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H. Hao, D. S. Kim, K. Johnson, C. Zang, K. Cui, J. Gregorski, F. Yang, K. Zhao, A. Swaroop; Global Target Analysis of Nrl, the Key Transcriptional Regulator of Photoreceptor Differentiation and Homeostasis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5952.
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Rod and cone photoreceptors are highly metabolically active post-mitotic neurons that are susceptible to changes in gene expression or function. The neural retina leucine zipper (NRL), a transcription factor of the Maf-family, is specifically expressed in the rod photoreceptors and the pineal gland. NRL plays a key role in regulating photoreceptor development and homeostasis. Abnormal NRL expression or activity can lead to changes in gene regulation and consequently blinding diseases. Understanding the NRL transcriptional "targetome" that dictates photoreceptor development and homeostasis will provide insights into the etiology of retinal degenerative diseases and may suggest targets for therapeutic interventions. Our goal is to identify direct NRL target genes in adult mouse retina.
NRL target genes were identified by chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-Seq) using both Illumina and ABI SOLID sequencing platforms. Candidate targets were validated by ChIP-qPCR. Their physiological relevance is being examined by sub-retinal injection of siRNA and in vivo electroporation.
Through ChIP-Seq, we identified 5041 significant peaks for in vivo NRL binding sites in genomic DNA from adult mouse retina. ChIP-qPCRs were performed for 20 gene regions to confirm NRL binding to the ChIP-seq peak regions. The ChIP-seq peaks are significantly enriched in the proximal promoter regions (14.41% for Illumina and 20.32% for ABI) as the proximal promoter regions account for 2.3% of the mouse genome. ChIP-seq peaks were also enriched in sequences similar to known NRL binding motifs. However, our ChIP-seq peak sequences further refined the NRL binding motif. We found that 302 genes that were altered in Nrl-/- mouse retina have NRL binding ChIP-Seq peaks within 10kb from transcription start site. Among these, 203 were down-regulated and 99 were up-regulated. We are evaluating NRL-binding DNA fragments as enhancers in transfected HEK 293 T cells. We are testing the functional pertinence of 15 candidate genes by sub-retinal injection of siRNA and in vivo electroporation.
Genome-wide analysis of NRL targets in the mouse retina will contribute to establishing transcriptional regulatory networks that dictate photoreceptor development and function.
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