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Hyun-Jin Yang, Anand Swaroop; Molecular Mechanisms Underlying the Delay of Rhodopsin Expression in Immature Rods. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6005.
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The proper formation and function of rod photoreceptors largely depend on rhodopsin, a structural and phototransduction protein. Although differentiating rods begin to express transcription activators of rhodopsin shortly after their final mitosis, the onset of rhodopsin expression exhibits five to ten days of delay in mouse. The purpose of this study is to identify regulatory sequences and transcription repressor(s), which control rhodopsin expression in the developing retina.
Multi-species alignment was employed to identify conserved elements of the rhodopsin regulatory region. Luciferase reporter constructs were generated with a full length or a series of 5’ truncated rhodopsin regulatory region. P0 mouse retinas were then electroporated in vivo with each rhodopsin reporter gene, and the reporter activity was assessed either at P2 or P6, before or after the normal onset of rhodopsin expression.
The regulatory sequences that negatively regulate rhodopsin expression in immature rod precursors were identified using in vivo reporter gene assays. Five conserved elements including the previously characterized rhodopsin proximal promoter region and rhodopsin enhancer region were identified by in silico analysis. In vivo reporter gene assays show that a 100 bp sequence located about 1.5 kb upstream of the rhodopsin transcription start site negatively regulates reporter gene expression at P2 but not at P6. In silico analysis and gene expression profile analysis were employed to identify candidate transcriptional repressors, which have a binding site within the 100 bp region and are down-regulated after the onset of rhodopsin expression. The transcription factors that meet both criteria are now being validated by biochemical analysis.
Our data show that a 100 bp region about 1.5 kb upstream of transcription start site imposes a negative regulatory effect on rhodopsin expression selectively at P2. Our findings should provide insights into mechanisms that trigger maturation of rod cells.
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