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Patchy Interspecific Sequence Similarities Efficiently Identify Positive cis-Regulatory Elements in the Sea Urchin

Yuh, Chiou-Hwa and Brown, C. Titus and Livi, Carolina B. and Rowen, Lee and Clarke, Peter J. C. and Davidson, Eric H. (2002) Patchy Interspecific Sequence Similarities Efficiently Identify Positive cis-Regulatory Elements in the Sea Urchin. Developmental Biology, 246 (1). pp. 148-161. ISSN 0012-1606. doi:10.1006/dbio.2002.0618.

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We demonstrate that interspecific sequence conservation can provide a systematic guide to the identification of functional cis-regulatory elements within a large expanse of genomic DNA. The test was carried out on the otx gene of Strongylocentrotus purpuratus. This gene plays a major role in the gene regulatory network that underlies endomesoderm specification in the embryo. The cis-regulatory organization of the otx gene is expected to be complex, because the gene has three different start sites (X. Li, C.-K. Chuang, C.-A. Mao, L. M. Angerer, and W. H. Klein, 1997, Dev. Biol. 187, 253–266), and it is expressed in many different spatial domains of the embryo. BAC recombinants containing the otx gene were isolated from Strongylocentrotus purpuratus and Lytechinus variegatus libraries, and the ordered sequence of these BACs was obtained and annotated. Sixty kilobases of DNA flanking the gene, and included in the BAC sequence from both species, were scanned computationally for short conserved sequence elements. For this purpose, we used a newly constructed software package assembled in our laboratory, “FamilyRelations.” This tool allows detection of sequence similarities above a chosen criterion within sliding windows set at 20–50 bp. Seventeen partially conserved regions, most a few hundred base pairs long, were amplified from the S. purpuratus BAC DNA by PCR, inserted in an expression vector driving a CAT reporter, and tested for cis-regulatory activity by injection into fertilized S. purpuratus eggs. The regulatory activity of these constructs was assessed by whole-mount in situ hybridization (WMISH) using a probe against CAT mRNA. Of the 17 constructs, 11 constructs displayed spatially restricted regulatory activity, and 6 were inactive in this test. The domains within which the cis-regulatory constructs were expressed are approximately consistent with results from a WMISH study on otx expression in the embryo, in which we used probes specific for the mRNAs generated from each of the three transcription start sites. Four separate cis-regulatory elements that specifically produce endomesodermal expression were identified, as well as ubiquitously active elements, and ectoderm-specific elements. We confirm predictions from other work with respect to target sites for specific transcription factors within the elements that express in the endoderm.

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Brown, C. Titus0000-0001-6001-2677
Additional Information:© 2002 Elsevier Science. Received for publication December 23, 2001. Revised February 7, 2002. Accepted February 7, 2002. Available online 23 May 2002. We thank Prof. Ellen Rothenberg of Caltech for a thoughtful and careful critique of this manuscript. This work was supported by NIH Grants GM-61005 and HD-37105 and by the Caltech Beckman Institute. C.T.B. is a participant in the Initiative in Computational Molecular Biology, which is funded by an award from the Burroughs Wellcome Fund Interfaces program. C. B. L. was supported by the Gordon Ross Fellowship and the Walter and Sylvia Treadway Foundation.
Funding AgencyGrant Number
Caltech Beckman InstituteUNSPECIFIED
Burroughs Wellcome FundUNSPECIFIED
Gordon Ross FellowshipUNSPECIFIED
Walter and Sylvia Treadway FoundationUNSPECIFIED
Subject Keywords:otx gene; FamilyRelations; cis-regulatory sequence; computational genomics; sea urchin embryo
Issue or Number:1
Record Number:CaltechAUTHORS:20160121-071017802
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Official Citation:Chiou-Hwa Yuh, C.Titus Brown, Carolina B. Livi, Lee Rowen, Peter J.C. Clarke, Eric H. Davidson, Patchy Interspecific Sequence Similarities Efficiently Identify Positive cis-Regulatory Elements in the Sea Urchin, Developmental Biology, Volume 246, Issue 1, 1 June 2002, Pages 148-161, ISSN 0012-1606, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:63818
Deposited By: Ruth Sustaita
Deposited On:21 Jan 2016 19:14
Last Modified:10 Nov 2021 23:21

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