Linking substance use genetics to circadian function using high-throughput genomics - SUMMARY Substance use disorders, including opioid use disorder, are rising across the United States have devastating consequences on those affected as well as their family and friends. There is growing evidence that addiction and sleep are tightly linked at the genetic, molecular, and behavioral levels. At the genetic level, there have been hundreds of loci confidently linked with addiction phenotypes and sleep phenotypes. Genetic variants associated with both sleep and addiction show enrichment in distal open chromatin regions of many of the same striatal neuron subtypes. Despite these associations, it remains unexplored whether genetic variants that impact the predisposition to addiction or sleep phenotypes also directly impact the circadian regulation of gene expression. The difficulty in understanding the gene regulatory mechanisms underlying candidate addiction- and sleep-associated genetic variants results from an inability to perform controlled experiments in an intact human brain. Here, we leverage a combination of machine learning and genomic technologies to make inferences about how genetic variants impact the circadian regulation of gene expression by altering the combinatorial code of transcription factors at distal enhancer regions. In Aim 1, we conduct controlled single cell genomic experiments in the mouse striatum, which serve as training data for convolutional neural network models. These models then make inferences across the entire human genome about which genetic variants are likely to modulate circadian gene regulation. In Aim 2, we synthesize hundreds of candidate human addiction- and sleep- associated enhancers and then measure their circadian regulation in in vivo massively parallel reporter assays conducted in the mouse striatum. In Aim 3, we apply comparative genomics to find enhancers that are likely to be conserved in function between human and mouse. The result of disrupting those enhancers in a CRISPR-KRAB mouse strain is measured using a variety of behavioral assays for addiction and sleep phenotypes. The result of these aims will be predictions and measurements of the circadian function across hundreds of enhancers that contain addiction- and sleep-associated genetic variants. These experiments will allow us to test the hypothesis that addiction-associated genetic variants impact predisposition by influencing circadian gene regulation. More broadly, we will provide a framework to understand the genetic mechanisms of addiction and sleep across other brain regions and tissues as well as in other disease contexts.
