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Elife. 2016 Mar 14;5:e12242. doi: 10.7554/eLife.12242.

Rhodopsin targeted transcriptional silencing by DNA-binding.

Author information

1
Telethon Institute of Genetics and Medicine, Napoli, Italy.
2
Department of Veterinary Medical Sciences, University of Bologna, Bologna, Italy.
3
Multidisciplinary Department of Medical, Surgical and Dental Sciences, Eye Clinic, Second University of Naples, Naples, Italy.
4
Department of Translational Medicine, University of Naples Federico II, Naples, Italy.

Abstract

Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector-mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of-function mutations.

KEYWORDS:

AAV; gene expression; gene therapy; human biology; medicine; mouse; neurodegeneration; neuroscience; retina; rhodopsin; transcription; transcription factors; zinc finger

PMID:
26974343
PMCID:
PMC4805542
DOI:
10.7554/eLife.12242
[Indexed for MEDLINE]
Free PMC Article

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