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Janina Gregorski, Hong Hao, Douglas Kim, Bin Zhang, Anand Swaroop; Lman1, Encoding ER-Golgi Transport Protein ERGIC-53, is a Direct Transcriptional Target of the Photoreceptor Transcription Factor NRL. Invest. Ophthalmol. Vis. Sci. 2011;52(14):910.
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© ARVO (1962-2015); The Authors (2016-present)
The neural retinal leucine zipper (NRL), a bZIP transcription factor of the Maf subfamily, is a key regulator of rod photoreceptor differentiation and homeostasis. As transcripts of Lman1, encoding ERGIC-53, are decreased in Nrl-/- mice, we wanted to examine the regulation and function of Lman1 in rod photoreceptors.
Chromatin immunoprecipitation followed by real-time quantitative polymerase chain reaction (ChIP-qPCR) was used to detect interaction of Lman1 promoter sequenceswith NRL. Lman1 enhancer element, the genomic fragment bound by NRL in vivo, was cloned in front of a luciferase reporter gene. The enhancer construct was cotransfected with an empty vector or mNRL expression plasmid in HEK293T cells. Physiological relevance of Lman1 was tested by in vivo electroporation of Lman1 shRNA and examining the retina of Lman1-/- mice.
ChIP-qPCR detected in vivo association of NRL with Lman1 promoter sequences. Cotransfection of the Lman1 enhancer construct with mNRL expression plasmid showed a 2-fold increase in luciferase reporter activity in HEK293T cells compared to cells cotransfected with an empty vector. Knockdown of Lman1 expression by in vivo electroporation of shRNA resulted in an abnormal morphology of transfected retinal cells, including shorter photoreceptor outer segments and abnormal cell body location. The abnormal morphology could be rescued by cotransfection of shRNA-resistant Lman1 cDNA, suggesting that the phenotype is caused by the specific knockdown of Lman1 expression. No gross change in morphology has been observed in Lman1-/- retina at 5 weeks of age. We will be evaluating the retina of Lman1-/- mice at later time points by ERG and immunohistochemistry.
ChIP-qPCR and enhancer-reporter assays revealed that Lman1 is a novel, direct transcriptional target of NRL. The abnormal phenotype observed by in vivo knock down and cDNA rescue experiments suggest that Lman1 plays an important role in rod photoreceptor homeostasis.
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