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Identification and characterization of early photoreceptor cis-regulatory elements and their relation to Onecut1
Nathalie Jean-Charles1,2, Diego F Buenaventura1,3, Mark M Emerson4,5
1Department of Biology, The City College of New York, City University of New York, New York, NY, 10031, USA.
Background:
Cone and rod photoreceptors are two of the primary cell types affected in human retinal disease. Potential strategies to combat these diseases are the use of gene therapy to rescue compromised photoreceptors or to generate new functional photoreceptors to replace those lost in the diseased retina. Cis-regulatory elements specific to cones, rods, or both types of photoreceptors are critical components of successful implementation of these two strategies. The purpose of this study was to identify and characterize the cell type specificity and activity of cis-regulatory elements active in developing photoreceptors.
Methods:
Cis-regulatory elements were introduced into the developing chicken and mouse retina by electroporation. Characterization of reporter activity in relation with cell type markers was determined using confocal microscopy. In addition, two high-throughput flow cytometry assay were developed to assess whether these elements were downstream of Onecut1 in the photoreceptor specification network.
Results:
The majority of cis-regulatory elements were active in both cone and rod photoreceptors and were largely uninfluenced by a Onecut1 dominant-negative construct. Elements associated with the Thrb, Nr2e3, and Rhodopsin genes showed highly enriched activity in cones or rods, and were affected by interference in Onecut1 signaling. Rhodopsin promoter activity was the most highly influenced by Onecut1 activity and its induction could be modulated by the Maf family transcription factor L-Maf. Nr2e3 elements were observed to have activity in cone photoreceptors and Nr2e3 protein was expressed in developing cone photoreceptors, suggesting a role for this predominant rod gene in cone photoreceptor development.
Conclusions:
The analysis presented here provides an experimental framework to determine the specificity and strength of photoreceptor elements within specific genetic networks during development. The Onecut1 transcription factor is one such factor that influences the gene regulatory networks specific to cones and rods, but not those that are common to both.
Insights
Researchers identified cis-regulatory elements in developing photoreceptors for gene therapy applications. Specific elements for cone and rod cells were characterized, revealing insights into the Onecut1 transcription factor
Area of Science:
- Retinal cell biology
- Molecular genetics
- Gene therapy
Background:
- Cone and rod photoreceptors are key cell types affected in human retinal diseases.
- Gene therapy strategies aim to rescue or replace these photoreceptors.
- Cell type-specific cis-regulatory elements are crucial for targeted gene therapy.
Purpose of the Study:
- To identify and characterize cis-regulatory elements active in developing photoreceptors.
- To determine the cell type specificity (cone vs. rod) of these elements.
- To understand their role in photoreceptor development and gene regulatory networks.
Main Methods:
- Electroporation of cis-regulatory elements into developing chicken and mouse retinas.
- Confocal microscopy to analyze reporter activity and cell type markers.
- Development of high-throughput flow cytometry assays to assess Onecut1 network involvement.
Main Results:
- Most identified cis-regulatory elements were active in both cone and rod photoreceptors.
- Elements linked to Thrb, Nr2e3, and Rhodopsin genes showed specific activity in cones or rods.
- Rhodopsin promoter activity was highly influenced by Onecut1, modulated by L-Maf; Nr2e3 elements showed cone activity.
Conclusions:
- An experimental framework was established to assess photoreceptor element specificity and strength.
- The Onecut1 transcription factor specifically influences cone and rod gene regulatory networks, not common ones.
- Findings support the development of targeted gene therapies for retinal diseases affecting photoreceptors.
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