriptional output

Abstract

In order for cells to survive, they must be able to interpret environmental and developmental signals. Often the critical targets of these signals are sequence-specific DNA-binding proteins that serve as transcriptional activators or repressors. These relatively small regulatory proteins bind DNA at their cognate promoters to modulate the specific patterns of gene expression needed to respond to particular signals. However, these transcription factors that bind promoter DNA are generally not able to potentiate transcriptional readouts on their own (Hoffman et al. 1990; Peterson et al. 1990; Ryu et al. 1999). Instead, the tight control of gene expression is often dependent on the ordered assembly of large multiprotein coregulators at the promoters of mRNA genes that respond to specific cellular signals (Dynlacht et al. 1991; Naar et al. 1998; Fondell et al. 1999; Rachez et al. 1999; Ryu et al. 1999).

These multiprotein complexes can be divided, albeit roughly, into several distinct classes based on their properties (Lemon and Tjian 2000; Naar et al. 2001; Taatjes et al. 2004). One class, the coactivators, interprets activator signals and conveys them to the basal transcription apparatus. Of these, TFIID and Mediator (MED) are known to be direct targets of sequence-specific DNA-binding proteins. TFIID has been shown to be required for activated transcription at many promoters (Naar et al. 2001). TFIID consists of the TATA-binding protein (TBP) and the TBP-associated factors (TAFs). The central components of the complex are conserved from yeast to man. However, the metazoan version of TFIID has expanded to include some important differences. For instance, the glutamine-rich amino terminus of TAF4, a common target of activators, is missing in the yeast homolog. Additionally, metazoans have developed tissue-specific versions of the TAFs that play a role in gonad function, further expanding the repertoire of coactivators (Dikstein et al. 1996; Freiman et al. 2001; Hiller et al. 2004). Likewise, many subunits of the MED complex are conserved from yeast to man. However, the MED complex has diverged over time. The regions of obvious similarity in pairwise alignments of the subunits are often small, with large insertions and expansions occurring in the metazoan homologs (Boube et al. 2002). Moreover, there are at least 11 metazoan-specific subunits, and recent analysis of the mammalian MED complex revealed that it too contains potentially gene-specific variant subunits, although the role of these subunits remains unclear (Sato et al. 2004).

Many coactivator complexes have been identified based on their ability to stimulate activated transcription in defined in vitro systems (Meisterernst et al. 1991; Kim et al. 1994; Naar et al. 1998; Rachez et al. 1999; Ryu et al. 1999). Interestingly, although both TFIID and MED are targets for activators, many promoters require both coactivators in vitro. This suggests that the complexes are not redundant but may instead perform nonoverlapping functions in potentiating transcription. Furthermore, in vitro experiments with a synthetic activator suggest that TFIID and MED might functionally interact (Johnson et al. 2002; Johnson and Carey 2003). Exactly what role each of the complexes plays remains an open question. Moreover, it is unclear whether these complexes also interact with each other to cross-talk during the process of transcriptional regulation in vivo and whether their function has diversified in different metazoan cells.

Here, we examine the potential functional interactions and specific role of the TFIID and MED complexes at the Drosophila metallothionein promoters in vivo by taking advantage of the efficient RNAi response in insect cells (Clemens et al. 2000). We have depleted various subunits of the MED and TFIID complex by RNAi and examined the effect of the loss of these subunits on both the level of mRNA synthesized and the physical recruitment of the transcriptional apparatus in conventional cell population experiments as well as in single cells. We find that the two coactivator complexes interact to meter the transcriptional response so that it is physiologically appropriate. Unexpectedly, we found that although depleting MED subunits compromises the metal inducible activation of transcription, depleting both TFIID and MED components restores much of the transcriptional activity of the metallothionein A gene, suggesting an intriguing functional relationship between the TAFs and MED coactivator complexes.

Acknowledgments

Footnotes

References