saccharomyces-cerevisiae-metabolic-labeling-with-4-thiouracil

<em>Saccharomyces cerevisiae</em> Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France

Centre National de la Recherche Scientifique, Illkirch, France

Institut National de la Santé et de la Recherche Médicale, Illkirch, France

université de strasbourg

The SAGA (Spt-Ada-Gcn5 acetyltransferase) coactivator complex contains distinct chromatin-modifying activities and is recruited by DNA-bound activators to regulate the expression of a subset of genes. Surprisingly, recent studies revealed little overlap between genome-wide SAGA-binding profiles and changes in gene expression upon depletion of subunits of the complex. As indicators of SAGA recruitment on chromatin, we monitored in yeast and human cells the genome-wide distribution of histone H3K9 acetylation and H2B ubiquitination, which are respectively deposited or removed by SAGA. Changes in these modifications after inactivation of the corresponding enzyme revealed that SAGA acetylates the promoters and deubiquitinates the transcribed region of all expressed genes. In agreement with this broad distribution, we show that SAGA plays a critical role for RNA polymerase II recruitment at all expressed genes. In addition, through quantification of newly synthesized RNA, we demonstrated that SAGA inactivation induced a strong decrease of mRNA synthesis at all tested genes. Analysis of the SAGA deubiquitination activity further revealed that SAGA acts on the whole transcribed genome in a very fast manner, indicating a highly dynamic association of the complex with chromatin. Thus, our study uncovers a new function for SAGA as a bone fide cofactor for all RNA polymerase II transcription. 

The SAGA coactivator complex acts on the whole transcribed genome and is required for RNA polymerase II transcription.

Prior studies suggested that SAGA and TFIID are alternative factors that promote RNA polymerase II transcription, with about 10% of genes in S. cerevisiae dependent on SAGA. We reassessed the role of SAGA by mapping its genome-wide location and role in global transcription in budding yeast. We find that SAGA maps to the UAS elements of most genes, overlapping with Mediator binding and irrespective of previous designations of SAGA- or TFIID-dominated genes. Disruption of SAGA through mutation or rapid subunit depletion reduces transcription from nearly all genes, measured by newly synthesized RNA. We also find that the acetyltransferase Gcn5 synergizes with Spt3 to promote global transcription and that Spt3 functions to stimulate TBP recruitment at all tested genes. Our data demonstrate that SAGA acts as a general cofactor required for essentially all RNA polymerase II transcription and is not consistent with the previous classification of SAGA- and TFIID-dominated genes.

Didier Devys

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity