Abstract
ABSTRACTEnhancer activity is determined by both the activity and occupancy of transcription factors as well as the specific sequences they bind. Experimental investigation of this dynamic requires the ability to manipulate components of the system, ideally in as close to an in vivo context as possible. Here we use electroporation of plasmid reporters to define critical parameters of a specific cis-regulatory element, ThrbCRM1, during retinal development. ThrbCRM1 is associated with cone photoreceptor genesis and activated in a subset of developing retinal cells that co-express the Otx2 and Onecut1 (OC1) transcription factors. Variation of reporter plasmid concentration was used to generate dose response curves and revealed an effect of binding site availability on the number and strength of cells with reporter activity. Critical sequence elements of the ThrbCRM1 element were defined using both mutagenesis and misexpression of the Otx2 and OC1 transcription factors in the developing retina. Additionally, these experiments suggest that the ThrbCRM1 element is co-regulated by Otx2 and OC1 even under conditions of sub-optimal binding of OC1.
Highlights
The rules and logic of cis-regulatory activity that underlie dynamic gene regulation during development are an area of great interest (Rickels and Shilatifard, 2018)
Cis-regulatory elements are a critical component of gene regulatory networks (GRNs) that have proven difficult to analyze at the quantitative level
This study shows that the concentration of reporter plasmid is an important variable in experimental design, though this aspect has been ignored by the vast majority of previously published electroporation studies
Summary
The rules and logic of cis-regulatory activity that underlie dynamic gene regulation during development are an area of great interest (Rickels and Shilatifard, 2018). Quantitative measurements are largely determined through highly reductionist approaches such as EMSAs or protein microarrays, while in vivo activity is qualitative, limiting the ability to correlate specific sequence elements with reporter output. Elucidation of this process during development is further complicated by temporal dynamics as cells have rapid shifts in active gene regulatory networks (GRNs). Identification of these networks provides insights into how transcription factor expression and activation are coordinated to direct cell fate choices during development.
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