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| Hereditary retinal degeneration associated with CRX gene mutation interference with the transcriptional expression of rhodopsin and NRL stability |
| ZHAO Na,LU Yasu,LU Qinkang,WANG Hongting,WANG Huiyun,TONG Qihu,ZHANG Xianning |
| Ophthalmology Center, Yinzhou People′s Hospital, Yinzhou Hospital Affiliated to Medical School of Ningbo University, Ningbo 315040, China; Institute of Biology, Zhejiang University, Hangzhou 310058, China |
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Abstract Objective To investigate the effect of cone-rod homeobox containing gene (CRX) mutation on the transcriptional expression of the rhodopsin gene and the stability of the neural retina leucine zipper (NRL) protein, both in the presence or absence of the NRL gene. Methods In this experimental study, different expression vectors carrying the wild-type (WT) or the c.C766T (p.Q256X) nonsense mutant CRX were constructed with the NRL and the bovine rhodopsin gene promoter sub-luciferase (pBR130-luc). The vectors were then transfected into 293T and ARPE-19 cell lines, respectively. Then, dual-luciferase assays and Western blotting analyses were performed. The housekeeping protein glyceraldehyde-3-phosphate dehydrogenase was used as an internal reference marker. Results The expression vector of CRX/WT caused a 5-fold increased activation of bovine rhodopsin promoter compared to a 25% decrease by CRX/c.C766T. Co-expression of CRX/WT and NRL increased activation by 30-fold compared to a 7.7% decrease by the co-expression of CRX/c.C766T and NRL. Also, expression of NRL protein increased with the co-expression of CRX/c.C766T and NRL. Conclusion The in vitro experiments suggest that the c.C766T (p.Q256X) mutant of the CRX gene down-regulated the transcriptional expression of rhodopsin and interfered with the homeostasis of CRX and NRL proteins.
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Received: 13 December 2016
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| Fund: Natural Science Foundation of Zhejiang Province (LY12H12001); Ningbo Social Livelihood Science and Technology Major Program (2014C50091); Zhejiang Provincial Medical and Health Science and Technology Project (2017KY616); Yinzhou District Science and Technology Project (Yinke 2013-90) |
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Corresponding Authors:
LU Qinkang, Email: 12556677@qq.com
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| [1] |
Furukawa T, Morrow EM, Cepko CL. Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation[J]. Cell,1997,91(4):531- 541.
|
| [2] |
Furukawa A, Koike C, Lippincott P, et al. The mouse Crx 5'-upstream transgene sequence directs cell-specific and developmentally regulated expression in retinal photoreceptor cells[J]. J Neurosci,2002,22(5):1640-1647.
|
| [3] |
Sorrentino FS, Gallenga CE, Bonifazzi C, et al. A challenge to the striking genotypic heterogeneity of retinitis pigmentosa: a better understanding of the pathophysiology using the newest genetic strategies[J]. Eye (Lond),2016. DOI:10.1038/eye.2016.197.
|
| [4] |
Farjo Q, Jackson AU, Xu J, et al. Molecular characterization of the murine neural retina leucine zipper gene, Nrl[J]. Genomics,1993,18(2):216-222. DOI:10.1006/geno.1993.1458.
|
| [5] |
Mears AJ, Kondo M, Swain PK, et al. Nrl is required for rod photoreceptor development[J]. Nat Genet,2001,29(4):447-452. DOI:10.1038/ng774.
|
| [6] |
Rehemtulla A, Warwar R, Kumar R, et al. The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression[J]. Proc Natl Acad Sci USA,1996,93(1):191-195.
|
| [7] |
Tran NM, Zhang A, Zhang X, et al. Mechanistically distinct mouse models for CRX-associated retinopathy[J]. PLoS Genet, 2014,10(2):e1004111. DOI:10.1371/journal.pgen.1004111.
|
| [8] |
Lu QK, Zhao N, Lv YS, et al. A novel CRX mutation by whole-exome sequencing in an autosomal dominant cone-rod dystrophy pedigree[J]. Int J Ophthalmol,2015,8(6):1112-1117. DOI:10.3980/j.issn.2222-3959.2015.06.06.
|
| [9] |
Viets K, Eldred KC, Johnston RJ. Mechanisms of Photoreceptor Patterning in Vertebrates and Invertebrates[J]. Trends Genet, 2016,32(10):638-659. DOI:10.1016/j.tig.2016.07.004.
|
| [10] |
Chaitankar V, Karakülah G, Ratnapriya R, et al. Next generation sequencing technology and genomewide data analysis: Perspectives for retinal research[J]. Prog Retin Eye Res,2016. DOI:10.1016/j.preteyeres.2016.06.001.
|
| [11] |
Choudhary M, Malek G. Rethinking Nuclear Receptors as Potential Therapeutic Targets for Retinal Diseases[J]. J Biomol Screen,2016. DOI:10.1177/1087057116659856.
|
| [12] |
Akagi T, Mandai M, Ooto S, et al. Otx2 homeobox gene induces photoreceptor-specific phenotypes in cells derived from adult iris and ciliary tissue[J]. Invest Ophthalmol Vis Sci,2004, 45(12):4570-4575. DOI:10.1167/iovs.04-0697.
|
| [13] |
Fei Y, Hughes TE. Nuclear trafficking of photoreceptor protein crx: the targeting sequence and pathologic implications[J]. Invest Ophthalmol Vis Sci,2000,41(10):2849-2856.
|
| [14] |
Gao M, Zhang S, Liu C, et al. Whole exome sequencing identifies a novel NRL mutation in a Chinese family with autosomal dominant retinitis pigmentosa[J]. Mol Vis,2016,22:234-242.
|
| [15] |
Brzezinski JA, Reh TA. Photoreceptor cell fate specification in vertebrates[J]. Development,2015,142(19):3263-3273. DOI: 10.1242/dev.127043.
|
| [16] |
Freund CL, Gregory-Evans CY, Furukawa T, et al. Cone-rod dystrophy due to mutations in a novel photoreceptor-specific homeobox gene (CRX) essential for maintenance of the photoreceptor[J]. Cell,1997,91(4):543-553.
|
| [17] |
Barrera LA, Vedenko A, Kurland JV, et al. Survey of variation in human transcription factors reveals prevalent DNA binding changes[J]. Science,2016,351(6280):1450-1454. DOI:10.1126/ science.aad2257.
|
| [18] |
Wang L, Shi P, Xu Z, et al. Up-regulation of VEGF by retinoic acid during hyperoxia prevents retinal neovascularization and retinopathy[J]. Invest Ophthalmol Vis Sci,2014,55(7):4276-4287. DOI:10.1167/iovs.14-14170.
|
| [19] |
Hamel CP. Cone rod dystrophies[J]. Orphanet J Rare Dis,2007,2:7. DOI:10.1186/1750-1172-2-7.
|
| [20] |
Lek M, Karczewski KJ, Minikel EV, et al. Analysis of protein-coding genetic variation in 60,706 humans[J]. Nature,2016,536(7616):285-291. DOI:10.1038/nature19057.
|
| [21] |
Nichols LL, Alur RP, Boobalan E, et al. Two novel CRX mutant proteins causing autosomal dominant Leber congenital amaurosis interact differently with NRL[J]. Hum Mutat,2010, 31(6):E1472-1483. DOI:10.1002/humu.21268.
|
| [22] |
Huang L, Xiao X, Li S, et al. Molecular genetics of cone-rod dystrophy in Chinese patients: New data from 61 probands and mutation overview of 163 probands[J]. Exp Eye Res,2016, 146:252-258. DOI:10.1016/j.exer.2016.03.015.
|
| [23] |
Tran NM, Chen S. Mechanisms of blindness: animal models provide insight into distinct CRX-associated retinopathies[J]. Dev Dyn,2014,243(10):1153-1166. DOI:10.1002/dvdy.24151.
|
|
|
|
