Gcn4 binding in coding regions can activate internal and canonical 5’ promoters in yeast [ChIP-seq]

Gcn4 is a yeast transcriptional activator induced by amino acid starvation. ChIP-seq analysis revealed 546 genomic sites occupied by Gcn4 in starved cells, representing ~30% of all Gcn4 binding-motifs. Deviation from the consensus motif and nucleosome occupancy are key negative determinants of Gcn4 binding. Surprisingly, only ~40% of the bound sites are in promoter regions, and only ~50-67% of these activate transcription, indicating extensive negative control over Gcn4 function. Most of the remaining ~300 Gcn4-bound sites are within coding sequences (CDS), with ~75 representing the only bound sites near Gcn4-induced genes. Many such unconventional sites map between divergent antisense and sub-genic sense transcripts induced within CDS, adjacent to induced TBP peaks—consistent with Gcn4 activation of cryptic, bidirectional internal promoters. Mutational analysis confirms that Gcn4 sites within CDS can activate sub-genic and full-length transcripts from the same or adjacent genes, showing that functional Gcn4 binding is not confined to promoters. Immunopurified sonicated DNA fragments were subjected to paired-end sequencing. Paired-end sequencing study of (1) ChIP samples for Gcn4 (Affinity purified Gcn4 antibody was described in Zhang et al., Mol Cell Biol. 2008 Nov;28(20), (2) ChIP samples for the Rpb3 subunit of Pol II, (3) ChIP samples for the TBP(SPT15)-myc13.