Rhodopsin (RHO) mutations cause autosomal recessive and autosomal dominant retinitis pigmentosa (arRP and adRP, respectively). Rho dominant mutations account for 25-50% of the total adRP and no treatment is currently available. Therefore, the development of therapeutic represents an urgent medical need. RHO is the most abundantly expressed gene in the retinal photoreceptors, accounting for about 90% of all proteins in the outer segment region. Rho transcriptional machinery including the epigenetics status is poorly known and explored thus far. In particular, key epigenetics signatures controlling histones methylation status of Rho locus are not yet investigated. As a first step towards the characterization of RHO locus epigenetics, we focus on Histone3 lysine9 trimethylation (H3K9me3), and histone3 lysine27 trimethylation, which represents well-established repressive marks. In order to assess H3K9me3 and H3K27me3 modifications into Rho locus we used the porcine retinae. As anticipated, considering the high levels of Rho transcriptional activity, we found that H3K9me3 modification is absent in the porcine Rho promoter. However, intriguingly, we found that the Rho gene body is progressively enriched in the H3K9me3 along its length (from 5' to 3' of the gene). This preliminary result is in agreement with the notion that the H3K9me3 signature is present in the gene body of highly expressed genes, promoting the precise transcriptional start and elongation steps through the positive interaction with RNA polymerase II elongation machinery. Conversely using H3K27me3 antibody we found a significant enrichment both on the promoter and on the gene body of rhodopsin. Regarding this unexpected result, we can hypothesize that H3K27me3 could be a repressive mark important for the repression of rhodopsin in non-rods cells of the retina. We are working to isolate rods from other cells of the retina and perform the same analysis on homogeneous sample. Besides the physiological epigenetic state of Rho, we are currently assessing the effects of targeting the RHO promoter with an engineered DNA-binding protein carrying an effector domain which induces transcriptional repression by epigenetic modifications, (Zinc-finger DNA-binding domain [ZF6] coupled to Kruppel-associated box domain, KRAB) towards the development of transcriptional gene silencers as therapeutics for retinal and non-retinal inherited diseases.

Exploit Transcriptional Machinery to Develop Therapeutics

Della Ragione F;Matarazzo MR;
2013

Abstract

Rhodopsin (RHO) mutations cause autosomal recessive and autosomal dominant retinitis pigmentosa (arRP and adRP, respectively). Rho dominant mutations account for 25-50% of the total adRP and no treatment is currently available. Therefore, the development of therapeutic represents an urgent medical need. RHO is the most abundantly expressed gene in the retinal photoreceptors, accounting for about 90% of all proteins in the outer segment region. Rho transcriptional machinery including the epigenetics status is poorly known and explored thus far. In particular, key epigenetics signatures controlling histones methylation status of Rho locus are not yet investigated. As a first step towards the characterization of RHO locus epigenetics, we focus on Histone3 lysine9 trimethylation (H3K9me3), and histone3 lysine27 trimethylation, which represents well-established repressive marks. In order to assess H3K9me3 and H3K27me3 modifications into Rho locus we used the porcine retinae. As anticipated, considering the high levels of Rho transcriptional activity, we found that H3K9me3 modification is absent in the porcine Rho promoter. However, intriguingly, we found that the Rho gene body is progressively enriched in the H3K9me3 along its length (from 5' to 3' of the gene). This preliminary result is in agreement with the notion that the H3K9me3 signature is present in the gene body of highly expressed genes, promoting the precise transcriptional start and elongation steps through the positive interaction with RNA polymerase II elongation machinery. Conversely using H3K27me3 antibody we found a significant enrichment both on the promoter and on the gene body of rhodopsin. Regarding this unexpected result, we can hypothesize that H3K27me3 could be a repressive mark important for the repression of rhodopsin in non-rods cells of the retina. We are working to isolate rods from other cells of the retina and perform the same analysis on homogeneous sample. Besides the physiological epigenetic state of Rho, we are currently assessing the effects of targeting the RHO promoter with an engineered DNA-binding protein carrying an effector domain which induces transcriptional repression by epigenetic modifications, (Zinc-finger DNA-binding domain [ZF6] coupled to Kruppel-associated box domain, KRAB) towards the development of transcriptional gene silencers as therapeutics for retinal and non-retinal inherited diseases.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.14243/280361
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