Abstract
The cannabinoid-1 receptor (CB1) plays a critical role in a number of biological processes including nutrient intake, addiction and anxiety-related behaviour. Numerous studies have shown that expression of the gene encoding CB1 (CNR1) is highly dynamic with changes in the tissue specific expression of CNR1 associated with brain homeostasis and disease progression. However, little is known of the mechanisms regulating this dynamic expression. To gain a better understanding of the genomic mechanisms modulating the expression of CNR1 in health and disease we characterised the role of a highly conserved regulatory sequence (ECR1) in CNR1 intron 2 that contained a polymorphism in linkage disequilibrium with disease associated SNPs. We used CRISPR/CAS9 technology to disrupt ECR1 within the mouse genome. Disruption of ECR1 significantly reduced CNR1 expression in the hippocampus but not in the hypothalamus. These mice also displayed an altered sex-specific anxiety-related behavioural profile (open field test), reduced ethanol intake and a reduced hypothermic response following CB1 agonism. However, no significant changes in feeding patterns were detected. These data suggest that, whilst not all of the expression of CNR1 is modulated by ECR1, this highly conserved enhancer is required for appropriate physiological responses to a number of stimuli. The combination of comparative genomics and CRISPR/CAS9 disruption used in our study to determine the functional effects of genetic and epigenetic changes on the activity of tissue-specific regulatory elements at the CNR1 locus represent an important first step in gaining a mechanistic understanding of cannabinoid regulatory pharmacogenetics.
Highlights
The cannabinoid-1 receptor (CB1) is extensively expressed in areas of the nervous system, where it plays a critical role in appetite regulation (Pomorska et al, 2016), the reward system and anxiety-related behaviour (Akirav, 2011)
Our previous studies used a combination of comparative genomics and linkage disequilibrium analysis to identify a highly conserved polymorphic sequence (ECR1; rs9444584) within intron 2 of the CNR1 gene that was in strong LD with two other single nucleotide polymorphisms (SNPs) (Fig. 1A, rs2023239 and rs9450898) that were previously associated with various pathologies (Benzinou et al, 2008; Chen et al, 2008; Hutchison et al, 2008; Icick et al, 2015; Ketcherside et al, 2017; Pava and Woodward, 2012; Schacht et al, 2012; Suarez-Pinilla et al, 2015) and which displayed allele specific differential enhancer activity in different tissues (Nicoll et al, 2012)
In the current study we asked whether this enhancer could be disrupted using CRISPR genome editing and what effects disrupting this enhancer would have on the expression of the endogenous CNR1 gene in different tissues as well as a broad analysis of food and alcohol intake, cannabinoid response, movement and anxiety-related behaviour
Summary
The cannabinoid-1 receptor (CB1) is extensively expressed in areas of the nervous system, where it plays a critical role in appetite regulation (Pomorska et al, 2016), the reward system and anxiety-related behaviour (Akirav, 2011). There is growing evidence that functional enhancer regions are often highly conserved (Montalbano et al, 2017) so we previously used a combination of Psychoneuroendocrinology 109 (2019) 104407 comparative genomics, molecular biology and primary cell based studies to identify a highly conserved enhancer element in intron 2 of the CNR1 gene that we called evolutionary conserved region 1 (ECR1) (Nicoll et al, 2012) and which contained a SNP (rs9444584) that was in strong linkage disequilibrium (LD) with rs2023239 and rs9450898. We analysed aspects of mouse behaviour including cannabinoid response, alcohol intake, locomoter activity and thigmotaxis; an indicator of anxiety-related behaviour. The significance of these observations is discussed in the context of cannabinoid pharmacogenomics and disease
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