Allele-specific Differences in Activity of a Novel Cannabinoid Receptor 1 (CNR1) Gene Intronic Enhancer in Hypothalamus, Dorsal Root Ganglia, and Hippocampus

Abstract

Background: Intron 2 of CNR1 gene contains multiple disease-associated SNPs.Results: Allelic variants of a novel enhancer in CNR1 intron 2 affect its MAPK response in hypothalamus and hippocampus.Conclusion: Alleles of enhancer may be functionally linked to obesity and addictive behavior.Significance: Understanding the effects of CNR1 polymorphisms on gene regulation will accelerate understanding of human disease.

Genetic, epigenetic and pharmacological influences modulating tissue specific regulation of the cannabinoid receptor-1 gene (CB1); implications for cannabinoid pharmacogenetics

“…pECR1C/TLuc. The ECR1 enhancer sequence was also recovered from pGEM-T easy (Nicoll et al 2012) and the minor T allele of ECR1 was re-created using site-directed mutagenesis where the template DNA was a previously established pECR1(C)Gem-Teasy construct, primers for site-directed mutagenesis were:…”

“…Hippocampal or hypothalamic tissues were dissected into ice cold Neurobasal-A medium (Life Technologies). Cells were dissociated from pooled tissues to reduce variability and cultured in poly-d-lysine coated 24-well plates as previously described (Nicoll et al, 2012) at a density of 180,000 cells per well as assessed using a Biorad TC10 cell viability counter. Cells were maintained at 37°C in 5% CO2 for up to 7 days in vitro in Neurobasal-A medium supplemented with 2% B27, 2mM L-glutamine, 50 μg/ml Streptomycin and 50U Penicillin (Life Technologies) with the medium changed on the first day after plating and then every 3 days.…”

“…Because this LD block was non-coding we used comparative genomics to identify functional sequences based on the hypothesis that high (sequence-sequence) and deep (conservation through evolution) conservation reflects functionality (Dogan et al, 2015;Visel et al, 2007). We identified a sequence that demonstrated high levels of conservation in all higher vertebrates (Fig 1A and B) and which contained a SNP (rs9444584) in high LD with both rs2023239 and rs9450898 ( Fig 1B) (Nicoll et al, 2012). To determine the possible function of this sequence we used CRISPR genome editing to disrupt the most highly conserved core region of ECR1 from the mouse genome ( Fig 3A) by microinjecting single guide RNA (gRNA; gRNAECR1a and gRNAECR1b; Fig 3A) molecules, designed to disrupt the core region of ECR1, centred on a sequence homologous to that containing the human rs9444584 polymorphism ( Fig 3A), together with CAS9 mRNA into the cytoplasm of fertilised single cell mouse embryos.…”

Exploring the effects of polymorphisms on cis-regulatory signal transduction response

“…For example, the major polymorphic variant of a highly conserved CRS within intron 2 of the CNR1 (cannabinoid receptor 1 gene) gene, which encodes the cannabinoid receptor, was inactive in primary hippocampal neurons. However, the minor variant, which was in strong LD with another SNP associated with obesity and addictive behavior, was highly active in hippocampal neurons and, in contrast to the major variant, responded robustly to the activation of MAP kinase pathways [43].…”

“…In addition to determining the causes of disease, GWA studies have also been used to find the causes of drug response stratification. Clues to the possible mechanisms through which polymorphisms in the noncoding genome affect drug response can be seen in the significant differences observed between the C and T allele of the CNR1 ECR1 enhancer; the T allele responds much more strongly to treatment using angiotensin II [43]. GWAS-based approaches to determining the parts of the genome responsible for response stratification include the Genome-based Therapeutic Drugs for Depression (GENDEP) study.…”