Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage

Abstract

We introduce cap analysis gene expression (CAGE), which is based on preparation and sequencing of concatamers of DNA tags deriving from the initial 20 nucleotides from 5 end mRNAs. CAGE allows high-throughout gene expression analysis and the profiling of transcriptional start points (TSP), including promoter usage analysis. By analyzing four libraries (brain, cortex, hippocampus, and cerebellum), we redefined more accurately the TSPs of 11-27% of the analyzed transcriptional units that were hit. The frequency of CAGE tags correlates well with results from other analyses, such as serial analysis of gene expression, and furthermore maps the TSPs more accurately, including in tissue-specific cases. The highthroughput nature of this technology paves the way for understanding gene networks via correlation of promoter usage and gene transcriptional factor expression.full-length cDNA ͉ transcriptome ͉ sequencing ͉ cap-trapping E ven the comparison of mammalian genome draft sequences (1) has left many unanswered questions with regard to the exact identification of expressed genes, their promoter elements, and the network of promoter͞transcriptional factor usage that underlies gene expression. Partial identification of the promoter sites has been provided by gene discovery programs based on the sequencing of full-length cDNA libraries (2-4); these have been instrumental in identifying the sequence of promoter regions, including potentially different promoters (5). Several thousand promoters can be determined by sequencing 5Ј ends from full-length cDNA libraries and mapping the sequences to the genome, thus determining which correspond to coding and regulatory regions, respectively. These analyses can produce statistics on transcriptional start sites derived from large numbers of 5Ј end sequences. However, these methods lack the throughput to provide significantly abundant data for intermediately͞lowly expressed genes, chiefly because the comprehensive sequencing of cDNA libraries is prohibitively expensive. On the other hand, microarrays for high-throughput tissue expression analysis do exist (6), but these cannot determine transcription starting points and therefore cannot be used to accurately identify the cis regulatory elements that will be essential for computing gene networks. Another limitation of microarrays is that the only genes͞transcripts that can be studied are those that have already been identified by the sequencing, which is far from completion (2). Serial analysis of gene expression (SAGE) allows partial sequence information of short tags at the 3Ј ends of mRNAs (7) to be obtained. Although the information is partial, it is amenable to relatively cheap high-throughput digital data collection, because it is based on the cloning and subsequent sequencing of concatamers of short DNA fragments derived from 3Ј ends of multiple mRNAs (http:͞͞cgap.nci.nih.gov͞ SAGE). This method was further improved on by Long-SAGE, which allows for the cloning of 20-nt SAGE tags (8), which mainly identify single loci on the ge...

Promoter architectures and developmental gene regulation

“…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.…”

“…CAGE is a high-throughput method for transcriptome analysis [23] that takes advantage of the 7-methylguanosine cap structure found at 5' ends of RNAPII transcripts to map precise transcription start sites (Fig. 2).…”

Genome-wide analysis of promoter architecture in Drosophila melanogaster

“…Subsequently, improved approaches such as rapid amplification of cDNA ends (RACE) (Frohman et al 1988) and cap-trapped 59 expressed sequence tag (EST) sequencing (Carninci et al 1996) were developed. More recently, cap analysis of gene expression (CAGE), a high-throughput method for promoter discovery, has been used in mouse and human to characterize capped transcript ends (Shiraki et al 2003;Carninci et al 2006). In Drosophila, TSSs have been defined on a modest scale by sequencing and annotation of 59 ESTs generated from cap-trapped cDNA clones (Misra et al 2002;Stapleton et al 2002).…”

Advancing transcriptome platforms

“…The conventional SAGE has been further optimized. LongSAGE (Saha et al 2002), CAGE (Shiraki et al 2003) and SuperSAGE (Matsumura et al 2003) were developed by using different tagging enzymes to release longer tags, which would provide higher specificity for transcript identification. Recently, a so-called SuperSAGE array was At present, a few disadvantages remain with the technique (Table 1).…”