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Fig. 2
Two ZNF423 Mutations Have Dominant Negative Characteristics, ZNF423 Mutation Abrogates Interaction with PARP1, and ZNF423 Directly Interacts with the NPHP-RC Protein CEP290/NPHP6 (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 wild-type or mutant ZNF423. (B) Representative field of cells transfected with wild-type 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 < 105, ANOVA with post-hoc Tukey honestly significant difference [HSD]). (E) ZNF423 interacts with PARP1. P19 cells were cotransfected with expression constructs for N terminally FLAG-tagged human full-length ZNF423 and V5-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 (arrowhead) 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).
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