Presentation
Expression and Purification of Retina-Specific Transcription Factors CRX and NRL
James Madison University College of Science and Math Summer Symposium
(2016)
Abstract
The vertebrate retina is a neuronal tissue of the eye containing rod and cone photoreceptors that make vision possible. Highly regulated gene expression controls differentiation in the developing retina. DNA methylation is an epigenetic modification inversely correlated with gene expression. Currently in our lab, the relationship between DNA methylation and the ability of two retina-specific transcription factors to bind in the genome is being studied. These transcription factors, known as cone-rod homeobox (CRX) and neural leucine zipper (NRL), have been shown to act synergistically to control photoreceptor expression. Preliminary data has supported the hypothesis that DNA methylation is critical for modulating cell-specific binding of CRX and NRL to target recognition sites. Full length coding sequences and DNA binding domain sequences of the human CRX and NRL proteins were cloned into expression vectors, were transformed into competent E. coli cells, and transformants were selected for induction of protein expression. Batch cultures of expressed proteins will be affinity purified for downstream biochemical analysis including gel shift assays to determine the ability of CRX and NRL to bind to in vitro unmethylated and methylated oligonucleotides as well as structural analysis of transcription factors bound to their respective binding motifs. Collectively, these studies will contribute to a better understanding of how epigenetic modifications influence the development, homeostasis and pathology of human retinal neurons.
Keywords
- human retina,
- CRX,
- NRL,
- protein expression
Disciplines
Publication Date
July, 2016
Citation Information
Courtney Stout^*, Nicholas Dunham*, Chris Berndsen and Raymond A Enke. "Expression and Purification of Retina-Specific Transcription Factors CRX and NRL" James Madison University College of Science and Math Summer Symposium (2016) Available at: http://works.bepress.com/raymond_enke/77/