Systems genetics analysis of the LXS recombinant inbred mouse strains:Genetic and molecular insights into acute ethanol tolerance

Abstract

We have been using the Inbred Long- and Short-Sleep mouse strains (ILS, ISS) and a recombinant inbred panel derived from them, the LXS, to investigate the genetic underpinnings of acute ethanol tolerance which is considered to be a risk factor for alcohol use disorders (AUDs). Here, we have used RNA-seq to examine the transcriptome of whole brain in 40 of the LXS strains 8 hours after a saline or ethanol "pretreatment" as in previous behavioral studies. Approximately 1/3 of the 14,184 expressed genes were significantly heritable and many were unique to the pretreatment. Several thousand cis- and trans-eQTLs were mapped; a portion of these also were unique to pretreatment. Ethanol pretreatment caused differential expression (DE) of 1,230 genes. Gene Ontology (GO) enrichment analysis suggested involvement in numerous biological processes including astrocyte differentiation, histone acetylation, mRNA splicing, and neuron projection development. Genetic correlation analysis identified hundreds of genes that were correlated to the behaviors. GO analysis indicated that these genes are involved in gene expression, chromosome organization, and protein transport, among others. The expression profiles of the DE genes and genes correlated to AFT in the ethanol pretreatment group (AFT-Et) were found to be similar to profiles of HDAC inhibitors. Hdac1, a cis-regulated gene that is located at the peak of a previously mapped QTL for AFT-Et, was correlated to 437 genes, most of which were also correlated to AFT-Et. GO analysis of these genes identified several enriched biological process terms including neuron-neuron synaptic transmission and potassium transport. In summary, the results suggest widespread genetic effects on gene expression, including effects that are pretreatment-specific. A number of candidate genes and biological functions were identified that could be mediating the behavioral responses. The most prominent of these was Hdac1 which may be regulating genes associated with glutamatergic signaling and potassium conductance.

Fig 1. Heritability (H²) of expression for individual genes in the LXS RI panel.

Only genes that were significantly heritable in the saline and/or ethanol (Et) pretreatment are shown (FDR<0.05; n=5,054). Each dot represents a single gene. Red dots: significant in both pretreatment groups; green dots: significant in saline only; blue dots: significant in ethanol only.

Fig 2. Genes that were differentially expressed (DE) due to ethanol (EtOH) pretreatment in the LXS RI panel (FDR<0.05).

(A) Number of DE genes by RI strain. Strains 76, 115, and 122 did not have any DE genes. (B) Distribution of the fold-change in expression due to ethanol pretreatment (log2; n=1,230).

Fig 3. UpSet plot of the number of eQTLs in the LXS RI panel following saline or ethanol (EtOH) pretreatment.

Set Size is the total number of eQTLs in each group. Interaction Size represents the number of eQTLs in the intersections of the groups as indicated by the black dots below the bars. The black portion of the bars in the Interaction panel represents the number of pretreatment-unique eQTLs that are “golden” (see text). The number of those “golden” eQTLs is shown in parentheses.

Fig 4. UpSet plot of the number of genes correlated to AFT or ST in the LXS RI panel following saline or ethanol (EtOH) pretreatment.

See Fig 3 legend for additional details.

Fig 5. REVIGO plots of significantly enriched GO Biological Process terms.

(A) Genes correlated to ST, ethanol (Et) pretreatment. (B) Genes correlated to AFT, ethanol (Et) pretreatment. (C) DE genes. Fold enrichment is the proportion of term genes found in the input list compared to the proportion of total term genes found in the background. The size of the bubble is inversely proportional to the number of genes in the term; i.e., the larger the bubble, the more specific the term.

Fig 6. Circos plot of genes correlated to Hdac1 in the ethanol pretreatment group (nominal p<0.01; n=446).

Colored lines connect Hdac1 to the location of the genes to which it is correlated. Black bars for each gene show the absolute value of the Pearson product-moment correlation coefficient (r) between the gene’s expression and AFT-Et (range of r values: <0.01 to 0.67). The red line over the black bars indicates the nominal p<0.05 cutoff for the correlation between gene expression and AFT-Et (r ≅ 0.31).