Regulatory characterisation of the schizophrenia-associated CACNA1C proximal promoter and the potential role for the transcription factor EZH2 in schizophrenia aetiology

Genomic wide association studies identified the CACNA1C locus as genetically associated with both schizophrenia and bipolar affective disorder. CACNA1C encodes Cav1.2, one of four subunits of L-type voltage gated calcium channels. Variation resides in non-coding regions of CACNA1C which interact with the promoter and are validated expression quantitative trait loci. Using reporter gene constructs we demonstrate the CACNA1C promoter is a major mediator of inducible regulation of CACNA1C activity in the SH-SY5Y neuroblastoma cell line. Exposure of SH-SY5Y cells to lithium and cocaine modulated both the endogenous CACNA1C gene and the promoter in reporter gene constructs. Deletion analysis of the promoter demonstrated the actions of both lithium and cocaine were mediated by the proximal promoter. Initial interrogation of ENCODE ChIP-seq data over the CACNA1C promoter indicated binding of the transcription factor ‘Enhancer of zeste homolog 2’ (EZH2), which was consistent with our data that overexpression of EZH2 repressed CACNA1C promoter reporter gene expression. Array data from the Human Brain Transcriptome demonstrated that EZH2 was highly expressed across the developing brain, but subsequently maintained at low levels after birth and adulthood. RNA-seq data obtained from PD_NGSAtlas, a reference database for epigenomic and transcriptomic data for psychiatric disorders, demonstrated a 3-fold increase in EZH2 expression in the anterior cingulate cortex of individuals with schizophrenia compared to controls. We propose that EZH2 may contribute to schizophrenia risk at two distinct time points either through disruption in development leading to neurodevelopmental changes, or through anomalous reactivation of expression in the adult brain.