Sleep is nearly ubiquitous throughout the animal kingdom, with deficiencies in sleep having been linked to a wide range of human disorders and diseases. While genome wide association studies (GWAS) in humans have been identified loci robustly associated with several heritable diseases or traits, little is known about the functional roles of the underlying causal variants in regulating sleep duration or quality. We applied an ATAC-seq/promoter focused Capture C methodology in iPSC-derived neural progenitors to carry out a variant-to-gene mapping campaign that identified 88 candidate sleep effector genes connected to relevant GWAS signals. To functionally validate the role of the implicated effector genes in sleep regulation, we performed a neuron-specific RNAi screen in the fruit fly, Drosophila melanogaster. This approach identified a number of genes that regulated sleep, including phosphatidylinositol N-acetylglucosaminyltransferase subunit Q (PIG-Q). This gene encodes an enzyme involved in the first step of glycosylphosphatidylinositol (GPI)-anchor biosynthesis. We show that flies deficient for PIG-Q have longer sleep during both the day and night due to an increase in the total number of sleep bouts. Subsequent systematic investigation of other PIG-family genes identified increased sleep in flies for multiple different genes within the PIG pathway. We then mutated the PIG-Q locus in zebrafish and identified similar increases in sleep to those observed in Drosophila, confirming deep homology of PIG-Q mediated sleep regulation. These results provide the first physical variant-to-gene mapping of human sleep genes, and reveals a novel and conserved role for GPI-anchor biosynthesis in sleep regulation.

Chemosensory systems are critical for evaluating the caloric value and potential toxicity of food. While animals can discriminate between thousands of odors, much less is known about the discriminative capabilities of taste systems. Fats and sugars represent calorically potent and attractive food sources that contribute to hedonic feeding. Despite the differences in nutritional value between fats and sugars, the ability of the taste system to discriminate between different rewarding tastants is thought to be limited. In Drosophila, taste neurons expressing the ionotropic receptor 56d (IR56d) are required for reflexive behavioral responses to the medium-chain fatty acid, hexanoic acid. Here, we tested whether flies can discriminate between different classes of fatty acids using an aversive memory assay. Our results indicate that flies are able to discriminate medium-chain fatty acids from both short- and long-chain fatty acids, but not from other medium-chain fatty acids. While IR56d neurons are broadly responsive to short-, medium-, and long-chain fatty acids, genetic deletion of IR56d selectively disrupts response to medium-chain fatty acids. Further, IR56d+ GR64f+ neurons are necessary for proboscis extension response (PER) to medium-chain fatty acids, but both IR56d and GR64f neurons are dispensable for PER to short- and long-chain fatty acids, indicating the involvement of one or more other classes of neurons. Together, these findings reveal that IR56d is selectively required for medium-chain fatty acid taste, and discrimination of fatty acids occurs through differential receptor activation in shared populations of neurons. Our study uncovers a capacity for the taste system to encode tastant identity within a taste category.

Genome-wide association studies (GWAS) in humans have identified loci robustly associated with several heritable diseases or traits, yet little is known about the functional roles of the underlying causal variants in regulating sleep duration or quality. We applied an ATAC-seq/promoter focused Capture C strategy in human iPSC-derived neural progenitors to carry out a “variant-to-gene” mapping campaign that identified 88 candidate sleep effector genes connected to relevant GWAS signals. To functionally validate the role of the implicated effector genes in sleep regulation, we performed a neuron-specific RNA interference screen in the fruit fly, Drosophila melanogaster , followed by validation in zebrafish. This approach identified a number of genes that regulate sleep including a critical role for glycosylphosphatidylinositol (GPI)–anchor biosynthesis. These results provide the first physical variant-to-gene mapping of human sleep genes followed by a model organism–based prioritization, revealing a conserved role for GPI-anchor biosynthesis in sleep regulation.

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Study objectives: Insomnia is a pervasive sleep disorder with heritability estimates ranging from 22-59 percent. Human genome-wide association studies (GWAS) have identified more than 200 genome-wide significant loci associated with the pathogenesis of insomnia; however, few of the implicated effector genes at these loci have undergone functional characterization, and little is known of their relevance to sleep dysfunction in insomnia. High-throughput phenotyping in model organisms, especially for conserved traits like sleep, provides an opportunity for enhanced gene prioritization to improve mechanistic understanding of this complex polygenic disorder and to yield strong genetic candidates for therapeutic intervention. Methods: Leveraging existing datasets, we performed a functional screen of candidate insomnia-associated genes in zebrafish. Our previous 3D-genomics approach, which integrated ATAC-seq RNA-seq and high-resolution promoter-focused Capture C in neural progenitor cells, revealed putatively causal variants and corresponding effector genes at insomnia GWAS loci. We went on to report first-pass neuron-specific RNA interference screening in Drosophila to implicate high-confidence effector genes at these genetic signals. This current study builds on those initial observations by leveraging CRISPR/Cas9 in a diurnal vertebrate model, namely larval zebrafish (5-7 days post fertilization), to perform functional validation of six top candidates to determine conserved regulators of sleep behaviors relevant to insomnia. Results: CRISPR mutation of three high-confidence insomnia-implicated effector genes (MEIS1, SKIV2L, and ARFGAP2) resulted in robust changes to sleep and activity patterns in larval zebrafish. Specifically, we observed phenotypes that reflect insomnia-like behaviors, including altered sleep duration, poor sleep consolidation, and impaired latency to sleep onset after lights off. Moreover, mutation of ARFGAP2 also impacted development resulting in a significant motor phenotype. Conclusions: Our results reveal the utility of CRISPR/Cas9 in F0 larval zebrafish for high-throughput screening of GWAS-implicated candidate effector genes. We validated three of the six strongest candidate effector genes identified in our previous combined 3D Genomic and Drosophila screen, two of which were 3D-mapped to different genes rather than the nearest gene reported in the original insomnia GWAS study. Together, we demonstrate the necessity for functional validation of GWAS-implicated effector genes, and we provide evidence for two novel genes producing sleep-wake phenotypes not previously suggested through positional mapping alone.