Proteins called transcription factors bind to sections of DNA known as regulatory elements to activate or deactivate nearby genes. In animals, transcription factors typically have two sections: a “DNA-binding domain” that attaches to DNA, and an “effector domain” that is responsible for interacting with other proteins to regulate the gene’s expression.

Rhodopsin is a gene that encodes the instructions needed to make a light-sensitive protein in the eyes of humans and other animals. Botta et al. have now used this gene as an example to investigate whether proteins that contain a DNA-binding domain – but not an effector domain – can repress gene expression.

The experiments show that only a small section of the regulatory elements in the human Rhodopsin gene is actually required for the gene to be expressed. Botta et al. designed an artificial protein – referred to as ZF6-DB – that is able to bind to this section of DNA. The binding of ZF6-DB to this short DNA section was sufficient to switch off a Rhodopsin gene in living pig cells, and, unlike conventional transcription factors, seemed to have minimal impact other genes.

Next, Botta et al. used a virus to insert both the gene that encodes ZF6-DB and a normal copy of Rhodopsin into pigs. In these animals, ZF6-DB switched off the existing copy of Rhodopsin, but not the inserted copy so the cells produced a working form of the light-sensitive protein. Further experiments were carried out in mice that have both a faulty version and a normal copy of the Rhodopsin gene. ZF6-DB switched off the faulty Rhodopsin gene, which allowed the normal Rhodopsin gene to work without any interference from the faulty copy.

Mutations in Rhodopsin can cause an eye disease that leads to severe loss of vision in humans. These new findings could now guide future efforts to develop treatments for people with this condition. It will also be important to investigate how ZF6-DB binds to the regulatory elements in the Rhodopsin gene and whether a similar strategy could be used to alter the expression of other genes.

Transcription factors (TFs) operate by entangling their DNA-binding and transcriptional activation or repression functions (Ptashne, 2014). However, in eukaryotes TF DNA binding and effector activities are typically structurally modular (Brent, 1985) consisting of a DNA-binding domain (DBD) controlling the TF topology on genomic targets and an effector domain (ED) (Brent, 1985; Kadonaga, 2004) that recruits co-activator or co-repressor complexes (Malik and Roeder, 2010; Perissi et al., 2010) resulting in either transcriptional activation or repression of gene regulatory networks (GRNs) (Neph et al., 2012). Engineered TFs mimic the design of natural TFs (Pavletich and Pabo, 1991; Beerli and Barbas, 2002). To generate target specificity the DBD module is engineered to recognize unique genome sites (Beerli and Barbas, 2002), whereas the transcriptional activation or repressor properties are conferred by the selection of the ED (Konermann et al., 2013). To silence gain-of-function mutations, while studying the features of genomic DNA-TF interactions, here we investigated the hypothesis that engineered DNA-binding proteins without canonical ED activity possess transcriptional repression properties. As a transcriptional repression target we selected the G-protein-coupled Receptor Rhodopsin (RHO) gene whose gain-of-function mutations are those most commonly associated with autosomal dominant retinitis pigmentosa (adRP), an incurable form of blindness (Dryja et al., 1990).

We generated a DNA-binding protein targeted to a cis-regulatory element (CRE) of the human proximal RHO promoter region by deconstructing an engineered TF (synthetic) composed of a DBD (ZF6-DNA-binding protein, ZF6-DB) and the ED (Kruppel-associated box, KRAB repressor domain, KRAB), which we have shown to be effective in repressing specifically the human RHO transgene carried in an adRP mouse model (Mussolino et al., 2011a). The deletion of the ED resulted in a protein, ZF6-DB targeting 20 base pairs of genomic CRE, here named ZF6-cis, found at -84 bp to -65 bp from the transcription start site (TSS) of the human RHO gene (Figure 1a; Mitton et al., 2000). Genomic ZF6-cis is without apparent photoreceptor-specific endogenous transcription factor-binding sites (TFBS; Figure 1a), as reported (Kwasnieski et al., 2012). To study the CRE features of ZF6-cis that ZF6-DB would interfere with upon binding in the absence of KRAB-mediated co-repressor recruitment, we deleted the 20 bp genomic ZF6-cis sequence and assessed its function by eGFP reporter assay (Kwasnieski et al., 2012) in living porcine retina by AAV delivery. The 776 bp-long RHO promoter fragment carrying the ZF6-cis deletion upstream of the eGFP reporter gene (AAV-RHO-cis-del-EGFP), after delivery to the porcine retinal photoreceptor, showed loss of eGFP expression compared to the control vector (AAV-RHO-EGFP) (Figure 1b,c). This suggests that ZF6-cis CRE is necessary for RHO expression (at least for the 776 bp region used in the assay) and that binding of the synthetic ZF6-DB trans-acting counterpart of ZF6-cis, may indeed repress RHO transcription.

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Chromatin immunoprecipitation (ChIP) experiments to evaluate binding to the ZF6-cis target genomic sequence showed occupancy by the DNA-binding protein ZF6-DB (Figure 1—figure supplement 1b). To evaluate whether ZF6-DB represses transcription of the RHO gene in a physiological genomic context, we used the porcine retina (Mussolino et al., 2011b), which shares 19 out of 20 DNA bp with the human genomic ZF6-cis sequence (Figure 1—figure supplement 1a). Subretinal delivery of a low AAV8 vector dose (1x10¹⁰ genome copies; gc) of ZF6-DB (AAV8-CMV-ZF6-DB) resulted in a 45% decrease of porcine Rho transcript levels at 15 days post-injection (Figure 1d). Immunofluorescence analysis showed depletion of Rho protein and consequent collapse of the rod outer segments in ZF6-DB positive cells (Figure 1e,f). Despite the lack of detectable Rho expression in most of the transduced rods, rows of photoreceptor nuclei were preserved from degeneration at this time point (Figure 1e). To further evaluate the extent of Rho silencing in rod photoreceptors by ZF6-DB, we performed FACS analysis on eGFP-labelled rod cells. Rod cells were isolated from porcine retina that had received a subretinal injection of an AAV vector encoding eGFP under the control of a rod-specific promoter (human Guanine Nucleotide Binding Protein1, GNAT1 promoter elements (Lee et al., 2010); AAV8-GNAT1-eGFP; dose 1x10¹² gc) with or without the vector encoding ZF6-DB (5x10¹⁰ gc). Fifteen days after injection, the retina were disaggregated and FACS-sorted. The retina co-transduced with eGFP and ZF6-DB vector showed virtually a 'somatic knock-out' of Rho expression (~85% decrease of Rho transcript levels; Figure 1g,h).

To evaluate genome–wide transcriptional specificity, we analyzed the porcine retinal transcriptome by RNA sequencing (RNA-Seq) from retina harvested 15 days after subretinal injection of the AAV8 vector encoding ZF6-DB (Figure 2). For comparison we used the engineered TF with the ED, KRAB (AAV8-CMV-ZF6-KRAB). The low vector doses delivered to the porcine retina (1x10¹⁰ gc) resulted in about twenty-fold lower expression levels of the ZF6-DB and ZF6-KRAB transgenes compared to Crx and Nrl, two retina-specific TFs (Swaroop et al., 2010) (Figure 2a). Of note, despite these low expression levels, we observed robust Rho transcriptional repression (Figure 2b). We then analyzed the transcriptional perturbation in response to the AAV retinal gene transfer of ZF6-DB by determining the differentially expressed genes (DEGs). Remarkably, in vivo the ZF6-DB protein generated about ten-fold less transcriptional perturbation compared with the ZF6-KRAB protein (19 vs. 222 DEGs; Figure 2e). Notably, this magnitude of perturbation is twenty five-fold lower than that induced by the ablation of an endogenous rod-specific TF (NRL, 500 DEGs vs 19 DEGs, ZF6-DBD; [Roger et al., 2014]). Retinal-specific pathway analysis of DEGs showed that ZF6-DB–induced down-regulation is restricted to the Rho biochemical interactor Gnat1 (Palczewski, 2012), and the up-regulation of 2 genes associated with acute phase inflammatory response, alpha-2-macroglobulin (A2m) and glial fibrillary acidic protein (Gfap) (Figure 2c). ZF6-KRAB induced the de-regulation of 17 retinal network associated genes (Figure 2—figure supplement 1). These results suggest that both ZF6-DB and the consequent Rho down-regulation marginally interferes with photoreceptor specific pathways, apart from Gnat1 repression, and that the up-regulation of the inflammatory response genes may be due to the collapse of the retinal scaffold caused by Rho depletion. The intersection of retinal transcriptome changes between ZF6-DB and ZF6-KRAB showed that both drive similar perturbation in the expression of 16 genes, which represent 84% of the entire pool of ZF6-DB DEGs (Figure 2e). Consistently, both ZF6-DB and ZF6-KRAB generated similar functional effects, i.e. concordant up- or down- differential expression of these 16 shared genes (Figure 2d). These results suggest that both ZF6-DB and ZF6-KRAB bind to similar genomic targets. We next studied whether the differential transcriptional repression induced by ZF6-DB and ZF6-KRAB was due to similar biochemical binding properties for the ZF6-cis DNA target. Both ZF6-DB and ZF6-KRAB proteins bind the ZF6-cis RHO DNA target site with similar affinities (Figure 2—figure supplement 2). These data suggest that, despite the presence of an active canonical repressor domain, ZF6-KRAB generated Rho silencing by DNA binding. Indeed, ZF6-DB, being exclusively a DNA-binding protein identical to the DBD of ZF6-KRAB, showed similar Rho silencing effects but far less retinal transcriptional perturbations. This indicates that the specificity is conferred by both the engineered design of the DNA-binding on a genome-specific target (Beerli and Barbas, 2002) and the lack of the ED. In addition, this finding supports the notion that that ZF6-cis CRE is necessary for Rho expression genome-wide.

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To test the functional activity of ZF6-DB in an adRP animal model, we used the RHO-P347S mouse (Li et al., 1996), which harbors the P347S RHO human mutant allele including the 20 DNA base pairs of the human genomic ZF6-cis sequence, whereas the murine Rho promoter sequence lacks the ZF6-Cis target (Figure 1—figure supplement 1a) (Li et al., 1996). Therefore, human-specific P347S RHO silencing by ZF6-DB (which does not affect murine Rho expression) may result in preservation of retinal function (Mussolino et al., 2011a). Strikingly, AAV8 vector delivery of ZF6-DB resulted in significantly higher functional protection in both the A- and B-wave components of electroretinography (ERG analysis) compared to ZF6-KRAB and AAV-GFP controls (Figure 3a). In addition, injection of either ZF6-KRAB or ZF6-DB in C57Bl/6 wild type retina did not produce detectable functional detrimental effects (Figure 3b). Thus, the higher ERG responses observed in ZF6-DB- compared to ZF6-KRAB-treated P347S mice should be further investigated.

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To determine the therapeutic potential of DNA-binding-mediated silencing, we carried out the silencing-replacement strategy (Kiang et al., 2005) by coupling ZF6-DB with RHO replacement (human RHO, hRHO CDS) in order to complement Rho transcriptional repression in porcine retina (Figure 4—figure supplement 1). To achieve simultaneous photoreceptor transduction of both ZF6-DB and hRHO, we cloned the two expression cassettes into a single vector (DNA-binding repression and replacement, DBR-R, construct; Figure 4a). The key variables to achieve highly differential expression required for balanced simultaneous RHO repression and replacement are the vector dose and promoter strength. Indeed, ZF6-DB (~200 counts; RNA-seq expression levels) generated a decrease of about 100,000 Rho RNA-seq counts after transduction (~250,000 counts in controls vs. ~150,000 after treatment; Figure 2a,b). Thus, to ensure high and rod-specific hRHO replacement, we opted for a high vector dose and the strength of the GNAT1 promoter (Figure 1h; [Lee et al., 2010]). To decrease ZF6-DB expression levels at high vector dose, while keeping rod-specificity, we both shortened the human RHO promoter and deleted the 5’ sequence of the ZF6-DB target ZF6-Cis (Figure 4—figure supplement 2). We used 1x10¹² gc of vector of DBR-R (AAV8-RHOΔ-ZF6-DB-GNAT1-hRHO) to administer to porcine retina. As an internal control the contralateral eye received the previously used ZF6-DB vector (AAV8-CMV-ZF6-DB; at 1x10¹⁰ gc; Figure 1). AAV8-CMV-eGFP was co-administrated to label the transduced area. Administration of the DBR-R vector resulted in rod-specific transcriptional repression of the porcine Rho (38%) and in concomitant replacement of the exogenous hRHO (68%), as assessed by transcripts, protein expression levels, and integrity of photoreceptor outer segments (Figure 4b–d and Figure 4—figure supplement 3). Notably, Gnat1 transcript and protein (data not shown) levels demonstrated complementation, supporting a secondary down-regulation of Gnat1 associated with Rho repression (Figure 4b).

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In this study, we showed that photoreceptor genomic binding of a 20 bp-long DNA sequence by a synthetic DNA-binding protein dramatically reduces Rho expression. The combination of Rho transcriptional silencing and the restricted transcriptome perturbation induced by ZF6-DB, without the intrinsic repression activity contained in an ED, indicate that the local binding to ZF6-cis per se is the determinant of transcriptional repression, whereas the high specificity observed may result by both DNA-binding specificity (biochemical affinity) of ZF6-DB and the rod genomic context. The transcriptional repression mechanism of ZF6-DB binding likely relies on the interference occurring between TFs and local DNA sequence features within the RHO proximal promoter region (Mitton et al., 2000), which we showed here to be necessary to control Rho expression at the genomic level. The lack of known TFBSs and the low level of expression of ZF6-DB expressed in the photoreceptors, which was twenty-fold below the levels of photoreceptor specific TFs (Crx and Nrl), suggest that the molecular determinant of silencing may not be the simple displacement of key RHO TFs (Mao et al., 2011). We propose a model in which the molecular features of the DNA (loop, twisting, bending, for instance) may contribute to Rhodopsin transcriptional output. In this context, the DNA may be envisaged as not being exclusively the source of storage of functional information (protein coding and non-coding transcripts) or an inert DNA-binding protein harbor (i.e. positional information for TFs DNA binding), but also as an intrinsically active operator of the transcriptional function. This contribution of DNA is supported by both the transcriptional repression upon the ZF6-cis deletion of 20 bp of DNA and intereference in trans by using a synthetic DNA binding protein that, not being encoded by the genome, may occupy a protein-free portion of the genome. It follows that in terms of signaling, the information source, the DNA, generates an output signal the RNA and eventually a protein (TF), whose final output functional activity is completed back by the DNA. Thus the information source, the DNA, becomes also integral part of the signaling (Rhodopsin transcriptional output). In this vision, to act (interfere) upstream the DNA, an external function is necessary, which is, in this study, the synthetic ZF6-DB carried by the vector.

From a therapeutic prospective, a relevant property of ZF6-DB DNA binding interference is the high rate of transcriptional silencing observed after in vivo gene transfer, which is consistent with canonical TFs mode of action (Kiang et al., 2005). DNA binding interference via ZF6-DB in transduced retina generated 45% Rho transcriptional repression, which reached 85% when rods were sorted, supporting its use for diseases requiring correction of a large number of affected cells, such as adRP and other Mendelian disorders due to gain-of-function mutations. Furthermore, Rho transcriptional silencing and its complete RHO replacement support in principle the use of the DBR-R constructs for treatment of any RHO mutation including those caused by a dominant negative mechanism (Mao et al., 2011). However, the 38% silencing and replacement observed may not yet be sufficient to achieve therapeutic efficacy/benefit in patients with adRP. Therefore, further development of this proof-of-concept will include optimization of the design of the silencing and replacement double construct (tuning the strength of the promoter elements), vector selection and dose, and the surgical approach. In conclusion, in vivo retinal gene transfer of an AAV vector (Doria et al., 2013; Liang et al., 2001; Scatchard, 1949) carrying a 22 kDa orthogonal (Surace et al., 2005) and gene-extrinsic noise-resistant (the permissive rod photoreceptor cell-specific environment; [Li et al., 2011]) synthetic protein acts as a transcriptional repressor, which results in a potent and specific silencing of the Rho gene upon binding to an essential Rho DNA element.

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Author details

1. Salvatore Botta

   Telethon Institute of Genetics and Medicine, Napoli, Italy

   Contribution

   SB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Elena Marrocco

   Telethon Institute of Genetics and Medicine, Napoli, Italy

   Contribution

   EM, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Nicola de Prisco

   Telethon Institute of Genetics and Medicine, Napoli, Italy

   Contribution

   NdP, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Fabiola Curion

   Telethon Institute of Genetics and Medicine, Napoli, Italy

   Contribution

   FC, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Mario Renda

   Telethon Institute of Genetics and Medicine, Napoli, Italy

   Contribution

   MR, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

6. Martina Sofia

   Telethon Institute of Genetics and Medicine, Napoli, Italy

   Contribution

   MS, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

7. Mariangela Lupo

   Telethon Institute of Genetics and Medicine, Napoli, Italy

   Contribution

   ML, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

8. Annamaria Carissimo

   Telethon Institute of Genetics and Medicine, Napoli, Italy

   Contribution

   AC, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

9. Maria Laura Bacci

   Department of Veterinary Medical Sciences, University of Bologna, Bologna, Italy

   Contribution

   MLB, Coordinated the in vivo studies in large animals

   Competing interests

   The authors declare that no competing interests exist.

10. Carlo Gesualdo

    Multidisciplinary Department of Medical, Surgical and Dental Sciences, Eye Clinic, Second University of Naples, Naples, Italy

    Contribution

    CG, Assisted the subretinal injections in large animals

    Competing interests

    The authors declare that no competing interests exist.

11. Settimio Rossi

    Multidisciplinary Department of Medical, Surgical and Dental Sciences, Eye Clinic, Second University of Naples, Naples, Italy

    Contribution

    SR, Performed subretinal injections in large animals

    Competing interests

    The authors declare that no competing interests exist.

12. Francesca Simonelli

    Multidisciplinary Department of Medical, Surgical and Dental Sciences, Eye Clinic, Second University of Naples, Naples, Italy

    Contribution

    FS, Supervised the subretinal injections and provided reagents for large animal studies

    Competing interests

    The authors declare that no competing interests exist.

13. Enrico Maria Surace

    1. Telethon Institute of Genetics and Medicine, Napoli, Italy
    2. Department of Translational Medicine, University of Naples Federico II, Naples, Italy

    Contribution

    EMS, Conception and design, Drafting or revising the article

    For correspondence

    surace@tigem.it

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-2975-942X

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