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S.S. Deeb, M. Dorschner, A. Shafer, T. Kutyavin, J. Stamatoyannopolous; Novel Regulatory Regions of the Human L/M Photopigment Gene Locus. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):654.
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Purpose: The genes encoding the long–wave–sensitive (L) and middle–wave–sensitive (M) cone photopigments are arranged in a head–to–tail tandem array on the long arm of the X–chromosome. These genes are expressed in cones in a mutually exclusive fashion. The regulatory mechanism by which this is accomplished is unknown. The purpose of this study is to delineate all cis–regulatory sequences of the L/M pigment gene locus. Methods: An "open" chromatin conformation appears to be necessary for high–level gene expression, and is associated with increased sensitivity of the involved DNA region to digestion by DNase I. We developed a novel high throughput and high–resolution method to map DNase I hypersensitive (HS) sites and used it on nuclei of the human retinoblastoma cell line WERI that expresses both the L and M pigment genes. Quantitative real–time PCR amplification of 250 bp amplicons was used to quantify the degree of DNase I sensitivity across 36.4 kb of the L/M locus. Results: We observed 5 HS sites in WERI cells: one coincides with the promoter of the L–pigment gene, the second lies within the previously described locus control region (LCR). Three additional novel HS sites were discovered: two are located within 4 kb 5' of the LCR and the third is located in intron 4. The sequence of all these HS sites has been determined. Phylogenetic sequence comparisons indicated significant conservation at all HS sites except for the one in intron 4. Conclusions: The L/M pigment gene locus contains at least 5 HS sites, the physical interaction of which may lead to the formation of a transcriptionally permissive chromatin domain. The sequence of these sites may lead to identification of bound transcription factor complexes involved in ensuring mutually exclusive expression of these genes and in determining the L/M cone ratio
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