Effects of repeated alcohol abstinence on within-subject prefrontal cortical gene expression in rhesus macaques.

Male rhesus monkeys (n = 24) had a biopsy of prefrontal cortical area 46 prior to chronic ethanol self-administration (n = 17) or caloric control (n = 7). Fourteen months of daily self-administration (water vs. 4% alcohol, 22 h access/day termed "open-access") was followed by two cycles of prolonged abstinence (5 weeks) each followed by 3 months of open-access alcohol and a final abstinence followed by necropsy. At necropsy, a biopsy of Area 46, contralateral to the original biopsy, was obtained. Gene expression data (RNA-Seq) were collected comparing biopsy/necropsy samples. Monkeys were categorized by drinking status during the final post-abstinent drinking phase as light (LD), binge (BD), heavy (HD) and very heavy (VHD drinkers). Comparing pre-ethanol to post-abstinent biopsies, four animals that converted from HD to VHD status had significant ontology enrichments in downregulated genes (necropsy minus biopsy n = 286) that included immune response (FDR < 9 × 10-7) and plasma membrane changes (FDR < 1 × 10-7). Genes in the immune response category included IL16 and 18, CCR1, B2M, TLR3, 6 and 7, SP2 and CX3CR1. Upregulated genes (N = 388) were particularly enriched in genes associated with the negative regulation of MAP kinase activity (FDR < 3 × 10-5), including DUSP 1, 4, 5, 6 and 18, SPRY 2, 3, and 4, SPRED2, BMP4 and RGS2. Overall, these data illustrate the power of the NHP model and the within-subject design of genomic changes due to alcohol and suggest new targets for treating severe escalated drinking following repeated alcohol abstinence attempts.

Modeling Brain Gene Expression in Alcohol Use Disorder with Genetic Animal Models.

Animal genetic models have and will continue to provide important new information about the behavioral and physiological adaptations associated with alcohol use disorder (AUD). This chapter focuses on two models, ethanol preference and drinking in the dark (DID), their usefulness in interrogating brain gene expression data and the relevance of the data obtained to interpret AUD-related GWAS and TWAS studies. Both the animal and human data point to the importance for AUD of changes in synaptic transmission (particularly glutamate and GABA transmission), of changes in the extracellular matrix (specifically including collagens, cadherins and protocadherins) and of changes in neuroimmune processes. The implementation of new technologies (e.g., cell type-specific gene expression) is expected to further enhance the value of genetic animal models in understanding AUD.

Brain gene expression differences related to ethanol preference in the collaborative cross founder strains.

The collaborative cross (CC) founder strains include five classical inbred laboratory strains [129S1/SvlmJ (S129), A/J (AJ), C57BL/6J (B6), NOD/ShiLtJ (NOD), and NZO/HILtJ (NZO)] and three wild-derived strains [CAST/EiJ (CAST), PWK/PhJ (PWK), and WSB/EiJ (WSB)]. These strains encompass 89% of the genetic diversity available in Mus musculus and ∼10-20 times more genetic diversity than found in Homo sapiens. For more than 60 years the B6 strain has been widely used as a genetic model for high ethanol preference and consumption. However, another of the CC founder strains, PWK, has been identified as a high ethanol preference/high consumption strain. The current study determined how the transcriptomes of the B6 and PWK strains differed from the 6 low preference CC strains across 3 nodes of the brain addiction circuit. RNA-Seq data were collected from the central nucleus of the amygdala (CeA), the nucleus accumbens core (NAcc) and the prelimbic cortex (PrL). Differential expression (DE) analysis was performed in each of these brain regions for all 28 possible pairwise comparisons of the CC founder strains. Unique genes for each strain were identified by selecting for genes that differed significantly [false discovery rate (FDR) < 0.05] from all other strains in the same direction. B6 was identified as the most distinct classical inbred laboratory strain, having the highest number of total differently expressed genes (DEGs) and DEGs with high log fold change, and unique genes compared to other CC strains. Less than 50 unique DEGs were identified in common between B6 and PWK within all three brain regions, indicating the strains potentially represent two distinct genetic signatures for risk for high ethanol-preference. 338 DEGs were found to be commonly different between B6, PWK and the average expression of the remaining CC strains within all three regions. The commonly different up-expressed genes were significantly enriched (FDR < 0.001) among genes associated with neuroimmune function. These data compliment findings showing that neuroimmune signaling is key to understanding alcohol use disorder (AUD) and support use of these 8 strains and the highly heterogeneous mouse populations derived from them to identify alcohol-related brain mechanisms and treatment targets.

Sex Differences in the Brain Transcriptome Related to Alcohol Effects and Alcohol Use Disorder.

There is compelling evidence that sex and gender have crucial roles in excessive alcohol (ethanol) consumption. Here, we review some of the data from the perspective of brain transcriptional differences between males and females, focusing on rodent animal models. A key emerging transcriptional feature is the role of neuroimmune processes. Microglia are the resident neuroimmune cells in the brain and exhibit substantial functional differences between males and females. Selective breeding for binge ethanol consumption and the impacts of chronic ethanol consumption and withdrawal from chronic ethanol exposure all demonstrate sex-dependent neuroimmune signatures. A focus is on resolving sex-dependent differences in transcriptional responses to ethanol at the neurocircuitry level. Sex-dependent transcriptional differences are found in the extended amygdala and the nucleus accumbens. Telescoping of ethanol consumption is found in some, but not all, studies to be more prevalent in females. Recent transcriptional studies suggest that some sex differences may be due to female-dependent remodeling of the primary cilium. An interesting theme appears to be developing: at least from the animal model perspective, even when males and females are phenotypically similar, they differ significantly at the level of the transcriptome.

On the Use of Heterogeneous Stock Mice to Map Transcriptomes Associated With Excessive Ethanol Consumption.

We and many others have noted the advantages of using heterogeneous (HS) animals to map genes and gene networks associated with both behavioral and non-behavioral phenotypes. Importantly, genetically complex Mus musculus crosses provide substantially increased resolution to examine old and new relationships between gene expression and behavior. Here we report on data obtained from two HS populations: the HS/NPT derived from eight inbred laboratory mouse strains and the HS-CC derived from the eight collaborative cross inbred mouse strains that includes three wild-derived strains. Our work has focused on the genes and gene networks associated with risk for excessive ethanol consumption, individual variation in ethanol consumption and the consequences, including escalation, of long-term ethanol consumption. Background data on the development of HS mice is provided, including advantages for the detection of expression quantitative trait loci. Examples are also provided of using HS animals to probe the genes associated with ethanol preference and binge ethanol consumption.

Effect of chronic ethanol consumption in rhesus macaques on the nucleus accumbens core transcriptome.

The nucleus accumbens core (NAcc) has been repeatedly demonstrated to be a key component of the circuitry associated with excessive ethanol consumption. Previous studies have illustrated that in a nonhuman primate (NHP) model of chronic ethanol consumption, there is significant epigenetic remodeling of the NAcc. In the current study, RNA-Seq was used to examine genome-wide gene expression in eight each of control, low/binge (LD*), and high/very high (HD*) rhesus macaque drinkers. Using an FDR < 0.05, zero genes were significantly differentially expressed (DE) between LD* and controls, six genes between HD* and LD*, and 734 genes between HD* and controls. Focusing on HD* versus control DE genes, the upregulated genes (N = 366) were enriched in genes with annotations associated with signal recognition particle (SRP)-dependent co-translational protein targeting to membrane (FDR < 3 × 10-59 ), structural constituent of ribosome (FDR < 3 × 10-47 ), and ribosomal subunit (FDR < 5 × 10-48 ). Downregulated genes (N = 363) were enriched in annotations associated with behavior (FDR < 2 × 10-4 ), membrane organization (FDR < 1 × 10-4 ), inorganic cation transmembrane transporter activity (FDR < 2 × 10-3 ), synapse part (FDR < 4 × 10-10 ), glutamatergic synapse (FDR < 1 × 10-6 ), and GABAergic synapse (FDR < 6 × 10-4 ). Ingenuity Pathway Analysis (IPA) revealed that EIF2 signaling and mTOR pathways were significantly upregulated in HD* animals (FDR < 3 × 10-33 and <2 × 10-16 , respectively). Overall, the data supported our working hypothesis; excessive consumption would be associated with transcriptional differences in GABA/glutamate-related genes.

Phenotypic and gene expression features associated with variation in chronic ethanol consumption in heterogeneous stock collaborative cross mice.

Of the more than 100 studies that have examined relationships between excessive ethanol consumption and the brain transcriptome, few rodent studies have examined chronic consumption. Heterogeneous stock collaborative cross mice freely consumed ethanol vs. water for 3 months. Transcriptional differences were examined for the central nucleus of the amygdala, a brain region known to impact ethanol preference. Early preference was modestly predictive of final preference and there was significant escalation of preference in females only. Genes significantly correlated with female preference were enriched in annotations for the primary cilium and extracellular matrix. A single module in the gene co-expression network was enriched in genes with an astrocyte annotation. The key hub node was the master regulator, orthodenticle homeobox 2 (Otx2). These data support an important role for the extracellular matrix, primary cilium and astrocytes in ethanol preference and consumption differences among individual female mice of a genetically diverse population.

RNA-Seq Analysis of Genetic and Transcriptome Network Effects of Dual-Trait Selection for Ethanol Preference and Withdrawal Using SOT and NOT Genetic Models.

BACKGROUND: Genetic factors significantly affect alcohol consumption and vulnerability to withdrawal. Furthermore, some genetic models showing predisposition to severe withdrawal are also predisposed to low ethanol (EtOH) consumption and vice versa, even when tested independently in naïve animals. METHODS: Beginning with a C57BL/6J × DBA/2J F2 intercross founder population, animals were simultaneously selectively bred for both high alcohol consumption and low acute withdrawal (SOT line), or vice versa (NOT line). Using randomly chosen fourth selected generation (S4) mice (N = 18-22/sex/line), RNA-Seq was employed to assess genome-wide gene expression in ventral striatum. The MegaMUGA array was used to detect genome-wide genotypic differences. Differential gene expression and the weighted gene co-expression network analysis were implemented as described elsewhere (Genes Brain Behav 16, 2017, 462). RESULTS: The new selection of the SOT and NOT lines was similar to that reported previously (Alcohol Clin Exp Res 38, 2014, 2915). One thousand eight hundred and sixteen transcripts were detected as differentially expressed between the lines. For genes more highly expressed in the SOT line, there was enrichment in genes associated with cell adhesion, synapse organization, and postsynaptic membrane. The genes with a cell adhesion annotation included 23 protocadherins, Mpdz and Dlg2. Genes with a postsynaptic membrane annotation included Gabrb3, Gphn, Grid1, Grin2b, Grin2c, and Grm3. The genes more highly expressed in the NOT line were enriched in a network module (red) with annotations associated with mitochondrial function. Several of these genes were module hub nodes, and these included Nedd8, Guk1, Elof1, Ndufa8, and Atp6v1f. CONCLUSIONS: Marked effects of selection on gene expression were detected. The NOT line was characterized by higher expression of hub nodes associated with mitochondrial function. Genes more highly expressed in the SOT aligned with previous findings, for example, Colville and colleagues (Genes Brain Behav 16, 2017, 462) that both high EtOH preference and consumption are associated with effects on cell adhesion and glutamate synaptic plasticity.

Tetracycline derivatives reduce binge alcohol consumption in High Drinking in the Dark mice.

Alcohol use disorders (AUDs) are prevalent, and are characterized by binge-like drinking, defined by patterns of focused drinking where dosages ingested in 2-4 â€‹h reach intoxicating blood alcohol levels (BALs). Current medications are few and compliance with the relatively rare prescribed usage is low. Hence, novel and more effective medications are needed. We developed a mouse model of genetic risk for binge drinking (HDID: High Drinking in the Dark mice) by selectively breeding for high BALs after binge drinking. A transcriptional analysis of HDID brain tissue with RNA-Seq implicated neuroinflammatory mechanisms, and, more specifically extracellular matrix genes, including those encoding matrix metalloproteinases (MMPs). Prior experiments from other groups have shown that the tetracycline derivatives doxycycline, minocycline, and tigecycline, reduce binge drinking in inbred C57BL/6J mice. We tested these three compounds in female and male HDID mice and found that all three reduced DID and BAL. They had drug-specific effects on intake of water or saccharin in the DID assay. Thus, our results show that the effectiveness of synthetic tetracycline derivatives as potential therapeutic agents for AUDs is not limited to the single C57BL/6J genotype previously targeted, but extends to a mouse model of a population at high risk for AUDs.

Chronic Voluntary Ethanol Drinking in Cynomolgus Macaques Elicits Gene Expression Changes in Prefrontal Cortical Area 46.

BACKGROUND: Genome-wide profiling to examine brain transcriptional features associated with excessive ethanol (EtOH) consumption has been applied to a variety of species including rodents, nonhuman primates (NHPs), and humans. However, these data were obtained from cross-sectional samples which are particularly vulnerable to individual variation when obtained from small outbred populations typical of human and NHP studies. In the current study, a novel within-subject design was used to examine the effects of voluntary EtOH consumption on prefrontal cortex (PFC) gene expression in a NHP model. METHODS: Two cohorts of cynomolgus macaques (n = 23) underwent a schedule-induced polydipsia procedure to establish EtOH self-administration followed by 6 months of daily open access to EtOH (4% w/v) and water. Individual daily EtOH intakes ranged from an average of 0.7 to 3.7 g/kg/d. Dorsal lateral PFC area 46 (A46) brain biopsies were collected in EtOH-naïve and control monkeys; contralateral A46 biopsies were collected from the same monkeys following the 6 months of fluid consumption. Gene expression changes were assessed using RNA-Seq paired analysis, which allowed for correction of individual baseline differences in gene expression. RESULTS: A total of 675 genes were significantly down-regulated following EtOH consumption; these were functionally enriched for immune response, cell adhesion, plasma membrane, and extracellular matrix. A total of 567 genes that were up-regulated following EtOH consumption were enriched in microRNA target sites and included target sites associated with Toll-like receptor pathways. The differentially expressed genes were also significantly enriched in transcription factor binding sites. CONCLUSIONS: The data presented here are the first to use a longitudinal biopsy strategy to examine how chronic EtOH consumption affects gene expression in the primate PFC. Prominent effects were seen in both cell adhesion and neuroimmune pathways; the latter contained both pro- and antiinflammatory genes. The data also indicate that changes in miRNAs and transcription factors may be important epigenetic regulators of EtOH consumption.

Regional Analysis of the Brain Transcriptome in Mice Bred for High and Low Methamphetamine Consumption.

Transcriptome profiling can broadly characterize drug effects and risk for addiction in the absence of drug exposure. Modern large-scale molecular methods, including RNA-sequencing (RNA-Seq), have been extensively applied to alcohol-related disease traits, but rarely to risk for methamphetamine (MA) addiction. We used RNA-Seq data from selectively bred mice with high or low risk for voluntary MA intake to construct coexpression and cosplicing networks for differential risk. Three brain reward circuitry regions were explored, the nucleus accumbens (NAc), prefrontal cortex (PFC), and ventral midbrain (VMB). With respect to differential gene expression and wiring, the VMB was more strongly affected than either the PFC or NAc. Coexpression network connectivity was higher in the low MA drinking line than in the high MA drinking line in the VMB, oppositely affected in the NAc, and little impacted in the PFC. Gene modules protected from the effects of selection may help to eliminate certain mechanisms from significant involvement in risk for MA intake. One such module was enriched in genes with dopamine-associated annotations. Overall, the data suggest that mitochondrial function and glutamate-mediated synaptic plasticity have key roles in the outcomes of selective breeding for high versus low levels of MA intake.

Scn4b regulates the hypnotic effects of ethanol and other sedative drugs.

The voltage-gated sodium channel subunit ß4 (SCN4B) regulates neuronal activity by modulating channel gating and has been implicated in ethanol consumption in rodent models and human alcoholics. However, the functional role for Scn4b in ethanol-mediated behaviors is unknown. We determined if genetic global knockout (KO) or targeted knockdown of Scn4b in the central nucleus of the amygdala (CeA) altered ethanol drinking or related behaviors. We used four different ethanol consumption procedures (continuous and intermittent two-bottle choice (2BC), drinking-in-the dark and chronic intermittent ethanol vapor) and found that male and female Scn4b KO mice did not differ from their wild-type (WT) littermates in ethanol consumption in any of the tests. Knockdown of Scn4b mRNA in the CeA also did not alter 2BC ethanol drinking. However, Scn4b KO mice showed longer duration of the loss of righting reflex induced by ethanol, gaboxadol, pentobarbital and ketamine. KO mice showed slower recovery to basal levels of handling-induced convulsions after ethanol injection, which is consistent with the increased sedative effects observed in these mice. However, Scn4b KO mice did not differ in the severity of acute ethanol withdrawal. Acoustic startle responses, ethanol-induced hypothermia and clearance of blood ethanol also did not differ between the genotypes. There were also no functional differences in the membrane properties or excitability of CeA neurons from Scn4b KO and WT mice. Although we found no evidence that Scn4b regulates ethanol consumption in mice, it was involved in the acute hypnotic effects of ethanol and other sedatives.

Regional Differences and Similarities in the Brain Transcriptome for Mice Selected for Ethanol Preference From HS-CC Founders.

The high genetic complexity found in heterogeneous stock (HS-CC) mice, together with selective breeding, can be used to detect new pathways and mechanisms associated with ethanol preference and excessive ethanol consumption. We predicted that these pathways would provide new targets for therapeutic manipulation. Previously (Colville et al., 2017), we observed that preference selection strongly affected the accumbens shell (SH) genes associated with synaptic function and in particular genes associated with synaptic tethering. Here we expand our analyses to include substantially larger sample sizes and samples from two additional components of the "addiction circuit," the central nucleus of the amygdala (CeA) and the prelimbic cortex (PL). At the level of differential expression (DE), the majority of affected genes are region-specific; only in the CeA did the DE genes show a significant enrichment in GO annotation categories, e.g., neuron part. In all three brain regions the differentially variable genes were significantly enriched in a single network module characterized by genes associated with cell-to-cell signaling. The data point to glutamate plasticity as being a key feature of selection for ethanol preference. In this context the expression of Dlg2 which encodes for PSD-93 appears to have a key role. It was also observed that the expression of the clustered protocadherins was strongly associated with preference selection.

Cross-species molecular dissection across alcohol behavioral domains.

This review summarizes the proceedings of a symposium presented at the "Alcoholism and Stress: A Framework for Future Treatment Strategies" conference held in Volterra, Italy on May 9-12, 2017. Psychiatric diseases, including alcohol-use disorders (AUDs), are influenced through complex interactions of genes, neurobiological pathways, and environmental influences. A better understanding of the common neurobiological mechanisms underlying an AUD necessitates an integrative approach, involving a systematic assessment of diverse species and phenotype measures. As part of the World Congress on Stress and Alcoholism, this symposium provided a detailed account of current strategies to identify mechanisms underlying the development and progression of AUDs. Dr. Sean Farris discussed the integration and organization of transcriptome and postmortem human brain data to identify brain regional- and cell type-specific differences related to excessive alcohol consumption that are conserved across species. Dr. Brien Riley presented the results of a genome-wide association study of DSM-IV alcohol dependence; although replication of genetic associations with alcohol phenotypes in humans remains challenging, model organism studies show that COL6A3, KLF12, and RYR3 affect behavioral responses to ethanol, and provide substantial evidence for their role in human alcohol-related traits. Dr. Rob Williams expanded upon the systematic characterization of extensive genetic-genomic resources for quantifying and clarifying phenotypes across species that are relevant to precision medicine in human disease. The symposium concluded with Dr. Robert Hitzemann's description of transcriptome studies in a mouse model selectively bred for high alcohol ("binge-like") consumption and a non-human primate model of long-term alcohol consumption. Together, the different components of this session provided an overview of systems-based approaches that are pioneering the experimental prioritization and validation of novel genes and gene networks linked with a range of behavioral phenotypes associated with stress and AUDs.

Gender-Specific Effects of Selection for Drinking in the Dark on the Network Roles of Coding and Noncoding RNAs.

BACKGROUND: Transcriptional differences between heterogeneous stock mice and high drinking-in-the-dark selected mouse lines have previously been described based on microarray technology coupled with network-based analysis. The network changes were reproducible in 2 independent selections and largely confined to 2 distinct network modules; in contrast, differential expression appeared more specific to each selected line. This study extends these results by utilizing RNA-Seq technology, allowing evaluation of the relationship between genetic risk and transcription of noncoding RNA (ncRNA); we additionally evaluate sex-specific transcriptional effects of selection. METHODS: Naïve mice (N = 24/group and sex) were utilized for gene expression analysis in the ventral striatum; the transcriptome was sequenced with the Illumina HiSeq platform. Differential gene expression and the weighted gene co-expression network analysis were implemented largely as described elsewhere, resulting in the identification of genes that change expression level or (co)variance structure. RESULTS: Across both sexes, we detect selection effects on the extracellular matrix and synaptic signaling, although the identity of individual genes varies. A majority of nc RNAs cluster in a single module of relatively low density in both the male and female network. The most strongly differentially expressed transcript in both sexes was Gm22513, a small nuclear RNA with unknown function. Associated with selection, we also found a number of network hubs that change edge strength and connectivity. At the individual gene level, there are many sex-specific effects; however, at the annotation level, results are more concordant. CONCLUSIONS: In addition to demonstrating sex-specific effects of selection on the transcriptome, the data point to the involvement of extracellular matrix genes as being associated with the binge drinking phenotype.

On the relationships in rhesus macaques between chronic ethanol consumption and the brain transcriptome.

This is the first description of the relationship between chronic ethanol self-administration and the brain transcriptome in a non-human primate (rhesus macaque). Thirty-one male animals self-administered ethanol on a daily basis for over 12 months. Gene transcription was quantified with RNA-Seq in the central nucleus of the amygdala (CeA) and cortical Area 32. We constructed coexpression and cosplicing networks, and we identified areas of preservation and areas of differentiation between regions and network types. Correlations between intake and transcription included largely distinct gene sets and annotation categories across brain regions and between expression and splicing; positive and negative correlations were also associated with distinct annotation groups. Membrane, synaptic and splicing annotation categories were over-represented in the modules (gene clusters) enriched in positive correlations (CeA); our cosplicing analysis further identified the genes affected only at the exon inclusion level. In the CeA coexpression network, we identified Rab6b, Cdk18 and Igsf21 among the intake-correlated hubs, while in the Area 32, we identified a distinct hub set that included Ppp3r1 and Myeov2. Overall, the data illustrate that excessive ethanol self-administration is associated with broad expression and splicing mechanisms that involve membrane and synapse genes.

Alignment of the transcriptome with individual variation in animals selectively bred for High Drinking-In-the-Dark (HDID).

Among animals at risk for excessive ethanol consumption such as the HDID selected mouse lines, there is considerable individual variation in the amount of ethanol consumed and the associated blood ethanol concentrations (BECs). For the HDID lines, this variation occurs even though the residual genetic variation associated with the DID phenotype has been largely exhausted and thus is most likely associated with epigenetic factors. Here we focus on the question of whether the genes associated with individual variation in HDID-1 mice are different from those associated with selection (risk) (Iancu et al., 2013). Thirty-three HDID-1 mice were phenotyped for their BECs at the end of a standard DID trial, were sacrificed 3 weeks later, and RNA-Seq was used to analyze the striatal transcriptome. The data obtained illustrate that there is considerable overlap of the risk and variation gene sets, both focused on the fine-tuning of synaptic plasticity.

Diversity Outbred Mice at 21: Maintaining Allelic Variation in the Face of Selection.

Multi-parent populations (MPPs) capture and maintain the genetic diversity from multiple inbred founder strains to provide a resource for high-resolution genetic mapping through the accumulation of recombination events over many generations. Breeding designs that maintain a large effective population size with randomized assignment of breeders at each generation can minimize the impact of selection, inbreeding, and genetic drift on allele frequencies. Small deviations from expected allele frequencies will have little effect on the power and precision of genetic analysis, but a major distortion could result in reduced power and loss of important functional alleles. We detected strong transmission ratio distortion in the Diversity Outbred (DO) mouse population on chromosome 2, caused by meiotic drive favoring transmission of the WSB/EiJ allele at the R2d2 locus. The distorted region harbors thousands of polymorphisms derived from the seven non-WSB founder strains and many of these would be lost if the sweep was allowed to continue. To ensure the utility of the DO population to study genetic variation on chromosome 2, we performed an artificial selection against WSB/EiJ alleles at the R2d2 locus. Here, we report that we have purged the WSB/EiJ allele from the drive locus while preserving WSB/EiJ alleles in the flanking regions. We observed minimal disruption to allele frequencies across the rest of the autosomal genome. However, there was a shift in haplotype frequencies of the mitochondrial genome and an increase in the rate of an unusual sex chromosome aneuploidy. The DO population has been restored to genome-wide utility for genetic analysis, but our experience underscores that vigilant monitoring of similar genetic resource populations is needed to ensure their long-term utility.

Cosplicing network analysis of mammalian brain RNA-Seq data utilizing WGCNA and Mantel correlations.

Across species and tissues and especially in the mammalian brain, production of gene isoforms is widespread. While gene expression coordination has been previously described as a scale-free coexpression network, the properties of transcriptome-wide isoform production coordination have been less studied. Here we evaluate the system-level properties of cosplicing in mouse, macaque, and human brain gene expression data using a novel network inference procedure. Genes are represented as vectors/lists of exon counts and distance measures sensitive to exon inclusion rates quantifies differences across samples. For all gene pairs, distance matrices are correlated across samples, resulting in cosplicing or cotranscriptional network matrices. We show that networks including cosplicing information are scale-free and distinct from coexpression. In the networks capturing cosplicing we find a set of novel hubs with unique characteristics distinguishing them from coexpression hubs: heavy representation in neurobiological functional pathways, strong overlap with markers of neurons and neuroglia, long coding lengths, and high number of both exons and annotated transcripts. Further, the cosplicing hubs are enriched in genes associated with autism spectrum disorders. Cosplicing hub homologs across eukaryotes show dramatically increasing intronic lengths but stable coding region lengths. Shared transcription factor binding sites increase coexpression but not cosplicing; the reverse is true for splicing-factor binding sites. Genes with protein-protein interactions have strong coexpression and cosplicing. Additional factors affecting the networks include shared microRNA binding sites, spatial colocalization within the striatum, and sharing a chromosomal folding domain. Cosplicing network patterns remain relatively stable across species.

Splicing landscape of the eight collaborative cross founder strains.

BACKGROUND: The Collaborative Cross (CC) is a large panel of genetically diverse recombinant inbred mouse strains specifically designed to provide a systems genetics resource for the study of complex traits. In part, the utility of the CC stems from the extensive genome-wide annotations of founder strain sequence and structural variation. Still missing, however, are transcriptome-specific annotations of the CC founder strains that could further enhance the utility of this resource. RESULTS: We provide a comprehensive survey of the splicing landscape of the 8 CC founder strains by leveraging the high level of alternative splicing within the brain. Using deep transcriptome sequencing, we found that a majority of the splicing landscape is conserved among the 8 strains, with ~65% of junctions being shared by at least 2 strains. We, however, found a large number of potential strain-specific splicing events as well, with an average of ~3000 and ~500 with ≥3 and ≥10 sequence read coverage, respectively, within each strain. To better understand strain-specific splicing within the CC founder strains, we defined criteria for and identified high-confidence strain-specific splicing events. These splicing events were defined as exon-exon junctions 1) found within only one strain, 2) with a read coverage ≥10, and 3) defined by a canonical splice site. With these criteria, a total of 1509 high-confidence strain-specific splicing events were identified, with the majority found within two of the wild-derived strains, CAST and PWK. Strikingly, the overwhelming majority, 94%, of these strain-specific splicing events are not yet annotated. Strain-specific splicing was also located within genomic regions recently reported to be over- and under-represented within CC populations. CONCLUSIONS: Phenotypic characterization of CC populations is increasing; thus these results will not only aid in further elucidating the transcriptomic architecture of the individual CC founder strains, but they will also help in guiding the utilization of the CC populations in the study of complex traits. This report is also the first to establish guidelines in defining and identifying strain-specific splicing across different mouse strains.