Characterising the function of ZNF804A: A top genome-wide association study hit for schizophrenia

Schizophrenia is a debilitating psychiatric disorder with high heritability. Genome wide association studies (GWASs) have identified association between schizophrenia and an intronic single nucleotide polymorphism (SNP) in zinc finger domain containing 804A (ZNF804A). The biological functions of ZNF804A are largely unknown. Thus, the aim of this thesis was to determine the function of ZNF804A. Yeast two-hybrid (Y2H) screens were used to determine the protein binding partners of ZNF804A in the brain. This identified ZNF804A interacted with transcription factors (zinc finger protein 40 (ZNF40); trans-acting transcription factor 1 (Sp1) and basic helix-loop-helix family, member e40 (Bhlhe40)) and regulators of pre-mRNA splicing, including RNA binding protein, fox-1 and -2 (Rbfox1 and RBFOX2) and neuro-oncological ventral antigen 2 (NOVA2). Therefore, we hypothesised that, via interactions with its protein binding partners, ZNF804A may have a role in regulating transcription and pre-mRNA splicing. Exon arrays were used to determine the effects of ZNF804A knockdown on gene expression in SH-SY5Y neuroblastoma cells. Enrichment analysis on the differentially expressed or spliced genes indicated a significant effect of ZNF804A knockdown on genes involved in nervous system development, particularly synaptic contact and axon guidance. Among the most significantly differentially expressed genes were the known schizophrenia susceptibility genes reelin (RELN) and neuropeptide Y (NPY). Several alternative splicing events were confirmed empirically, including increased exclusion of exon 11a of enabled homolog (ENAH). Consistent with our hypothesis, splicing of exon 11a of ENAH is known to be regulated by RBFOX2. In complementary experiments, exon arrays were used to identify differentially expressed genes in a stable cell line expressing myc-ZNF804A. Enrichment analysis on the differentially expressed genes indicated an over-representation of genes involved in the regulation of epithelial to mesenchymal transition and receptor-mediated axon growth repulsion. Among the genes with the largest fold changes in expression was a gene implicated in synapse development: secreted protein acidic and rich in cysteine (SPARC). Enrichment analysis of the alternatively spliced genes indicated a significant effect of myc-ZNF804A over-expression on genes involved in cell-matrix interactions. These data suggest that ZNF804A regulates the expression of genes implicated in processes underlying schizophrenia pathology, and provide the first evidence that ZNF804A may be involved in the regulation of alternative splicing