Abstract
Purpose :
The retina-specific transcription factor, CRX, regulates photoreceptor gene expression and development. Mutations in the homeodomain lead to retinal degeneration. Here, we compared the binding kinetics of several CRX homeodomain mutants using biolayer interferometry (BLI) with their transcriptional activation.
Methods :
Human CRX homeodomain and mutants R41Q and R90W were expressed and purified in E. coli . BLI was used to measure protein-DNA interactions to DNA attached to sensor tips. Mutants were also prepared in full-length human CRX plasmids (pcDNA3.1) for assays in HEK293T cell transfections using PEI. Both the RHO and GNAT1 promoters were cloned into the pGL4 (Promega). Activity was measured using the Nano-Glo Dual-Luciferase Reporter Assay (Promega). Data was fit to saturable binding equations with 95% confidence intervals. Statistical comparisons were evaluated using two-way ANOVA (α = 0.05, n=3-4 for BLI and n=16-24 for transfections).
Results :
The kinetics of CRXHD binding to short DNA cis-regulatory sequences from the rhodopsin promoter (BAT1) bound rapidly with biphasic kinetics and dissociated quickly also with biphasic kinetics. Both R41Q and R90W had reduced binding to DNA. R41Q exhibited a single binding site with ~20-fold reduction in steady state apparent binding constant-both on and off rates were altered. R90W showed less severe effects on binding (~5-10-fold reduction in steady state) with a two-state model, both on and off rates were altered. Transfection activity showed that R41Q mutation had 2-4 fold reduced activation activity both by itself and in combination with NRL. By contrast, the CRX-R90W mutant had basal levels of activity by itself or in combination with NRL at all concentrations of mutant DNA transfected.
Conclusions :
These experiments show that while R41Q and R90W can bind to DNA, the R90W mutation renders the protein unable to activate transcription, suggesting a specific conformation(s) or orientation(s) of the homeodomain on cis-regulatory elements is essential for activating transcription. These results also indicate that such conformations could contribute to differential effects of disease-causing mutations in patients.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.