Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling

Abstract

Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina, and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as "ciliopathies." However, disease mechanisms remain poorly understood. Here, we identify by whole-exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164, and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. Our findings link degenerative diseases of the kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.

Figure 1. Identification of recessive mutations in MRE11, ZNF423 and CEP164 in NPHP-RC using homozygosity mapping and WER

Data regarding homozygosity mapping and mutations are shown for family A3471 with MRE11 mutation (A–B), family F874 with ZNF423 mutation (C–D), and family KKESH001 with CEP164 mutation (E–F). (A, C, E) Non-parametric lod scores (NPL) are plotted across the human genome in 3 families (A3471, F874 and KKESH001) with NPHP-RC (see also Table 1). The x-axis shows SNP positions on human chromosomes concatenated from p-ter (left) to q-ter (right). Genetic distance is given in cM. Maximum NPL peaks (Hildebrandt et al., 2009b) (red circles) indicate candidate regions of homozygosity by descent. The genes MRE11, ZNF423 and CEP164 are positioned (arrow heads) within one of the maximum NPL peaks. (B, D, F) Homozygous mutations detected in families with NPHP-RC. Family number (underlined), mutation (arrowheads) and predicted translational changes (in parenthesis) are indicated (see also Table 1). Sequence traces are shown for mutations above normal controls. (For additional mutations in other families see also Table 1 and Figure S2).

Figure 2. Two ZNF423 mutations have dominant negative characteristics (A–D), ZNF423 mutation abrogates interaction with PARP1 (E), and ZNF423 directly interacts with the NPHP-RC protein CEP290/NPHP6 (F–G)

(A) Amino acid residues altered by NPHP-RC mutations in ZNF423 are drawn in relation to functional annotation of its 30 Zn-fingers. (B–D) S-phase index assay (fraction of transfected cells incorporating BrdU) for P19 cells transfected with either wildtype or mutant ZNF423. (B) Representative field of cells transfected with wildtype ZNF423 shows high frequency of BrdU⁺ FLAG⁺ double-positive cells. (C) ZNF423–H1277Y transfected cells exhibits fewer FLAG–positive cells and a lower proportion that are double positive. (D) S-phase index measured in duplicate transfections for each of three DNA preparations per construct. A GFP construct was used as a nonspecific control. Constructs with P506fsX43 and H1277Y mutations detected in NPHP-RC show significantly reduced S-phase index (p>10⁻⁵, ANOVA with post-hoc TukeyHSD). (E) ZNF423 interacts with PARP1. P19 cells were co-transfected with expression constructs for N-terminally V5-tagged human full-length ZNF423 and FLAG-tagged human full length PARP1. Comparable amounts of both proteins were present in all lysates (lower panels). Proteins were precipitated, using anti-V5 (upper panels) and anti-Flag antibodies (middle panels), respectively. Reciprocal coIP demonstrates interaction between ZNF423 and PARP1. Note that the ZFN423 mutation P506fsX43 abrogates this interaction (arrow head) and that mutation H1277Y diminishes ZNF423 multimerization (arrow). (F–G) ZNF423 directly interacts with CEP290/NPHP6. (F) A human fetal brain yeast two-hybrid library screened with human CEP290/NPHP6 (JAS2; aa 1917–2479) fused to the DNA-binding domain of GAL4 (pDEST32) identified ZNF423 as a direct interaction partner of CEP290/NPHP6. The interaction was confirmed using direct yeast two-hybrid assay where 1 and 2 represent colony growth of CEP290 bait with ZNF423 prey. a–e are controls for colony growth on medium deficient in histidine, leucine and tryptophan. (G) HEK293T were cotransfected with human V5-tagged partial human V5-CEP290 clone and GFP-tagged full-length human ZNF423 clone. Immunoprecipitation with anti-V5 (lane 2), but not control IgG (lane 3) precipitated both the V5-tagged CEP290 (arrowhead) as well as gfp-tagged ZNF423 (arrow).

Figure 3. (A–D) Expression of mutant CEP164 in renal epithelial cells abrogates localization to centrosomes

(A) Immunofluorescence using α-SDCCAG8/NPHP10-CG antibody, labels both centrioles, whereas α-CEP164-ENR antibody demonstrates CEP164 staining at the mother centriole only. (B) Inducible overexpression of N-terminally GFP-tagged human full-length CEP164 isoform 1 (NGFP-CEP164-WT) in IMCD3 cells demonstrates, in addition to a cytoplasmic expression pattern, localization at one of the two centrioles (inset, arrow heads) consistent with selective localization to the mother centriole (Graser et al., 2007). Both centrioles are stained with an anti-γ-tubulin antibody. (C) In contrast, the centrosomal signal is abrogated upon overexpression of an N-terminally GFP-tagged truncated CEP164 construct representing the mutation p.Q525X. (D) The number of centrosomes positive for CEP164 is reduced upon overexpression of C-terminally GFP-tagged human full-length CEP164 isoform 1 (CGFP-hCEP164-WT), which mimics the mutation p.X1460fs57 that causes a read-through of the stop-codon X1460, adding 57 aberrant amino acid residues to the C-terminus of CEP164. Similar data was obtained upon CEP164 expression in hTERT-RPE cells (see also Figure S3B–D). IMCD3 cells were stably transfected with the respective CEP164 constructs in a retroviral vector for doxycyclin-inducible expression (pRetroX-Tight-Pur). Scale bars represent 10 μm. (E–H) Knockdown of Cep164 disrupts ciliary frequency. (E) Depletion of Cep164 by siRNA (F) causes a ciliary defect in 3D spheroid growth assays. IMCD3 cells transfected with either siCtrl or siCep164 were grown to spheroids in 72 hours and immunostained for acetylated tubulin (red). DAPI stains nuclei (blue). Doxycycline induced stably transfected NGFP-hCEP164-WT (green). Space bar represents 5 μm. (G) Nuclei and cilia were scored within a single spheroid to generate ciliary frequencies. siCep164 transfected cells manifest lower cilia frequencies (33%) compared to control transfected IMCD3 cells (49%). Induction of NGFP-hCEP164-WT in siCep164 transfected cells rescues this ciliary defect (57%). 50 spheroids per condition were analysed in three independent experiments. Error bars represent SEM, n=3, ^*p-value <0.0002. (H) Ciliary frequency is not rescued by mutant CEP164. Ciliary frequencies are reduced in siCep164 transfected IMCD3 cells (39%) compared to control siCtrl transfected IMCD3 cells (54%). Induction of NGFP-hCEP164-Q525X does not rescue this ciliary defect (34%). 50 spheroids per condition were analyzed. Error bars represent SEM, ^***p-value <0.0002. See also Figure S3.

Figure 4. (A–P) Colocalization upon immunofluorescence of the NPHP-RC proteins SDCCAG8/NPHP10, ZNF423 and CEP164 to nuclear foci that are positive for the DDR signaling proteins SC35, TIP60 and Chk1 in hTERT-RPE cells

(A–G) Colocalization of NPHP-RC proteins with SC35 in nuclear foci. SDCCAG8/NPHP10 (A–C) and ZNF423 (D) fully colocalize to nuclear foci with SC35, and (E) CEP164 partially colocalizes with SC35. SDCCAG8/NPHP10 also colocalizes with the newly identified NPHP-RC proteins ZNF423 (F) and CEP164 (G). (H–J) Colocalization of NPHP-RC proteins with the DDR protein TIP60 I nuclear foci. (H) TIP60 fully colocalizes with SC35. (I) TIP60 partially colocalizes with CEP164. (J) Chk1 fully colocalizes with SC35/SRSF2. DNA is stained in blue with DAPI. Scale bars represent 5μm. (K–P) Colocalization of DDR and NPHP proteins upon induction of DDR by UV radiation in HeLa cells. (K) Following irradiation of HeLa cells with UV light at 20 J/m² a strong immunofluorescence signal of an anti-γH2AX antibody indicates activation of DDR. (L–M) Upon irradiation with UV light, CEP164-positive nuclear foci condense and colocalize with newly appearing TIP60 foci of similar size. (N–O) In untreated cells (N) a pattern of broad CEP164 speckles, which are CHK1-negative and locate to DAPI-negative domains, changes to a pattern of multiple smaller foci (O) that are double-positive for both, CEP164-N11 and CHK1. (P) p317-CHK1 fully colocalizes with TIP60 to nuclear foci and to the centrosome (arrow head). See also Figures S4, S5.

Figure 5. Knockdown of Cep164 causes anaphase lag and retarded cell growth

(A–B) Knockdown of CEP164 causes anaphase lag. siCep164 knockdown in IMCD3 cells increased anaphase lag incidence from 1% after siCtrl to 21% after siCep164-treated cells (n >250 anaphases, five independent experiments). CREST antiserum (red) and DAPI (blue) confirmed the presence of incomplete mitotic congression and unattached kinetochores during late anaphase (white arrows). Doxycycline-inducible expression of WT-CEP164 during Cep164 siRNA knockdown reduced the incidence of anaphase lag to 4%, whereas untransfected IMCD3 cells had no detectable anaphase lag (0%) (B). Bars represent SEM, p values (student T-test) are indicated above the bar graph. (C–D) Transient knockdown of Cep164 inhibits proliferation, which is rescued by wild type but not mutant CEP164. In clonally selected and doxycycline (Dox)-inducible mouse IMCD3 cells siRNA knockdown was performed. (C) IMCD3 cells depleted of murine Cep164 grew more slowly (siRNA, green line) than non-depleted cells (control, blue line) or the non-depleted cells induced to express human wild type CEP164 (Dox, red line). Expression of WT Cep164 in siRNA-depleted cells rescued the slow growth phenotype of Cep164 depletion (siRNA+dox, purple line). (D) As in C, except mutant Cep164 cDNA (CEP164-Q525X) was expressed under doxycyclin control. Expression of this allele itself had a negative impact on cell growth (green line), suggesting a dominant negative effect. An even greater negative effect was seen when the endogenous Cep164 was depleted in cells expressing CEP164-Q525X (siRNA+dox, purple line). The average counts are plotted with standard deviations. Asterisks indicates significant differences by unpaired Student’s t-test (P< 0.05). (E–H) The effect of roscovitine on UV-induced DDR. Cells were UV-irradiated with 30 J/m² and analyzed 1 h post UV-irradiation. Where indicated, cells were pre-incubated for 24 h with the CDK inhibitor roscovitine (80 μM). (E–F) Immunofluorescence analysis showed that roscovitine triggered uniform nuclear distribution of γH2AX (activated H2AX phosphorylated at Ser139) in non UV-irradiated cells suggesting partial DDR activation (E). UV radiation caused enhanced γH2Ax staining with a prominent nuclear foci pattern, characteristic of strong DDR activation (F). (G–H) The effect of roscovitine on UV-triggered subcellular localization of CEP164 and Chk1. CEP164 and Chk1 proteins, along with nuclear marker Sam68 and cytoplasmic marker 14.3.3 were analyzed by western blot. Roscovitine decreased the amount of CEP164 present in control and UV-irradiated cells (G). This was most likely due to translocation of CEP164 into the nucleus upon roscovitine treatment as shown by subcellular fractionation (H). As expected, UV radiation increased phosphorylation of Chk1 at Ser317 (p-Chk1) (G), and roscovitine decreased Chk1 protein expression and abrogated UV-induced p-Chk1 in both cytoplasm and nucleus (G–H). Proteins 14.3.3 and Sam68 serve as controls for cytoplasmic vs. nuclear fraction, respectively. See also Figure S6.

Figure 6. Knockdown of cep164 in zebrafish embryos results in ciliopathy phenotypes, and knockdown of Cep164 or Zfp423(Znf423) causes sensitivity to DNA damage

A morpholino-oligonucleotide (cep164 MO) targeting the exon 7 splice donor site of zebrafish cep164 was injected into fertilized eggs at the one to four-cells stage together with p53 MO (0.2 mM) to minimize non-specific MO effects. (A–E) Whereas p53 MO injection (n=67) did not produce any phenotype (A), coinjection of cep164 MO at 28 hpf caused the mild ciliopathy phenotype of ventral body axis curvature in 48% of embryos (60/125) (B). 50% of embryos (62/125) showed severe cell death throughout the body as judged by grey-appearing cells in the head region (C). Embryos with severe cell death also showed increased expression of phosphorylated γH2AX (D) compared to p53 MO control (E). Most embryos with massive cell death did not survive beyond 48 hpf. (F–I) At 48 hpf, surviving cep164 morphants displayed the ciliopathy phenotype of laterality defects. Whereas p53 MO did not cause any abnormal heart looping (F,G), cep164 MO caused inverted heart looping (H) or ambiguous heart looping (I). (A, atrium; L, left; V, ventricle) (J–M) At 72 hpf, embryos developed further ciliopathy phenotypes. When compared to p53 MO controls (J), pronephric tubules (arrow heads) exhibited cystic dilation (K, asterisks) in 25% (7/28) of embryos. In addition, when compared to p53 MO controls (L), 0% (0/67) of embryos showed kidney cysts, hydrocephalus (asterisk), or retinal dysplasia (brackets) (M). (N) At 0.2 mM, cep164 MO knockdown effectively altered mRNA processing as revealed by RT-PCR. The wild type (WT) mRNA product is 339 bp. A shorter aberrantly spliced mRNA product appeared in cep164 morphants (asterisks), and the normal mRNA product was significantly reduced. p53 MO alone did not affect cep164 mRNA processing. See also Figure S7. (O) Quantification of γ-H2AX levels in cep164 MO morphants. Whole fish lysates were prepared from morphants injected with control MO (p53 0.2 mM) or cep164 MO (p53 0.2 mM, cep164 0.2 mM). Injection of cep164-targeting MO causes upregulation of γ-H2AX in cep164 kd embryos signifying perturbed DDR. γ-H2AX levels correlate with the phenotypic severity of the cep164 morphants (see panels A–C). Anti-α-tubulin antibody was used to show equal loading. (P–Q) Cep164-deficient IMCD3 cells exhibit radiation sensitivity. In IMCD3 cells transduced with shRNA retrovirus, Cep164 expression was suppressed by shRNA knockdown to about 20% of control as judged by qPCR (P). Cep164 knockdown resulted in a dose-dependent increase of γH2AX positive cells in a FACS assay, signifying increased radiation sensitivity to IR and perturbed DDR. See also Figure S8. In (Q) the level of significance of two-tailed t-test (p<0.001) is indicated by an asterisk. (R–T) Zfp423(Znf423)-deficient P19 cells exhibit radiation sensitivity. P19 cells transduced with shRNA lentivirus were exposed to the indicated level X-irradiation. Zfp423 and γH2AX immunofluorescence was quantified in matched replicate cultures for each virus 2h after irradiation. (R) Representative images illustrate dose-responsiveness of γH2AX and effective knockdown of Zfp423 expression. (S) γH2AX intensity normalized to DAPI⁺ nuclei is increased following IR at 0.5 and 1.0 Gy, signifying increased IR sensitivity and perturbed DDR (2 fields from each of 6 replicate cultures per condition). Asterisks, uncorrected pair-wise p<0.05, Mann-Whitney U test, 2 tails. (T) Histogram shows average γH2AX intensity per cell in 16 additional replicate cultures for each shRNA at 1.0 Gy exposure. P=0.018, Mann-Whitney U test, 2 tails. See also Figure S8.