BioProject

Promoter architecture, shape and usage play an important role in the regulation of eukaryotic gene expression. More...

Promoter architecture, shape and usage play an important role in the regulation of eukaryotic gene expression. Several promoter profiling techniques have been developed to detect transcription start sites and alternative promoter usage between tissues or during development. However promoter profiling has not been utilized in studies that demand quantification of expression changes between developmental stages, tissues and conditions. In this study, we combine promoter profiling and differential expression analysis in a single setup, using a fast and simple protocol, MAPCap (Multiplexed Affinity Purification of Capped RNA) along with a new software “icetea” (https://bioconductor.org/packages/icetea). The use of random molecular barcodes and spike-ins in MAPCap enables accurate quantification of expression from promoters. We detected sex-specific promoter usage in the brains of Drosophila melanogaster larvae. Comparison of male and female mutant flies of the male-less helicase (MLE), a protein known to be essential for the dosage compensation of the male X-chromosome, indicated that the sensitivity of promoters to dosage compensation depends on their genomic location. Our results expand the scope of promoter profiling methods to differential expression analysis and provide quantitative insights into promoter usage during dosage compensation. Overall design: To evaluate MAPCap data and compare with other protocols, we performed MAPCap on stage 15 Drosophila embryos in four replicates, 5uG RNA per-replicate. Files are multiplexed in the same fastq. Further, for analysis of dosage compensation, we performed MAPCap on RNA isolated from brains of maleless (MLE) RNA helicase mutant (also referred as knock-outs/KOs) Drosophila Melanogaster L3 larvae, in three replicates each (5uG RNA per replicate). For the L3 larvae, we also independently performed a ribo-depleted Illumina RNA-seq. To evaluate MAPCap TSS enrichment at various RNA concentration, we performed MAPCap on RNA isolated from S2 cells, at starting RNA concentrations of 5ug, 1ug, 500ng, and 100ng. To compare MAPCap with nAnTiCAGE, we performed MAPCap from 5ug RNA isolated from female mouse ESCs where the nAnTiCAGE data was available online Less...