CRISPR Deletion of a SVA Retrotransposon Demonstrates Function as a cis-Regulatory Element at the TRPV1/TRPV3 Intergenic Region.

SINE-VNTR-Alu (SVA) retrotransposons are a subclass of transposable elements (TEs) that exist only in primate genomes. TE insertions can be co-opted as cis-regulatory elements (CREs); however, the regulatory potential of SVAs has predominantly been demonstrated using bioinformatic approaches and reporter gene assays. The objective of this study was to demonstrate SVA cis-regulatory activity by CRISPR (clustered regularly interspaced short palindromic repeats) deletion and subsequent measurement of direct effects on local gene expression. We identified a region on chromosome 17 that was enriched with human-specific SVAs. Comparative gene expression analysis at this region revealed co-expression of TRPV1 and TRPV3 in multiple human tissues, which was not observed in mouse, highlighting key regulatory differences between the two species. Furthermore, the intergenic region between TRPV1 and TRPV3 coding sequences contained a human specific SVA insertion located upstream of the TRPV3 promoter and downstream of the 3' end of TRPV1, highlighting this SVA as a candidate to study its potential cis-regulatory activity on both genes. Firstly, we generated SVA reporter gene constructs and demonstrated their transcriptional regulatory activity in HEK293 cells. We then devised a dual-targeting CRISPR strategy to facilitate the deletion of this entire SVA sequence and generated edited HEK293 clonal cell lines containing homozygous and heterozygous SVA deletions. In edited homozygous ∆SVA clones, we observed a significant decrease in both TRPV1 and TRPV3 mRNA expression, compared to unedited HEK293. In addition, we also observed an increase in the variability of mRNA expression levels in heterozygous ∆SVA clones. Overall, in edited HEK293 with SVA deletions, we observed a disruption to the co-expression of TRPV1 and TRPV3. Here we provide an example of a human specific SVA with cis-regulatory activity in situ, supporting the role of SVA retrotransposons as contributors to species-specific gene expression.

The Role of SINE-VNTR-Alu (SVA) Retrotransposons in Shaping the Human Genome.

Retrotransposons can alter the regulation of genes both transcriptionally and post-transcriptionally, through mechanisms such as binding transcription factors and alternative splicing of transcripts. SINE-VNTR-Alu (SVA) retrotransposons are the most recently evolved class of retrotransposable elements, found solely in primates, including humans. SVAs are preferentially found at genic, high GC loci, and have been termed "mobile CpG islands". We hypothesise that the ability of SVAs to mobilise, and their non-random distribution across the genome, may result in differential regulation of certain pathways. We analysed SVA distribution patterns across the human reference genome and identified over-representation of SVAs at zinc finger gene clusters. Zinc finger proteins are able to bind to and repress SVA function through transcriptional and epigenetic mechanisms, and the interplay between SVAs and zinc fingers has been proposed as a major feature of genome evolution. We describe observations relating to the clustering patterns of both reference SVAs and polymorphic SVA insertions at zinc finger gene loci, suggesting that the evolution of this network may be ongoing in humans. Further, we propose a mechanism to direct future research and validation efforts, in which the interplay between zinc fingers and their epigenetic modulation of SVAs may regulate a network of zinc finger genes, with the potential for wider transcriptional consequences.

Treating the "E" in "G × E": Trauma-Informed Approaches and Psychological Therapy Interventions in Psychosis.

Despite advances in genetic research, causal variants affecting risk for schizophrenia remain poorly characterized, and the top 108 loci identified through genome-wide association studies (GWAS) explain only 3.4% of variance in risk profiles. Such work is defining the highly complex nature of this condition, with omnigenic models of schizophrenia suggesting that gene regulatory networks are sufficiently interconnected such that altered expression of any "peripheral" gene in a relevant cell type has the capacity to indirectly modulate the expression of "core" schizophrenia-associated genes. This wealth of associated genes with small effect sizes makes identifying new druggable targets difficult, and current pharmacological treatments for schizophrenia can involve serious side effects. However, the fact that the majority of schizophrenia genome-wide associated variants fall within non-coding DNA is suggestive of their potential to modulate gene regulation. This would be consistent with risks that can be mediated in a "gene × environment" (G × E) manner. Stress and trauma can alter the regulation of key brain-related pathways over the lifetime of an individual, including modulation of brain development, and neurochemistry in the adult. Recent studies demonstrate a significant overlap between psychotic symptoms and trauma, ranging from prior trauma contributing to psychosis, as well as trauma in response to the experience of psychosis itself or in response to treatment. Given the known effects of trauma on both CNS gene expression and severity of psychosis symptoms, it may be that pharmacological treatment alone risks leaving individuals with a highly stressful and unresolved environmental component that continues to act in a "G × E" manner, with the likelihood that this would negatively impact recovery and relapse risk. This review aims to cover the recent advances elucidating the complex genetic architecture of schizophrenia, as well as the long-term effects of early life trauma on brain function and future mental health risk. Further, the evidence demonstrating the role of ongoing responses to trauma or heightened stress sensitivity, and their impact on the course of illness and recovery, is presented. Finally, the need for trauma-informed approaches and psychological therapy-based interventions is discussed, and a brief overview of the evidence to determine their utility is presented.

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.

Novel brain expressed RNA identified at the MIR137 schizophrenia-associated locus.

Genome-wide association studies (GWAS) have identified a locus on chromosome 1p21.3 to be highly associated with schizophrenia. A microRNA, MIR137, within this locus has been proposed as the gene causally associated with schizophrenia, due to its known role as a regulator of neuronal development and function. However, the involvement of other genes within this region, including DPYD (dihydropyrimidine dehydrogenase), is also plausible. In this communication, we describe a previously uncharacterised, brain-expressed RNA, EU358092, within the schizophrenia-associated region at 1p21.3. As we observed for MIR137, EU358092 expression was modulated in response to psychoactive drug treatment in the human SH-SY5Y neuroblastoma cell line. Bioinformatic analysis of publically available CNS expression data indicates that MIR137 and EU358092 are often co-expressed in vivo. A potential regulatory domain for expression of EU358092 is identified by bioinformatic analysis and its regulatory function is confirmed by reporter gene assays. These data suggest a potentially important role for EU358092 in the aetiology of schizophrenia, either individually or in combination with other genes at this locus.

SVA retrotransposons as potential modulators of neuropeptide gene expression.

Many facets of human behaviour are likely to have developed in part due to evolutionary changes in the regulation of neuropeptide and other brain-related genes. This has allowed species-specific expression patterns and unique epigenetic modulation in response to our environment, regulating response not only at the molecular level, but also contributing to differences in behaviour between individuals. As such, genetic variants or epigenetic changes that may alter neuropeptide gene expression are predicted to play a role in behavioural conditions and psychiatric illness. It is therefore of interest to identify regulatory elements that have the potential to drive differential gene expression. Retrotransposons are mobile genetic elements that are known to be drivers of genomic diversity, with the ability to alter expression of nearby genes. In particular, the SINE-VNTR-Alu (SVA) class of retrotransposons is specific to hominids, and its appearance and expansion across the genome has been associated with the evolution of numerous behavioural traits, presumably through their ability to confer unique regulatory properties at the site of their insertion. We review the evidence for SVAs as regulatory elements, exploring how polymorphic variation within these repetitive sequences can drive allele specific gene expression, which would be associated with changes in behaviour and disease risk through the alteration of molecular pathways that are central to healthy brain function.

Identification and Potential Regulatory Properties of Evolutionary Conserved Regions (ECRs) at the Schizophrenia-Associated MIR137 Locus.

Genome-wide association studies (GWAS) have identified a region at chromosome 1p21.3, containing the microRNA MIR137, to be among the most significant associations for schizophrenia. However, the mechanism by which genetic variation at this locus increases risk of schizophrenia is unknown. Identifying key regulatory regions around MIR137 is crucial to understanding the potential role of this gene in the aetiology of psychiatric disorders. Through alignment of vertebrate genomes, we identified seven non-coding regions at the MIR137 locus with conservation comparable to exons (>70 %). Bioinformatic analysis using the Psychiatric Genomics Consortium GWAS dataset for schizophrenia showed five of the ECRs to have genome-wide significant SNPs in or adjacent to their sequence. Analysis of available datasets on chromatin marks and histone modification data showed that three of the ECRs were predicted to be functional in the human brain, and three in development. In vitro analysis of ECR activity using reporter gene assays showed that all seven of the selected ECRs displayed transcriptional regulatory activity in the SH-SY5Y neuroblastoma cell line. This data suggests a regulatory role in the developing and adult brain for these highly conserved regions at the MIR137 schizophrenia-associated locus and further that these domains could act individually or synergistically to regulate levels of MIR137 expression.