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A protocol describing the principles of cis-regulatory analysis in the sea urchin

Nature Protocols volume 3, pages 710–718 (2008)Cite this article

Abstract

cis-Regulatory analysis (CRA) is the precise identification of the cis-acting genomic sequences regulating gene transcription. As such, CRA provides essential mechanistic insight into key biological processes such as development. The first phase of this protocol involves identification of a large (∼100 kb) clone of genomic material surrounding the gene of interest and use of this clone to establish a reliable and unambiguous reporter assay. In the second phase, phylogenetic footprinting is used to identify candidate regulatory modules; these genomic sequences are then recursively tested for reporter activity. In the final phase, potential transcription factor binding sites are identified and disrupted in reporter constructs for individual testing. The strengths of this method reflect the use of large clones containing all relevant genomic regulatory sequences to establish a reporter assay with high fidelity. Given these foundational elements, predicted or suspected regulatory inputs can be rigorously tested and novel regulatory inputs identified. Although the expected time line varies greatly with the depth of information required, results may be obtained in as little as 4–6 months, but more detailed analyses will require several years to complete.

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Figure 1: Gene network subcircuits which yield ambiguous or uninformative network perturbation data.
Figure 2: General work flow for cis-regulatory analysis (CRA).

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Acknowledgements

Many thanks to Eric H. Davidson in whose laboratory this protocol was developed and who provided the primary logical arguments underpinning this methodology. Many thanks also to members of the Eric H. Davidson laboratory group, past and present, for their contributions. The author thanks Jongmin Nam, Smadar Ben-Tabou De-Leon and Andrew Ransick for thoughtful discussions, insights and suggestions. Thanks also to Jenifer Croce and Andrew Ransick for a critical reading of the article. This work is supported by NIH GM-75089. The author is supported by a California Institute of Regenerative Medicine fellowship.

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  1. Division of Biology, California Institute of Technology, 1200 E. California Boulevard, Pasadena, 91125, California, USA

    Joel Smith

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Correspondence to Joel Smith.

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Smith, J. A protocol describing the principles of cis-regulatory analysis in the sea urchin. Nat Protoc 3, 710–718 (2008). https://doi.org/10.1038/nprot.2008.39

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