Patchy interspecific sequence similarities efficiently identify positive cis-regulatory elements in the sea urchin

Area of Science:

• Developmental Biology
• Genomics
• Molecular Biology

Background:

• The otx gene is critical for endomesoderm specification in sea urchin embryos.
• The otx gene exhibits complex cis-regulatory organization due to multiple transcription start sites and diverse expression domains.
• Identifying functional cis-regulatory elements (CREs) is essential for understanding gene regulation.

Purpose of the Study:

• To systematically identify functional CREs of the otx gene using interspecific sequence conservation.
• To investigate the cis-regulatory landscape governing otx gene expression in Strongylocentrotus purpuratus embryos.
• To validate the utility of comparative genomics and computational tools for discovering regulatory elements.

Main Methods:

• Isolation and sequencing of BAC recombinants containing the otx gene from Strongylocentrotus purpuratus and Lytechinus variegatus.
• Computational scanning of flanking genomic DNA for conserved sequence elements using the 'FamilyRelations' software.
• Functional testing of conserved regions by cloning into a CAT reporter vector, injection into embryos, and assessment via whole-mount in situ hybridization (WMISH).

Main Results:

• Seventeen conserved regions were identified, and 11 displayed significant cis-regulatory activity in embryonic expression assays.
• Identified CREs exhibited spatially restricted expression patterns consistent with known otx gene expression domains.
• Four novel endomesoderm-specific CREs were discovered, alongside ubiquitously active and ectoderm-specific elements.

Conclusions:

• Interspecific sequence conservation is a powerful and systematic approach for identifying functional CREs in complex gene loci.
• The study successfully identified multiple novel CREs controlling otx gene expression, including key elements for endomesoderm development.
• Comparative genomics combined with functional assays provides a robust strategy for dissecting gene regulatory networks.