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A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design

Robin P Smith12, Samantha J Riesenfeld3, Alisha K Holloway3, Qiang Li126, Karl K Murphy12, Natalie M Feliciano12, Lorenzo Orecchia4, Nir Oksenberg12, Katherine S Pollard235* and Nadav Ahituv12*

Author affiliations

1 Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 1550 4th St, San Francisco, CA 94158, USA

2 Institute for Human Genetics, University of California San Francisco, 1550 4th St, San Francisco, CA 94158, USA

3 Gladstone Institutes, University of California San Francisco, 1650 Owens St, San Francisco, CA 94158, USA

4 Division of Biostatistics, University of California San Francisco, 1650 Owens St, CA 94158, USA

5 Department of Mathematics, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA

6 Current address: Institute for Pediatrics, Translational Research Center for Development and Disease, Children's Hospital of Fudan University, Shanghai, 201102, China

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Citation and License

Genome Biology 2013, 14:R72  doi:10.1186/gb-2013-14-7-r72

Published: 18 July 2013

Abstract

Background

Large-scale annotation efforts have improved our ability to coarsely predict regulatory elements throughout vertebrate genomes. However, it is unclear how complex spatiotemporal patterns of gene expression driven by these elements emerge from the activity of short, transcription factor binding sequences.

Results

We describe a comprehensive promoter extension assay in which the regulatory potential of all 6 base-pair (bp) sequences was tested in the context of a minimal promoter. To enable this large-scale screen, we developed algorithms that use a reverse-complement aware decomposition of the de Bruijn graph to design a library of DNA oligomers incorporating every 6-bp sequence exactly once. Our library multiplexes all 4,096 unique 6-mers into 184 double-stranded 15-bp oligomers, which is sufficiently compact for in vivo testing. We injected each multiplexed construct into zebrafish embryos and scored GFP expression in 15 tissues at two developmental time points. Twenty-seven constructs produced consistent expression patterns, with the majority doing so in only one tissue. Functional sequences are enriched near biologically relevant genes, match motifs for developmental transcription factors, and are required for enhancer activity. By concatenating tissue-specific functional sequences, we generated completely synthetic enhancers for the notochord, epidermis, spinal cord, forebrain and otic lateral line, and show that short regulatory sequences do not always function modularly.

Conclusions

This work introduces a unique in vivo catalog of short, functional regulatory sequences and demonstrates several important principles of regulatory element organization. Furthermore, we provide resources for designing compact, reverse-complement aware k-mer libraries.