Coactivator SAGA plays a crucial role in TBP recruitment for the Gcn4 transcriptome via subunits Spt3/Spt8 [RNA-seq]

Transcription initiation by RNA polymerase II is facilitated by coactivators that recruit general initiation factors to promoters. Coactivator complexes TFIID and SAGA recruit TATA-binding protein (TBP), while SAGA also enhances transcription by histone acetylation via the HAT Gcn5. It was proposed that most yeast genes depend exclusively on TFIID, with only ~10% requiring cumulative contributions by SAGA and TFIID for efficient transcription. It was further suggested that genes induced by Gcn4, transcriptional activator of amino acid biosynthetic genes induced by amino acid starvation, depend on the HAT but not the TBP-recruitment function of SAGA. At odds with this model, ChIP-sequencing of TBP and Pol II subunit Rpb1 revealed that deleting SPT3 or SPT8, but not GCN5, reduced TBP binding at many Gcn4 target genes. In contrast, deleting GCN5 but not SPT3 or SPT8 impaired promoter histone eviction at the highly remodeled subset of induced genes, whereas transcription was broadly reduced by all three SAGA mutations. Nuclear depletion of TFIID subunit Taf1 generally reduced TBP recruitment at these and most other SAGA-dependent genes only in cells lacking Spt3 or Spt8. We conclude that SAGA is crucial for TBP recruitment via Spt3/Spt8, beyond its role in histone acetylation, and functions non-redundantly with TFIID in the Gcn4 transcriptome of amino acid-starved cells. Total RNA isolation was carried out using Hot-Phenol Method.Followed by RNA-sequencing libraries preparation using the Illumina TrueSeq stranded mRNA Library Prep Kit. Ribo-depletion was performed to remove the rRNA using Qiagen FastSelect Yeast rRNA Depletion Kit, followed by paired-end sequencing using the Novaseq-X platform.