TRANSCRIPTOME 2002: From Functional Genomics to Systems
Systematic Screening of Tissue Specific Cis-regulatory Elements in Ciona intestinalis Embryo
Byung-in Lee1, David Keys2, Mei Wang1, Orsalem J. Kahsai1, Sylvia Ahn1, Qing Zhang1, David K. Engle1, Irma Rapier1, Jason Olivas1, Chris J. Detter1, Sharon Doyle1, Naoe Harafuji1, Anna Di Gregorio1, Trevor Hawkins1, Dan S. Rokhsar1, Michael Levine1,2, Paul M. Richardson1, 1DOE Joint Genome Institute, Walnut Creek, CA, 2Department of Molecular and Cellular Biology, University of California at Berkeley, Berkeley, CA
Regulatory DNA elements such as promoters and enhancers work by serving as docking sites for specific protein complexes. These complexes are comprised of cooperative groups of transcription factor proteins that recognize the target DNA sequences quite specifically and their presence or absence governs the off or on status of the target. Therefore understanding DNA regulatory elements is key to understanding the composition and function of the biochemical networks and pathways that carry out the essential processes of living organisms. To characterize gene regulatory networks, we used electroporation assays to screen genomic DNA fragments for tissue specific regulatory activities in Ciona intestinalis. The Ciona genome is one of the smallest and most compact of all chordate genomes and the Ciona tadpole represents the most simplified chordate body plan. Since, exogenous DNA can be introduced into the synchronously developing embryos via simple electroporation, we used this method with a vector containing the lacZ reporter to determine cis regulatory DNA modules that lead to the specification of key chordate tissues. We screened ~300kb of Ciona genomic DNA containing Hox genes for tissue specific enhancer elements using the shotgun approach, and found 30 clones (80kb) that were positive for cis-regulatory activity. Among 30 positives, 20 unique putative enhancer elements identified which indicate about one putative enhancer elements every 18kb with expression patterns in epidermis, tail muscles, cerebral vesicle, notochord, neural tube. Among 20 enhancers, 8 expression patterns may be associated with Hox genes. These are being confirmed by whole mount in-situ hybridization.
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