A paired-end sequencing strategy to map the complex landscape of transcription initiation

Abstract

Recent high-throughput sequencing protocols have uncovered the complexity of mammalian transcription by RNA polymerase II, helping to define several initiation patterns in which transcription start sites (TSSs) cluster within both narrow and broad genomic windows. Here, we describe a paired-end sequencing strategy, which enables more robust mapping and characterization of capped transcripts. This strategy was applied to explore the transcription initiation landscape in the Drosophila melanogaster embryo. Extending the previous findings in mammals, we found that fly promoters exhibit distinct initiation patterns, which are linked to specific promoter sequence motifs. Furthermore, we identified a large number of 5′ capped transcripts originating from coding exons; analyses support that they are unlikely the result of alternative TSSs, but rather the product of post-transcriptional modifications. Taken together, paired-end TSS analysis is demonstrated to be a powerful method to uncover the transcriptional complexity of eukaryotic genomes.

Promoter architectures and developmental gene regulation

“…When comparing across other Drosophila genomes, elements in broad promoters had lower levels of conservation than those in sharp promoters [93]. Furthermore, the distinct promoter classes in fly were associated with the same functional categories of genes and showed similar expression specificity patterns as in mammals [12,22,93], suggesting functional conservation of the observed promoter classes across Metazoa.…”

“…Unlike mammalian genome, the fly genome does not contain CpG islands; however, the two promoter classes were shown to be associated with distinct core promoter elements. The positionally restricted canonical core promoter elements, including TATA-box, Inr, DPE and MTE, were specifically enriched in sharp promoters [22,93]. When comparing across other Drosophila genomes, elements in broad promoters had lower levels of conservation than those in sharp promoters [93].…”

“…High resolution TSS mapping by PET [22] and CAGE [25] in Drosophila revealed analogous transcription initiation patterns, separating promoters into "sharp" and "broad" class. Unlike mammalian genome, the fly genome does not contain CpG islands; however, the two promoter classes were shown to be associated with distinct core promoter elements.…”

“…Similarly, full-length human cDNAs were used to annotate and functionally analyse human promoters [19,20]. More recently, several techniques that sequence short tags from the 5' end of cDNAs have been developed including 5' serial analysis of gene expression (5' SAGE) [21], oligo-capping [22] and cap analysis of gene expression (CAGE) [23], which when combined with high-throughput sequencing achieve higher coverage producing more reliable and quantitative mapping of 5' ends. These techniques allow genome-wide precise TSS mapping at single nucleotide resolution and provide the means for analysing promoterassociated features at high resolution.…”

“…Unlike CGI and non-CGI promoter classification, which is vertebrate-specific, the corresponding sharp and broad promoter classes defined based on transcription initiation patterns are conserved across Metazoa [12,22,93]. These promoter classes are significantly differentiated by nucleosome organization and chromatin structure in both fly and mammals.…”