Transcription Start Site Turnover in Drosophila using CAGE. Bradley J. Main, Hyosik Jang, Andrew Smith, Sergey Nuzhdin. MCB, Univ Southern California, Los Angeles, CA.

   Random mutations can give rise to new promoter sequences that are functionally redundant. These promoters and their associated transcription start sites (TSS) then experience relaxed selection at one or both copies, resulting in death or functional divergence of one TSS. Thus, functionally equivalent TSS may move, or turnover, via this birth and death process. TSS locations can be identified using sequencing methods like cap analysis for gene expression (CAGE) that anchor sequencing reads to the 5 end of mRNA. For this study, we developed a highly improved CAGE technique (Taq-ex CAGE) and employed it to assess TSS turnover among four Drosophila species: D. melanogaster (mel), D. simulans (sim), D. sechellia (sec), and D. pseudoobscura (Dpse). We identified 2849 high-confidence TSS in mel and found orthologs for the majority of them in each species (83%, 86%, and 55% for sim, sec, and Dpse, respectively). An appreciable number of mel TSS were unpaired due to extensive sequence divergence or lack of an ortholog within 500bp. These likely include distant turnover events, but may also include cases where the ortholog is expressed below detection. Overall, TSS were conserved, but ribosomal protein genes were highly enriched among genes with diverged TSS and turnover events were associated with divergence in expression. This suggests that TSS turnover is more common among certain types of genes and TSS changes contribute to cis-regulatory variation between Drosophila species.