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Our group recently characterized a cell-autonomous mammalian 12-h clock independent from the circadian clock, but its function and mechanism of regulation remain poorly understood. Here, we show that in mouse liver, transcriptional regulation significantly contributes to the establishment of 12-h rhythms of mRNA expression in a manner dependent on Spliced Form of X-box Binding Protein 1 (XBP1s). Mechanistically, the motif stringency of XBP1s promoter binding sites dictates XBP1s's ability to drive 12-h rhythms of nascent mRNA transcription at dawn and dusk, which are enriched for basal transcription regulation, mRNA processing and export, ribosome biogenesis, translation initiation, and protein processing/sorting in the Endoplasmic Reticulum (ER)-Golgi in a temporal order consistent with the progressive molecular processing sequence described by the central dogma information flow (CEDIF). We further identified GA-binding proteins (GABPs) as putative novel transcriptional regulators driving 12-h rhythms of gene expression with more diverse phases. These 12-h rhythms of gene expression are cell autonomous and evolutionarily conserved in marine animals possessing a circatidal clock. Our results demonstrate an evolutionarily conserved, intricate network of transcriptional control of the mammalian 12-h clock that mediates diverse biological pathways. We speculate that the 12-h clock is coopted to accommodate elevated gene expression and processing in mammals at the two rush hours, with the particular genes processed at each rush hour regulated by the circadian and/or tissue-specific pathways.

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Severe and persistent disruptions to sleep and circadian rhythms are common in people with opioid use disorder (OUD). Preclinical evidence suggests altered molecular rhythms in the brain modulate opioid reward and relapse. However, whether molecular rhythms are disrupted in the brains of people with OUD remained an open question, critical to understanding the role of circadian rhythms in opioid addiction. Using subjects’ times of death as a marker of time of day, we investigated transcriptional rhythms in the brains of subjects with OUD compared to unaffected comparison subjects. We discovered rhythmic transcripts in both the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc), key brain areas involved in OUD, that were largely distinct between OUD and unaffected subjects. Fewer rhythmic transcripts were identified in DLPFC of subjects with OUD compared to unaffected subjects, whereas in the NAc, nearly double the number of rhythmic transcripts was identified in subjects with OUD. In NAc of subjects with OUD, rhythmic transcripts peaked either in the evening or near sunrise, and were associated with an opioid, dopamine, and GABAergic neurotransmission. Associations with altered neurotransmission in NAc were further supported by co-expression network analysis which identified OUD-specific modules enriched for transcripts involved in dopamine, GABA, and glutamatergic synaptic functions. Additionally, rhythmic transcripts in DLPFC and NAc of subjects with OUD were enriched for genomic loci associated with sleep-related GWAS traits, including sleep duration and insomnia. Collectively, our findings connect transcriptional rhythm changes in opioidergic, dopaminergic, GABAergic signaling in the human brain to sleep-related traits in opioid addiction.

1 Though risk for cocaine use disorder is subject to considerable inter-individual variation, the 2 sources of that individual variation -including genetics and sex -are too often ignored in non-3 human animal studies of this phenomenon. Here, we studied both males and females of eight 4 different inbred mouse strains with reproducible genomes capturing 90% of the genetic 5 diversity mice. In these genetically diverse laboratory mice, individual differences explain a 6 substantial proportion -often the majority -of variance in important cocaine-related 7 behavioral, physiological, and striatum transcriptional responses traits. Individual mouse 8 genomes thus represent a missed opportunity for discovery and translation of addiction 9 mechanisms. 10

Circadian variability is driven by genetics and Diversity Outbred (DO) mice is a powerful tool for examining the genetics of complex traits because their high genetic and phenotypic diversity compared to conventional mouse crosses. The DO population combines the genetic diversity of eight founder strains including five common inbred and three wild-derived strains. In DO mice and their founders, we established a high-throughput system to measure cellular rhythms using in vitro preparations of skin fibroblasts. Among the founders, we observed strong heritability for rhythm period, robustness, phase and amplitude. We also found significant sex and strain differences for these rhythms. Extreme differences in period for molecular and behavioral rhythms were found between the inbred A/J strain and the wild-derived CAST/EiJ strain, where A/J had the longest period and CAST/EiJ had the shortest. In addition, we measured cellular rhythms in 329 DO mice, which displayed far greater phenotypic variability than the founders—80% of founders compared to only 25% of DO mice had periods of ~ 24 h. Collectively, our findings demonstrate that genetic diversity contributes to phenotypic variability in circadian rhythms, and high-throughput characterization of fibroblast rhythms in DO mice is a tractable system for examining the genetics of circadian traits.

Severe and persistent disruptions to sleep and circadian rhythms are common features of people with opioid use disorder (OUD). Preclinical findings suggest altered molecular rhythms in the brain are involved in opioid reward and dependence. However, whether molecular rhythms are disrupted in brains of people with OUD remained an open question, critical to understanding the role of circadian rhythms in opioid addiction. We previously used subjects' times of death (TOD) as a marker of time of day to investigate transcriptional rhythm alterations in psychiatric disorders. Using TOD and RNA sequencing, we discovered rhythmic transcripts in both the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc), key brain areas involved in opioid addiction, were largely distinct between OUD and unaffected comparison subjects. Further, fewer rhythmic transcripts were identified in DLPFC of OUD subjects compared to unaffected subjects, but nearly double the number of rhythmic transcripts were found in the NAc of OUD subjects. In OUD, rhythmic transcripts in the NAc peaked either in the evening or near sunrise, and were associated with dopamine, opioid, and GABAergic neurotransmission. Co-expression network analysis identified several OUD-specific modules in the NAc, enriched for transcripts involved in the modulation of dopamine and GABA synapses, including glutamatergic signaling and extracellular matrices. Integrative analyses with human GWAS revealed that rhythmic transcripts in DLPFC and NAc were enriched for genomic loci associated with sleep duration and insomnia. Overall, our results connect transcriptional rhythm changes in dopamine, opioid, and GABAergic synaptic signaling in human brain to sleep-related phenotypes and OUD.

Opioid craving and the vulnerability to relapse is associated with severe and persistent disruptions to sleep and circadian rhythms. Investigations into the cellular and molecular pathways in the human brain underlying the relationship between circadian rhythms and OUD remain limited. In human subjects with OUD, previous transcriptomics work implicated a role for circadian regulation of synaptic processes in key cognitive- and reward-related brain regions, dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc). To provide further insights into the synaptic alterations associated with OUD, we used mass-spectrometry based proteomics to deeply profile protein alterations in tissue homogenates and synaptosomes from both NAc and DLPFC of unaffected and OUD subjects. Between unaffected and OUD subjects, we identified 43 differentially expressed (DE) proteins in NAc homogenates and 55 DE proteins in DLPFC homogenates. In synaptosomes, we found 56 DE proteins in NAc of OUD subjects and 161 DE proteins in DLPFC. Examining synaptosome enrichment of specific proteins enabled us to identify brain region- and synapse-specific pathway alterations in NAc and DLPFC associated with OUD. Across both regions, we found OUD-associated protein alterations primarily in pathways involved in GABAergic and glutamatergic synaptic functions, as well as circadian rhythms. Using time-of-death (TOD) analyses, where the TOD of each subject is used as a time-point across a 24-hour cycle, we were able to map circadian-related changes in the synaptic proteomes in NAc and DLPFC associated with OUD. In OUD, TOD analysis revealed significant circadian changes in endoplasmic reticulum to Golgi vesicle-mediated transport and protein membrane trafficking in NAc synapses, accompanied by changes in platelet derived growth factor receptor beta signaling in DLPFC synapses. Together, our results lend further support for molecular disruption of circadian regulation of synaptic signaling in the human brain as a key factor in opioid addiction.