title: Characterisation and Functions of a Novel Cancer-Associated Gene, C1ORF112
Creators: name: Bell, Sarah
Publisher: name: University of Liverpool Repository
Year: 2022

description: C1ORF112 was first identified from bioinformatic searches for novel genes associated with aging; strong correlation (co-expression) with genes of the Fanconi anaemia (FA) pathway and the DNA damage response (DDR) was observed from the GeneFriends database. High C1ORF112 expression has been found in many cancers, often indicating poor prognosis. C1ORF112 has known roles in guiding accurate chromosome segregation via homologous recombination (HR) in both mitosis and meiosis, through its interactions with Polo-like kinase 1 (PLK1) and Fidgetin-like 1 (FIGNL1), respectively.

In silico analysis showed the C1ORF112 gene is conserved from fungi to Homo sapiens and BLAST analysis revealed a conserved domain of unknown function (DUF4487) in orthologous sequences across Opisthokonts and Embryophyta. Protein modelling suggested the DUF4487 is an important docking moiety for FIGNL1. Contact prediction, template homology, and AlphaFold methods indicated a protein structure composed of alpha helical repeats and a protein-protein interaction network generated from STRING predicted interactions with proteins of the DNA damage repair pathways, mitosis &amp; meiosis (HR), and development. GO-terms of functionally enriched biological pathways for C1ORF112 further supported this hypothesis.

The notion of C1ORF112 as a DDR gene was explored with two knockout cell lines, CRISPR-generated HAP1 cells harbouring frameshift mutations in exons 4 and 19, respectively. These cells were slow to proliferate, and hypersensitive to interstrand crosslink (ICL)-inducing agent mitomycin C (MMC), monofunctional alkylating agents, and poly adenosine diphosphate ribose polymerase inhibitors (PARPi), indicating a broader role for C1ORF112 the general response to DNA damage or replication stress (RS). Immunocytochemistry (ICC) and western blot (WB) analysis demonstrated increased phosphorylated histone H2A family member X (yH2AX) protein in mutants following MMC exposure indicating activation of the DDR. To explore the effect of C1ORF112 on the FA pathway, cell cycle analysis and chromosome metaphase analysis in MMC-treated cells suggested HR involvement rather than the classical FA pathway, with a small (but not significant) G2/M phase delay and significant chromatid exchanges in the knockout cells, respectively. Quantitative PCR (qPCR) of MMC treated mutant cell DNA was performed on all FA genes; all were downregulated with the exception of FANCD2 and FANCI (upregulated) and FANCB (no change). A FANCD2 WB also demonstrated upregulation of both FANCD2 isoforms in mutants, suggesting that C1ORF112 depletion results in upregulation of FANCD2/I complex ubiquitination and increased signalling for DNA repair by HR. This data suggests a role for C1ORF112 in the general response to DNA damage, and possibly in signalling for downstream FA pathway activation (HR) following stalled replication forks and unresolved ICLs. 

C1ORF112 is therefore a novel cancer-associated gene with a potential role in the downstream FA pathway (HR) and may therefore be a valid therapeutic target for the detection and early treatment of cancer.