Abstract
BackgroundHigh-resolution transcription start site (TSS) mapping in D. melanogaster embryos and cell lines has revealed a rich and detailed landscape of both cis- and trans-regulatory elements and factors. However, TSS profiling has not been investigated in an orthogonal in vivo setting. Here, we present a comprehensive dataset that links TSS dynamics with nucleosome occupancy and gene expression in the wandering third instar larva, a developmental stage characterized by large-scale shifts in transcriptional programs in preparation for metamorphosis.ResultsThe data recapitulate major regulatory classes of TSSs, based on peak width, promoter-proximal polymerase pausing, and cis-regulatory element density. We confirm the paucity of divergent transcription units in D. melanogaster, but also identify notable exceptions. Furthermore, we identify thousands of novel initiation events occurring at unannotated TSSs that can be classified into functional categories by their local density of histone modifications. Interestingly, a sub-class of these unannotated TSSs overlaps with functionally validated enhancer elements, consistent with a regulatory role for “enhancer RNAs” (eRNAs) in defining developmental transcription programs.ConclusionsHigh-depth TSS mapping is a powerful strategy for identifying and characterizing low-abundance and/or low-stability RNAs. Global analysis of transcription initiation patterns in a developing organism reveals a vast number of novel initiation events that identify potential eRNAs as well as other non-coding transcripts critical for animal development.
Highlights
High-resolution transcription start site (TSS) mapping in D. melanogaster embryos and cell lines has revealed a rich and detailed landscape of both cis- and trans-regulatory elements and factors
Start-seq signal correlates with nucleosome depletion, gene expression, and promoter proximal pausing To characterize the genome-wide landscape of gene expression, transcription initiation, and chromatin accessibility in third instar D. melanogaster larvae, we carried out rRNA-depleted total nuclear RNA-seq, Start-seq, which quantifies short, capped, nascent RNAs that represent newly initiated species [19, 21], and ATAC-seq, which quantifies transposase-accessible open chromatin [30], as previously described [31]
We found that bidirectional Previously observed TSS (obsTSS) had significantly more ATAC-seq signal than divergent or non-divergent obsTSSs, despite the expectation that ATAC-seq would correlate with the lower level of Start-seq signal at bidirectional obsTSSs (Additional file 1: Figure S5D)
Summary
High-resolution transcription start site (TSS) mapping in D. melanogaster embryos and cell lines has revealed a rich and detailed landscape of both cis- and trans-regulatory elements and factors. Prior to RNA polymerase II (RNA pol II) engaging with DNA at the TSS, nucleosome depletion directly upstream of the TSS facilitates assembly of the PIC and other general transcription factors. This stereotypical ‘minus-1’ nucleosome depleted region (NDR) is conserved across eukaryotes [10, 11], and is highly correlated with transcription initiation activity. Additional descriptive characteristics of transcription initiation activity, such as the breadth or distribution of initiating polymerases across a given domain [12, 13], correlate with gene expression outcomes It is not known whether these factors play a role in proper regulation of gene expression
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