Quantitative analysis of the ThrbCRM1-centered gene regulatory network

Enhancer 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 context as possible. Here we use...

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Bibliographic Details
Published in:Biology open 2019, Vol.8 (4)
Main Authors: Souferi, Benjamin, Emerson, Mark M
Format: Article
Language:English
Subjects:
Binding sites
Cis-regulatory elements
Cone photoreceptors
Electroporation
Flow cytometry
Gene regulatory networks
Mutagenesis
Otx2 protein
Quantitative analysis
Reporter assays
Retina
Retinal cells
Retinal development
Transcription factors
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Summary:Enhancer 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 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.
ISSN:2046-6390
2046-6390
DOI:10.1242/bio.039115