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Identification and Characterization of Cis-Regulatory Elements for Photoreceptor-Type-Specific Transcription in ZebraFish

  • Wei Fang
  • Yi Wen
  • Xiangyun WeiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2092)

Abstract

Tissue-specific or cell-type-specific transcription of protein-coding genes is controlled by both trans-regulatory elements (TREs) and cis-regulatory elements (CREs). However, it is challenging to identify TREs and CREs, which are unknown for most genes. Here, we describe a protocol for identifying two types of transcription-activating CREs—core promoters and enhancers—of zebrafish photoreceptor type-specific genes. This protocol is composed of three phases: bioinformatic prediction, experimental validation, and characterization of the CREs. To better illustrate the principles and logic of this protocol, we exemplify it with the discovery of the core promoter and enhancer of the mpp5b apical polarity gene (also known as ponli), whose red, green, and blue (RGB) cone-specific transcription requires its enhancer, a member of the rainbow enhancer family. While exemplified with an RGB-cone-specific gene, this protocol is general and can be used to identify the core promoters and enhancers of other protein-coding genes.

Key words

Gene expression regulation Transcription factors CREs Cis-regulatory elements Bioinformatics Zebrafish Teleost Photoreceptor Retina Apical polarity genes ponli nagie oko mpp5a mpp5b 

Notes

Acknowledgments

This work is supported by the National Institutes of Health (P30EY008098; EY025638; R21EY023665) as well as by the grants to the Department of Ophthalmology of the University of Pittsburgh from the Eye and Ear Foundation of Pittsburgh and Research to Prevent Blindness. The authors declare no competing financial interests. We thank Ms. Lynne Sunderman for proofreading the manuscript.

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Authors and Affiliations

  1. 1.Department of OphthalmologyUniversity of Pittsburgh School of MedicinePittsburghUSA
  2. 2.Department of Developmental BiologyUniversity of Pittsburgh, School of MedicinePittsburghUSA
  3. 3.Department of Microbiology and Molecular GeneticsUniversity of Pittsburgh, School of MedicinePittsburghUSA

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