Rhodopsin targeted transcriptional silencing by DNA-binding
- Telethon Institute of Genetics and Medicine, Italy
- University of Bologna, Italy
- Second University of Naples, Italy
- University of Naples Federico II, Italy
Figures
Figure 1 with 1 supplement
Delivery of ZF6-DB DNA-binding synthetic trans-acting factor targeted to a 20 bp of RHO cis-acting regulatory element (CRE) dramatically reduces Rho expression in photoreceptors.
(a) Schematic representation of the chromosomal location of the RHO locus and its proximal promoter elements indicating the transcription start site (in green, +1) and the location of ZF6-DB binding …
Figure 1—figure supplement 1
Chromatin Immunoprecipitation (ChIP) of ZF6-DB and ZF6-KRAB.
(a) Alignment of RHODOPSIN proximal promoter between human, mouse and pig DNA sequences; in bold ZF6-cis sequence, underlined the sequence differences present in ZF6-cis bound by ZF6-DB. (b) qPCR …
Figure 2 with 2 supplements
Photoreceptor delivery of ZF6-DB resulted in reduced genome-wide transcript perturbations.
(a) RNA-Seq expression levels (Mean Normalized Counts) comparison between 2 endogenous TFs (Crx and Nrl) and the expression levels resulting from transduction of AAV8-CMV-ZF6-DB and …
Figure 2—figure supplement 1
Ingenuity Pathway Analysis on DEGs of ZF6-KRAB treated retina.
(a) Delivery of AAV8-CMV-ZF6-KRAB resulted in up-regulation of 10 genes associated with inflammatory responses (red, up-regulation) and the down-regulation (green) of 7 genes associated with the rod …
Figure 2—figure supplement 2
Determination of the binding constants of ZF6-KRAB and ZF6-DB.
(a) Gel mobility shift titrations of ZF6-KRAB and ZF6-DB with the hRHO 65 bp oligonucleotide (see 'Materials and methods'). (b) In the saturation binding experiments the nanomolar concentration of …
Figure 3
ZF6-DB DNA-binding protein preserves retinal function of the P347S adRP mouse model.
(a) Electroretinography (ERG) analysis on P347S mice mice subretinally injected at post natal day 14 (PD14) with AAV8-CMV-ZF6-DB (n=10), AAV8-CMV-ZF6-KRAB (n=10), or AAV8-CMV-eGFP (n=10) and …
Figure 4 with 3 supplements
DNA-binding repression-replacement (DBR-R) of Rho in the porcine retina.
(a) AAV8-RHOΔ-ZF6-DB-GNAT1-hRHO DBR-R construct features, including the two expression cassettes, RHOΔ-ZF6-DB encoding for both the DNA-binding repressor ZF6-DB (orange), and GNAT1-hRHO for human RHO…
Figure 4—figure supplement 1
Outline of the DNA-binding repressor-replacement (DBR-R) strategy.
The DNA-binding protein, ZF6-DB, for transcriptional silencing of RHO is coupled to replacement, R, in the same AAV vector (left hexagon) to ensure simultaneous transduction of photoreceptors. The …
Figure 4—figure supplement 2
Strength and tissue specificity of RHOΔ promoter elements in murine retina.
(a) qReal Time PCR, mRNA levels (2^-ΔCT) on the adult murine retina 15 days after vector delivery of either AAV8-hRHO short-eGFP or AAV8-hRHO-s-ΔZF6-eGFP subretinally administered at a dose of 1x109,…
Figure 4—figure supplement 3
Cone morphological integrity after DNA-binding repression-replacement (DBR-R) subretinal delivery.
Rod-specific expression of DBR-R (AAV8-RHOΔ-ZF6-DB-GNAT1-hRHO) 2 month after vector delivery. Immunofluorescence double staining with human cone Arrestin 3 (hCAR; green) and HA-ZF6-DB (red) …
