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MAPCap allows high-resolution detection and differential expression analysis of transcription start sites.

ABSTRACT: The position, shape and number of transcription start sites (TSS) are critical determinants of gene regulation. Most methods developed to detect TSSs and study promoter usage are, however, of limited use in studies that demand quantification of expression changes between two or more groups. In this study, we combine high-resolution detection of transcription start sites and differential expression analysis using a simplified TSS quantification protocol, MAPCap (Multiplexed Affinity Purification of Capped RNA) along with the software icetea . Applying MAPCap on developing Drosophila melanogaster embryos and larvae, we detected stage and sex-specific promoter and enhancer activity and quantify the effect of mutants of maleless (MLE) helicase at X-chromosomal promoters. We observe that MLE mutation leads to a median 1.9 fold drop in expression of X-chromosome promoters and affects the expression of several TSSs with a sexually dimorphic expression on autosomes. Our results provide quantitative insights into promoter activity during dosage compensation.

SUBMITTER: Bhardwaj V

PROVIDER: S-EPMC6667505 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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MAPCap allows high-resolution detection and differential expression analysis of transcription start sites.

Bhardwaj Vivek V Semplicio Giuseppe G Erdogdu Niyazi Umut NU Manke Thomas T Akhtar Asifa A

Nature communications 20190730 1

The position, shape and number of transcription start sites (TSS) are critical determinants of gene regulation. Most methods developed to detect TSSs and study promoter usage are, however, of limited use in studies that demand quantification of expression changes between two or more groups. In this study, we combine high-resolution detection of transcription start sites and differential expression analysis using a simplified TSS quantification protocol, MAPCap (Multiplexed Affinity Purification ...[more]

PMID: 31363093

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Project description:Cap Analysis of Gene Expression (CAGE) in combination with single-molecule sequencing technology allows precision mapping of transcription start sites (TSSs) and genome-wide capture of promoter activities in differentiated and steady state cell populations. Much less is known about whether TSS profiling can characterize diverse and non-steady state cell populations, such as the approximately 400 transitory and heterogeneous cell types that arise during ontogeny of vertebrate animals. To gain such insight, we used the chick model and performed CAGE-based TSS analysis on embryonic samples covering the full 3-week developmental period. In total, 31,863 robust TSS peaks (>1 tag per million [TPM]) were mapped to the latest chicken genome assembly, of which 34% to 46% were active in any given developmental stage. ZENBU, a web-based, open-source platform, was used for interactive data exploration. TSSs of genes critical for lineage differentiation could be precisely mapped and their activities tracked throughout development, suggesting that non-steady state and heterogeneous cell populations are amenable to CAGE-based transcriptional analysis. Our study also uncovered a large set of extremely stable housekeeping TSSs and many novel stage-specific ones. We furthermore demonstrated that TSS mapping could expedite motif-based promoter analysis for regulatory modules associated with stage-specific and housekeeping genes. Finally, using Brachyury as an example, we provide evidence that precise TSS mapping in combination with Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-on technology enables us, for the first time, to efficiently target endogenous avian genes for transcriptional activation. Taken together, our results represent the first report of genome-wide TSS mapping in birds and the first systematic developmental TSS analysis in any amniote species (birds and mammals). By facilitating promoter-based molecular analysis and genetic manipulation, our work also underscores the value of avian models in unravelling the complex regulatory mechanism of cell lineage specification during amniote development.

Dataset Information

MAPCap allows high-resolution detection and differential expression analysis of transcription start sites.

Publications

MAPCap allows high-resolution detection and differential expression analysis of transcription start sites.

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