Identification and characterization of a dlx2b cis-regulatory element both sufficient and necessary for correct transcription during zebrafish tooth development

Abstract

Much remains to be learned about how cis-regulatory elements such as enhancers function, especially during vertebrate organ development. To increase knowledge in this area, we have examined the cis-regulation of the transcription factor dlx2b during zebrafish larval tooth formation. We have created a GFP knock-in line that replicates dlx2b expression during tooth development and have also isolated a minimal enhancer of dlx2b (MTE1) sufficient for activating most of the tooth germ expression pattern. We have found that four evolutionarily conserved predicted transcription factor binding sites are required for the function of this minimal enhancer in both contexts. When the conserved sequences are mutated in a transgene it eliminates the activity of the enhancer and when they are deleted at the dlx2b locus it causes a dramatic alteration in the expression pattern. We hypothesize that disabling this enhancer at the dlx2b locus may be enabling other nearby cis-regulatory elements to take control of the promoter. These experiments reveal details of how cis-regulatory elements are working to control gene expression during organogenesis and highlight how much remains to be learned by empirical studies of gene regulation.