CUR - The Council on Undergraduate Research

Bioinformatic and Experimental Evaluation of Transcription Factor Binding Site Conservation and Specificity in the Context of the Developing Retina

Mykel Barrett, Sacha Sulaiman, Denice Moran, Mark Emerson, Department of Biology, The City College of New York, 160 Convent Ave, New York, NY 10031

Retinal cell-fate specification is largely modulated by the interactions that take place between cis-regulatory DNA and transcription factors (TFs). A thorough biochemical understanding of how gene expression happens has not yet been achieved. Evolutionary conservation of transcription factor binding sites suggests that there is a higher level of specificity for the sequences that are required than is suggested from in vitro data. For example, in vivo experimental evidence suggests OTX2’s binding site in the ThrbCRM1 element is different from the ‘TAATCC’ motif derived from in vitro SELEX studies. To explore such instances of discord, a bioinformatic analysis of known binding sites, across species, and in different developmental contexts (i.e. hepatogenesis versus retinogenesis for HNF6), was done to identify where conservation suggests functional importance. This information was used to generate ‘sequence logos,’ which schematize how in vitro TF-DNA binding constraints do not always align with those observed in vivo. Site-directed mutagenesis experiments were performed to explore how single base substitutions alter the activity of cis-regulatory elements by assessing how well mutants recapitulate wild-type activity patterns. Flow cytometry analysis of retinal tissue electroporated with reporter vectors revealed that mutant activity patterns do indeed sometimes differ from wild-type expression patterns. This spotlights the limitations of in vitro assays, which don’t always fully recapitulate important molecular variables that affect the nature of TF-DNA interactions within the authentic physiological setting. The data reported herein may deepen understandings of photoreceptor evolution and the biochemistry underlying gene expression. This knowledge can be used to refine gene therapy techniques aimed at treating retinopathies.

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