Role of co-activators and promoter architecture in transcription of yeast protein-coding genes

Abstract

Transcription initiation is a complex, energy consuming process that requires the massive and coordinated activity of many proteins often organized in big multi-subunit complexes. The General Transcription Factors (GTFs) and the TATA-binding protein (TBP) orchestrate the formation of the so-called pre-initiation complex (PIC), a key step of transcription initiation required for RNA Polymerase II (RNAPII) association to promoters. Although the PIC is made by the GTFs, many other complexes generally referred to as transcriptional co-activators participate in the formation of the PIC. Transcriptional co-activator complexes include chromatin remodelers, Histone AcetylTransferases (HATs) and the Mediator complex. In yeast, it is has been reported that two main HAT complexes, NuA4 and SAGA, and the Mediator complex have a global role in transcription. However, though, it is well-known that histone acetylation is associated to high transcription and that Mediator plays a central function in PIC assembly, the deployment of NuA4, SAGA and Mediator at yeast genes is still elusive. Here, we investigate the contribution of NuA4, SAGA and Mediator in RNAPII association at all yeast genes through rapid nuclear depletion of key complex subunits. We reveal that Gcn5, the HAT of the SAGA complex, is modestly but equally involved in transcription of all yeast genes, while Esa1, the HAT of the NuA4 complex, is most strongly required for transcription of certain groups of genes. Curiously, we show that nuclear depletion of Med17, an essential subunit of the Mediator complex, strongly affects transcription of a small subset of genes characterized by a well-conserved TATA box, highlighting a previously underappreciated connection between Mediator complex and TBP. Our analysis reveals that three combinations of co-activator deployment are used to generate high transcription levels and that transcription of two groups of so called “house-keeping” or “growth-promoting genes” is particularly dependent on Tra1, a shared component of NuA4 and SAGA. Our work on Ribosomal Protein (RP) gene and Ribosome Biogenesis (RiBi) gene transcription discloses the importance of the transcription factors Ifh1 and Sfp1 in recruiting Tra1 at these promoters. 10 Furthermore, a deep investigation of transcription factors binding at RP gene promoters highlights the existence of a hierarchy, with Rap1 being required for the association of the other TFs. Our study also reveals that RP gene promoters, as well as the promoters of many other highly transcribed genes, are characterized by the presence of one or more unusually unstable MNase-sensitive nucleosomes referred to as fragile nucleosomes. Furthermore, our work discovers the main role of Rap1 and of other pioneer transcription factors in establishing nucleosome fragility and promoter architecture. Finally, we investigate the biophysical properties of fragile nucleosomes and provide evidence of their nucleosomal nature. Altogether the work presented in this thesis elucidates important features of yeast promoters and provides new insights into the mechanisms that drive eukaryotic transcription.