A mutation in ZNF513, a putative regulator of photoreceptor development, causes autosomal-recessive retinitis pigmentosa

Abstract

Retinitis pigmentosa (RP) is a phenotypically and genetically heterogeneous group of inherited retinal degenerations characterized clinically by night blindness, progressive constriction of the visual fields, and loss of vision, and pathologically by progressive loss of rod and then cone photoreceptors. Autosomal-recessive RP (arRP) in a consanguineous Pakistani family previously linked to chromosome 2p22.3-p24.1 is shown to result from a homozygous missense mutation (c.1015T>C [p.C339R]) in ZNF513, encoding a presumptive transcription factor. znf513 is expressed in the retina, especially in the outer nuclear layer, inner nuclear layer, and photoreceptors. Knockdown of znf513 in zebrafish reduces eye size, retinal thickness, and expression of rod and cone opsins and causes specific loss of photoreceptors. These effects are rescued by coinjection with wild-type (WT) but not p.C339R-znf513 mRNA. Both normal and p.C339R mutant ZNF513 proteins expressed in COS-7 cells localize to the nucleus. ChIP analysis shows that only the wild-type but not the mutant ZNF513 binds to the Pax6, Sp4, Arr3, Irbp, and photoreceptor opsin promoters. These results suggest that the ZNF513 p.C339R mutation is responsible for RP in this family and that ZNF513 plays a key role in the regulation of photoreceptor-specific genes in retinal development and photoreceptor maintenance.

Figure 1

ZNF513 Region, Pedigree, Gene, and Sequence (A) Haplotypes of the ZNF513 region of family 61115 showing the ZNF513 p.C339R mutation and surrounding microsatellite markers included in Table 1. The risk haplotype is shown in black. (B) Diagram of the ZNF513 gene showing the two nuclear localization signals (NLS), the p.C339R mutation, and the three zinc finger binding domains (ZF) with their positions. (C) Electropherograms show the affected sequence (left, individual 8), carrier sequence (middle, individual 12), and normal control sequence (right) surrounding the ZNF513 1015T>C mutation. (D) Amino acid sequence alignment around the ZNF513 C339 amino acid (red) in 31 species ranging from human to zebrafish.

Figure 2

Relative Expression of Znf513 in Mouse Eye Tissues at Various Ages and Distribution of ZNF513 mRNA in the Human Adult Retina (A) Expression of Znf513 (Zpf513) was measured in lens, ciliary body (CB), iris, cornea, optic nerve, retina, and RPE tissues by qRT-PCR at different time points during aging. Data represent the mean (±SD) on an arbitrary scale (y axis) representing expression relative to the housekeeping gene Rpl19 and were calculated from at least three independent experiments. The inset shows the expression pattern of Znf513 in the post natal-stage eye from P1 to P30. (B) In situ hybridization of ZNF513 probes in the retina of human eyes at 55°C–69°C. Hybridization with the antisense probe (left) shows signal (black arrows) in the ONL, INL, and GCL when contrasted to hybridization with the sense probe (right). Counter stain is nuclear fast red. RPE, retinal pigmented epithelia; OS, outer segment; ONL, outer nuclear layer; INL, inner nuclear layer; OPL, outer plexiform layer; IPL inner plexiform layer; GCL, ganglion cell layer.

Figure 3

Zebrafish Eye Development Disturbed by Knockdown of znf513 Expression and Rescue of znf513 Morphants Morpholino-sensitive 5′-modified EGFP mRNA (A, B, C) or native unmodified EGFP (D) was injected to single cell stage embryos. znf513-MO (B, D, F, I), MM-MO (C, G, H), or injection buffer (E) was injected into embryos. (A–D) Validation of the activity of znf513-MO on the expression of znf513. znf513-MO injection eliminated the fluorescence signal (green on the embryo body, arrowheads) from coinjected morpholino-sensitive 5′-modified EGFP mRNA (B), and it did not reduce the expression of native unmodified EGFP without the znf513 sequence (D). MM-MO had no effect on morpholino-sensitive GFP expression (C). Note that all embryos contain faint yellowish autofluorescence in the egg yolk not derived from EGFP. (E–G) 0.5 ng Znf513-MO (F) dramatically reduced eye size (black arrows) compared to MM-MO-injected (G) and buffer-injected (E) embryos. (H and I) Higher magnification of the heads from embryos shown in (G) and (F) showing reduced size of the retina in a znf513-MO-injected embryo relative to a MM-MO-injected embryo (l, lens; r, retina, white arrows). (J) Graph depicting proportions of embryos with abnormal phenotype (eye size) associated with znf513-MO injection and rescue by coinjected znf513 mut and WT mRNA. (A–D) 24 hpf, (E–I) 37 hpf. Scale bars represent 1 mm in (A)–(G) and 100 μm in (H) and (I).

Figure 4

Inhibition of Cone and Rod Photoreceptor Development by Knockdown of znf513 Gene Expression (A–D) Merged photos of frozen sections from MM-MO-injected (A, B) and znf513-MO-injected (C, D) zebrafish embryos stained for PKCβ1 (bipolar cells, green), Zpr-1 (cone photoreceptors, red, A and C), 1D1 (rod receptors, red, B and D), and DAPI (nuclei, blue). znf513-MO-injected embryos show decreased thickness of all layers except the OPL. (E–L) Staining of znf513-MO- and MM-MO-injected embryos for specific opsins. Frozen sections from MM-MO-injected (E, G, I, K) and znf513-MO-injected (F, H, J, L) embryos were stained with anti-blue opsin (E, F, green), anti-green opsin (G, H, green), anti-red opsin (I, J, green), anti-UV opsin (K, L, green), 1D4 (all, Rhodopsin, red), and DAPI (all, nuclei, blue). There is a decrease in each opsin in znf513-MO-injected embryos, with rhodopsin and red opsin being essentially absent. (M) Comparison of retina layer thicknesses in μm, eye perimeter, and cell number between the znf513-MO-treated (n = 5) and MM-MO-treated (n = 7) larvae. t is the unpaired Student's t test statistic. IN, inner nuclear layer cells. p < 0.05 was considered statistically significant. (A–L) 5 dpf. Scale bars represent 50 μm in (A)–(D) and 15 μm in (E)–(L). ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL ganglion cell layer.

Figure 5

Characterization of Mutant and Wild-Type GFP-ZNF513 Proteins by Western Blot Analysis and Localization of ZNF513 in COS-7 Cells by Immunofluorescence (A) Blot of COS-7 cell lysates probed with GFP antibodies. Lane 1, untransfected lysate; lane 2, transfected wild-type ZNF513-GFP lysate; lane 3, lysate transfected with mutant ZNF513-GFP; lane M, SeeBlue2 Plus molecular weight marker. Both WT and mutant proteins migrate at the predicted MW of 82 kDa. (B–I) COS-7 cells were transfected with pEGFPC1 fused in-frame with wild-type (B–E) or p.C339R mutant (F–I) ZNF513. Cells were immunostained with C23 antibody (red, nucleolus) or DAPI (4′,6-diamidino-2-phenylindole, blue, nucleus). Overlays of images from the first three columns are shown in (E) and (I). Scale bars represent 10 μm. Both wild-type (WT) and mutant (Mut) proteins localize to the nucleus in a speckled pattern.

Figure 6

Chromatin Binding and ChIP Analysis of ZNF513 (A) Agarose gel electrophoresis with the chromatin from NIH 3T3 cells transfected with wild-type GFP-ZNF513. (B) The DNA binding proteins present in the immunoprecipitated chromatin analyzed by western blotting via GFP antibody. (C) ChIP assays with NIH 3T3 cells transfected with wild-type GFP-ZNF513 (left) or p.C339R mutant GFP-ZNF513 (right) with GFP monoclonal antibody (+Ab). Normal rabbit IgG (–Ab) and no chromatin (Mock) samples serve as negative controls, whereas input (chromatin samples without IP) serve as positive controls. Arr3, cone arrestin; Rho 5′, rhodopsin 5′ regulatory region; IER, enhancer of interphotoreceptor retinoid binding protein (IRBP); Rho3′, exonic 3′ region of Rhodopsin; Nr2e3, nuclear receptor subfamily 2, group E, member 3; Nrl, neural retina leucine zipper; Sp4, Sp4 transcription factor; Pax2, paired box gene 2; Mop, M-cone opsin; Sop, S-cone opsin; Pax6, paired box gene 6. Specific bands are seen for Arr3, Rho5′, IER, Sp4, Mop, Sop, and Pax6 with the wild-type but not the p.C339R mutant GFP-ZNF513.