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SK Swamynathan, J Piatigorsky; Orientation-Dependency and Silencing of an Inter-Genic Enhancer in the Divergently Transcribed Mouse B-Crystallin and MKBP/HSPB2 Genes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2442.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Mouse αB-crystallin is expressed at high levels in the lens, at moderate levels in the skeletal muscle and heart, and at trace levels in several other tissues. The related MKBP/HSPB2 gene is located just 1kb upstream of the αB-crystallin gene and is divergently transcribed. MKBP/HSPB2 is expressed at moderate levels in the skeletal muscle and heart, and is not detectable in the lens. Here, we have attempted to assess the contribution of the inter-genic αB-crystallin enhancer to divergent transcription of these two related genes. Methods: Renilla and firefly luciferase reporter genes under the control of αB-crystallin or MKBP/HSPB2 promoters containing an intact, inverted or deleted enhancer (-258bp to –436bp, relative to transcriptional start site of αB-crystallin gene) were tested by transfections in αTN4 mouse lens epithelial cells, N/N1003 rabbit lens epithelial cells, and C2C12 mouse myoblasts. Enhancer blocking assays were performed to test the insulating ability of the –519bp to –836bp fragment by stable transformation of K562 erythroleukemia cells. Results: Deletion of the enhancer resulted in loss of transcription from both αB-crystallin and MKBP/HSPB2 promoters. When the enhancer was inverted, transcription from the αB-crystallin promoter was reduced by about 90%, while the transcription from the MKBP/HSPB2 promoter was reduced only by 20%. Enhancer blocking assays showed a four to five fold reduction in the activity of the enhancer when the –519bp to –836bp fragment was introduced between the mouse globin HS2 enhancer and the γ-globin promoter driving the NeoR gene. Conclusion: The inter-genic enhancer between αB-crystallin and MKBP/HSPB2 genes is orientation-dependent in its natural context, with regard to its ability to influence transcription from αB-crystallin promoter. MKBP/HSPB2 promoter is protected further from the influence of the strong αB-crystallin enhancer by the insulator between the MKBP/HSPB2 proximal promoter and the αB-crystallin enhancer.
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