High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression

Abstract

Many eukaryotic genes possess multiple alternative promoters with distinct expression specificities. Therefore, comprehensively annotating promoters and deciphering their individual regulatory dynamics is critical for gene expression profiling applications and for our understanding of regulatory complexity. We introduce RAMPAGE, a novel promoter activity profiling approach that combines extremely specific 59-complete cDNA sequencing with an integrated data analysis workflow, to address the limitations of current techniques. RAMPAGE features a streamlined protocol for fast and easy generation of highly multiplexed sequencing libraries, offers very high transcription start site specificity, generates accurate and reproducible promoter expression measurements, and yields extensive transcript connectivity information through paired-end cDNA sequencing. We used RAMPAGE in a genome-wide study of promoter activity throughout 36 stages of the life cycle of Drosophila melanogaster, and describe here a comprehensive data set that represents the first available developmental time-course of promoter usage. We found that >40% of developmentally expressed genes have at least two promoters and that alternative promoters generally implement distinct regulatory programs. Transposable elements, long proposed to play a central role in the evolution of their host genomes through their ability to regulate gene expression, contribute at least 1300 promoters shaping the developmental transcriptome of D. melanogaster. Hundreds of these promoters drive the expression of annotated genes, and transposons often impart their own expression specificity upon the genes they regulate. These observations provide support for the theory that transposons may drive regulatory innovation through the distribution of stereotyped cis-regulatory modules throughout their host genomes.

Paired-End Analysis of Transcription Start Sites in Arabidopsis Reveals Plant-Specific Promoter Signatures

“…Cap-trapping protocols such as CAGE (Carninci et al, 2005) and PEAT (Ni et al, 2010) aim to isolate pol-II mRNA transcripts and sequence the capped ends (TSS tags) with nucleotide resolution. Recent analyses using genome-scale TSS data sets produced with PEAT and CAGE in animal tissues (Ni et al, 2010;Batut et al, 2013;Nepal et al, 2013) have observed that TSSs are not simply located at one or even a few "alternative start site" locations upstream of each pol-II gene. When these TSSs are mapped back to the genome, they distribute spatially in a variety of different "TSS tag clusters" or "initiation patterns."…”

Characterization of regulatory sequences in alternative promoters of hypermethylated genes associated with tumor resistance to cisplatin

“…Recent findings support a prominent role for alternative promoters in cell type and human tissue type-specific gene expression (26). Considering that the transcription machinery utilizes alternative promoters for regulating differential transcription (10,16) and the aberrant use of one alternative promoter over another may result in disease, including cancer (11), we hypothesized that cisplatin resistance may be mediated by a differential usage of alternative promoters with variable regulatory sequences, TFBS and CGIs. Transcription factors and their binding sites in a given promoter are key elements in controlling the rate and extent of mRNA synthesis (19,27).…”

“…A promising approach to , RUIWEN ZHANG 6 and KALKUNTE S. SRIVENUGOPAL these questions appears to be the characterization of regulatory elements, CpG islands (CGIs) and transcription factor-binding sites (TFBS) in alternative promoters of genes that have been validated and their association with cisplatin resistance proven. It has been well established that alternative promoters are involved in the transcription of nearly half of all eukaryotic genes and are considered one of the main molecular characteristics of eukaryotic genomes (10,11). Overall, alternative promoters have been suggested to add significant flexibility and greater diversity to the regulation of gene expression (10)(11)(12)(13)(14)(15)(16).…”

“…It has been well established that alternative promoters are involved in the transcription of nearly half of all eukaryotic genes and are considered one of the main molecular characteristics of eukaryotic genomes (10,11). Overall, alternative promoters have been suggested to add significant flexibility and greater diversity to the regulation of gene expression (10)(11)(12)(13)(14)(15)(16). The functional significance of alternative promoters and their role in disease development and progression remains unclear, except for a few well characterized genes, such as the tumor suppressor p53, lymphoid enhancer-binding factor 1, insulin growth factor 2 and guanine nucleotide-binding protein α-stimulating genes, where these sequences are aberrantly activated, developmentally regulated or silenced (11).…”

Gappy TotalReCaller for RNASeq Base-Calling and Mapping

“…(a) Genes can be expressed with widely varying copy numbers that change rapidly, (b) the same gene can have multiple splice variants whose structures remain unannotated and are expressed in unknown and varying proportions, and (c) many genes belong to gene-families sharing high-degree of homology. See [11,2,22,8,7,6,4].…”