CEP164-null cells generated by genome editing show a ciliation defect with intact DNA repair capacity

Abstract

Primary cilia are microtubule structures that extend from the distal end of the mature, mother centriole. CEP164 is a component of the distal appendages carried by the mother centriole that is required for primary cilium formation. Recent data have implicated CEP164 as a ciliopathy gene and suggest that CEP164 plays some roles in the DNA damage response (DDR). We used reverse genetics to test the role of CEP164 in the DDR. We found that conditional depletion of CEP164 in chicken DT40 cells using an auxin-inducible degron led to no increase in sensitivity to DNA damage induced by ionising or ultraviolet irradiation. Disruption of CEP164 in human retinal pigmented epithelial cells blocked primary cilium formation but did not affect cellular proliferation or cellular responses to ionising or ultraviolet irradiation. Furthermore, we observed no localisation of CEP164 to the nucleus using immunofluorescence microscopy and analysis of multiple tagged forms of CEP164. Our data suggest that CEP164 is not required in the DDR.

Embryonic and foetal expression patterns of the ciliopathy geneCEP164

“…It is also well established that CEP164 is present at the basal body of retinal pigment epithelial cells, clarifying the results from our study [1, 2, 15, 17, 23]. It can be hypothesised that with abnormal functioning/loss of CEP164, there could be atypical outer segment formation, which could lead to the accumulation of phototransduction proteins that could trigger cell loss, leading to a retinal degeneration phenotype, as seen in some CEP164 NPHP-RC patients [1, 9, 44].…”

“…Multiple fusion protein studies have demonstrated that the CEP164 C-terminal domain is required for localisation of CEP164 to the distal appendages [1, 15]. Additionally, CEP164 has been localised to the nucleus [1, 14, 17, 27].…”

“…Upon initiation of ciliogenesis Rabin 11 imports the GTPase Rabin 8 to the basal body. CEP164 interacts with Rabin 8, facilitated by Chibby 1, to recruit Rab8 positive vesicles to the centrosome [15, 17, 19, 24, 30]. Vesicle docking is required for subsequent basal body anchoring to the plasma membrane and primary cilia development.…”

“…Several studies have indicated a potential role for CEP164 in ATM/ATR-mediated DNA damage response (DDR) and UV-induced nucleotide excision repair pathways, however results are conflicting [14, 17, 33]. Likewise, data supporting a role for CEP164 in cell-cycle regulation is inconsistent [1, 2, 14, 15, 25]; both of these CEP164 roles need to be further validated.…”

Open Sesame: How Transition Fibers and the Transition Zone Control Ciliary Composition

“…Mutations in two other TF genes, CEP164 and CEP83, can cause nephronophthisis (NPHP), a cystic kidney disease, sometimes accompanied by other signs such as intellectual disability (Chaki et al 2012; Failler et al 2014). Both proteins are required for ciliogenesis and Cep164 can also participate in the DNA damage response, a function it can share with other NPHP proteins (Graser et al 2007; Chaki et al 2012; Tanos et al 2013; Daly et al 2016). As noted above, C. elegans orthologs of the NPHP proteins Nphp1 and Nphp4 localize in both TFs and TZ (Hildebrandt et al 1997; Mollet et al 2002; Otto et al 2002; Jensen et al 2015).…”