Biology subjects

Zimmerman, A. J.

Publications and source records attributed to Zimmerman, A. J..

3 recordsLinked to original sources

bioRxiv2023

Perturbation of the insomnia WDR90 GWAS locus pinpoints rs3752495 as a causal variant influencing distal expression of neighboring gene, PIG-Q

Although genome wide association studies (GWAS) have been crucial for the identification of loci associated with sleep traits and disorders, the method itself does not directly uncover the underlying causal variants and corresponding effector genes. The overwhelming majority of such variants reside in non-coding regions and are therefore presumed to impact the activity of cis-regulatory elements, such as enhancers. Our previously reported variant-to-gene mapping effort in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs), combined with validation in both Drosophila and zebrafish, implicated PIG-Q as a functionally relevant gene at the insomnia WDR90 locus. However, importantly that effort did not characterize the corresponding underlying causal variant at this GWAS signal. Specifically, our genome-wide ATAC-seq and high-resolution promoter-focused Capture C datasets generated in this cell setting brought our attention to a shortlist of three tightly neighboring single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium in a candidate intronic enhancer region of WDR90 that contacted the open PIG-Q promoter. The objective of this study was to investigate the influence of the proxy SNPs collectively and then individually on PIG-Q modulation and to pinpoint the causal "regulatory" variant among the three SNPs. Starting at a gross level perturbation, deletion of the entire region harboring all three SNPs in human iPSC-derived neural progenitor cells via CRISPR-Cas9 editing and subsequent RNA sequencing revealed expression changes in specific PIG-Q transcripts. Results from more refined individual luciferase reporter assays for each of the three SNPs in iPSCs revealed that the intronic region with the rs3752495 risk allele induced a [~]2.5-fold increase in luciferase expression (n=10). Importantly, rs3752495 also exhibited an allele specific effect, with the risk allele increasing the luciferase expression by [~]2-fold compared to the non-risk allele. In conclusion, our variant-to-function approach and subsequent in vitro validation implicates rs3752495 as a causal insomnia risk variant embedded at the WDR90-PIG-Q locus.

genomics↗

bioRxiv2023

Screening effects of HCN channel blockers on sleep/wake behavior in zebrafish

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels generate electrical rhythmicity in various tissues although primarily heart, retina and brain. The HCN channel blocker compound, Ivabradine (Corlanor), is approved by the US Food and Drug Administration (FDA) as a medication to lower heart rate by blocking hyperpolarization activated inward current in the sinoatrial node. In addition, a growing body of evidence suggests a role for HCN channels in regulation of sleep/wake behavior. Zebrafish larvae are ideal model organisms for high throughput drug screening, drug repurposing and behavioral phenotyping studies. We leveraged this model system to investigate effects of three HCN channel blockers (Ivabradine, Zatebradine Hydrochloride and ZD7288) at multiple doses on sleep/wake behavior in wild type zebrafish. Results of interest included shorter latency to day time sleep at 0.1 M dose of Ivabradine (ANOVA, p:0.02), moderate reductions in average activity at 30 M dose of Zatebradine Hydrochloride (ANOVA, p:0.024) in daytime, and increased nighttime sleep at 4.5 M dose of ZD7288 (ANOVA, p:0.036). These differences support the hypothesis that compounds blocking HCN channels decreases wakefulness. HighlightsO_LIA drug screening study in which effects of HCN channel blocker compounds were tested displayed decreased wakefulwness in zebrafish. C_LIO_LIThere was modest evidence of these drugs on sleep and wake phenotypes including shorter latency to sleep, moderate reductions in average activity and increased sleep at different doses of three compounds compared to DMSO. C_LIO_LIWhile several specific doses of Ivabradine, Zatebradine hydrochloride or ZD7288 demonstrated some differences compared to DMSO, effects of these compounds was smaller than the effect of melatonin, a positive control. C_LI

pharmacology and toxicology↗

bioRxiv2022

Zebrafish screen of high-confidence targets at insomnia GWAS loci implicates MEIS1, SKIV2L, and ARFGAP2 as conserved regulators of sleep-wake behaviors

Recent large-scale human genome-wide association studies (GWAS) for insomnia have identified more than 200 significant loci. The functional relevance of these loci to the pathogenesis of insomnia is largely unknown. GWAS signals are typically non-coding variants, which are often arbitrarily annotated to the nearest protein-coding gene; however, due to 3D chromatin structure, variants can interact with more distal genes driving their function. The distal gene may, therefore, represent the true causal gene influencing the phenotype. By integrating our high-resolution chromatin interaction maps from neural progenitor cells with phenotypic data from a Drosophila RNAi screen, we prioritized candidate genes that we hypothesized would have deep phylogenetic conservation of sleep function. To determine the conservation of these candidate genes in the context of vertebrate sleep and their relevance to insomnia-like behaviors, we performed CRISPR-Cas9 mutagenesis in larval zebrafish for six highly conserved candidate genes and examined sleep-wake behaviors using automated video-tracking. CRISPR mutation of zebrafish orthologs of MEIS1 and SKIV2L produced insomnia-like behaviors, while mutation of ARFGAP2 impaired activity and development in our larval zebrafish model, demonstrating the importance of performing functional validation of GWAS-implicated effector genes to reveal genes influencing disease-relevant mechanisms.

genetics↗