Conserved noncoding cis elements associated with hibernation modulate metabolic and behavioral adaptations in mice

Abstract

Cis-regulatory elements (CREs) drive phenotypic diversity, yet how CREs are causally linked to function remains largely unclear. Our study elucidates functions for conserved ciselements associated with the evolution of mammalian hibernation and metabolic flexibility. Genomic analyses revealed topologically associated domains (TADs) enriched for convergent changes in hibernators, including the Fat Mass & Obesity (Fto) locus. In this TAD, we uncovered genetic circuits for metabolic responses and hibernation-linked ciselements forming regulatory contacts with neighboring genes. Deletions of individual ciselements in mice differentially altered Fto, Irx3, and Irx5 expression, reshaping downstream gene expression programs and affecting metabolism, torpor, obesogenesis, and foraging in distinct ways. Our findings show how convergent evolution in hibernators pinpoints functional genetic mechanisms of metabolic control, with multiple effects encoded in single CREs.

Fig. 1.. Convergent genomic changes in hibernators identify Fto-Irx TAD cis-elements for functional analysis in knockout mice to affect metabolic gene regulatory networks.

(A) Volcano plots show the odds ratio and p-values for TADs throughout the mouse genome to be enriched for hibernator (blue) and homeotherm (red) convergent genomic changes relative to background conserved regions (CRs). The results in the left plot for pHibARs (blue) and pHomeoARs (red) are based on a chi-square test of a contingency table of AR and CR counts (rows) by hibernator and homeotherm data (columns). The same analysis for pHibDELs versus pHomeoDELs is shown in the right plot. Significantly enriched TADs for hibernator (blue circles) or homeotherm (red circles) convergent genomic changes are shown (q-value<0.05). Other TADs are indicated by grey circles. The Fto-Irx TAD is among the top enriched TADs for both pHibARs and pHibDELs. (B) Barplots show the odds ratio for Irx3 and Irx5 binding site motifs to be located in adult female mouse hypothalamus ATAC-Seq peak sites of open chromatin (FDR 5%) that are specific to fed, 72-hr fasted, versus 12-hr refed conditions compared to peaks that are shared across all three conditions. The results show that Irx3 motifs are enriched in Fed, 72-hr fasted, and 12-hr refed specific peak sites, whereas Irx5 motifs are enriched in 72-hr fasted, and 12-hr refed specific sites. (C) Unsupervised hierarchical clustering (Poisson Dissimilarity Matrix, Ward’s method) on Irx3 Cut&Run binding site profiles in the mouse hypothalamus shows that replicates cluster according to fed, 72-hr fasted, and 12-hr refed condition, indicating metabolic state specific binding profiles (n=4 mice). Horizontal axis shows linkage Euclidean distance. (D) An example of a significant Irx3 binding site peak identified from Cut&Run across all replicates and conditions (q<0.0001, Genrich peak calling). (E) Number of significant differential Irx3 binding sites identified from fed vs. fasted, fasted vs refed and fed vs refed contrasts (q<0.1; EdgeR, n=4 mice). (F) A venn diagram of the numbers of genes contacted by Irx3 binding sites in fed, fasted, and refed conditions as revealed by Cut&Run and H3K27ac+ PLAC-Seq in the mouse hypothalamus. (G) Circos plot displaying genome-wide tracks of Cut&Run significant Irx3 binding sites in the hypothalamus for fed (yellow), fasted (blue) and refed (green) conditions. Arrows in the center connect the Irx3 locus to fed specific, fasting specific, refed specific or shared binding sites. See legend. (H) The plot displays multi-omics data for the Fto-Irx TAD focused on 5 cis-elements showing convergent genomics changes in hibernators (Fto-Irx::hibE1-5 cis-elements) that were selected for functional studies in knockout mice and the genomic size and coordinates for each cis-element. PLAC-Seq for H3K27ac shows the regulatory contacts made by each knocked out cis-element (E1, dark blue; E2, light blue; E3, red; E4, green; E5, purple) and reveals that each cis-element makes multiple long-distance contacts that affect Fto, Irx3, and Irx5, but not Rpgrip1l. Tracks (brown) are shown for the pHibARs and pHibDELs identified in the TAD and show the knockout targeted regions overlap with pHibARs (E1, E3, E4, E5) or pHibARs and pHibDELs (E2). The snATAC-Seq tracks show significant ATAC-Seq peaks in each targeted cis-element identified from single cell multi-omics profiling of fed, 72-hr fasted, and 12-hr refed adult mouse hypothalamus (FDR < 5%). The open chromatin peaks for each targeted cis-element in oligodendrocyte precursor cells (OPCs), astrocytes (Astro), endothelial cells (endo), GABAergic neurons (gaba), Glutamatergic neurons (Glut), Microglia (micro), Other neurons (neuron), and Oligodendrocytes (Oligo).

Fig. 2.. Gene expression alterations in the hypothalamus of Fto-Irx::hibE cis-element knockout mice under fed and 72-hr fasted conditions.

(A-E) The barplots show the RNA-Seq measured expression of Fto, Irx3, and Irx5 in the hypothalamus in each of the Fto-Irx::hibE knockout mouse lines (n=4 mice, two-tailed test (Fto) or One-way ANOVA (Irx3 and Irx5) with Tukey post-test). The H3K27ac+ PLAC-Seq detected regulatory contacts for each cis-element are shown above, along with tracks for pHibARs and the location of each knocked out cis-element (E). Data for mice in a fed (grey box) versus 72-hr fasted condition (orange box) are shown. CPM, counts per million. The results show how each element affects Fto, Irx3, and Irx5 expression in two different metabolic conditions. (F) The heatmap shows the expression of RNA-Seq detected differentially expressed genes (DEGs, FDR < 5%) in the hypothalamus for Fto-Irx::hibE3^−/− mice in fed and 72-hr fasted conditions relative to ^+/+ littermates. (G) The barplots show the numbers of significant DEGs (RNA-Seq, FDR<5%) detected in the hypothalamus of ^−/− versus ^+/+ mice for fed and 72-hr fasted conditions for all 5 different Fto-Irx::hibE cis-element knockout mice (E1-5). (H) The plot shows significant KEGG pathway enrichments from a gene set analysis of significant DEGs in Fto-Irx::hibE2^−/− , Fto-Irx::hibE3^−/− , and Fto-Irx::hibE4^−/− mice.

Fig. 3.. Distinct Metabolic and Thermoregulatory Response Profiles in Fto-Irx::hibE cis-Element Knockout Mice Across Fed-Fasted-Refed Stages of Fasting-Induced Torpor.

(A) The plot shows internal body temperature (Tbi) in mice over 7-days while placed in CLAMS cages for a metabolic assay of processes pertinent to hibernation. The assay has a 48-hr pre-torpor phase (red, ad libitum food + 25°C), 48-hr torpor phase (blue, food deprived + 18°C), and a 72-hr post-torpor ad libitum refeeding phase (orange, ad libitum food + 25°C). Mice show circadian changes with elevated Tbi during active periods (dark cycle, grey bars) and depressed Tbi during inactive periods (light cycle, colored bars). During the torpor phase, Tbi drops, which is indicative of torpor, and then rapidly recovers during refeeding. Dark green line shows the mean, whereas the shaded area shows S.E.M. (B-E) The plots show the metabolic rate (O₂ consumption ml/hr) measured from ^−/−(pink) versus ^+/+ (green) adult female mice in CLAMS cages during the metabolic assay. Mean metabolic rate results (solid line) are shown and S.E.M. (shaded area) for Fto-Irx::hibE1^−/− mice (n=11-14 mice) (B), Fto-Irx::hibE2^−/− mice (n=11 mice) (C), Fto-Irx::hibE3^−/− mice (n=11 mice) (D), and Fto-Irx::hibE4^−/− mice (n=11 mice) (E). Statistical significance in the pre-torpor, torpor, and refeeding phases was determined using a generalized linear regression analysis of the mean metabolic rate for each day and light-dark cycle and tested for a main effect of genotype and an interaction effect between genotype and day/light-dark cycle. Variance due to batch effects from independent CLAMS cohorts was accounted for by a “batch” term in the model. P-values in black show the genotype effect (significant p-values in bold). P-values in blue show the genotype x day/light-dark cycle interaction effect (only significant p-values shown; C, torpor phase). *p<0.05, **p<0.01, ***p<0.001

Fig. 4.. Distinct Effects of Fto-Irx::hibE Cis-Elements on Body Weight and High-Fat Diet-Induced Weight Gain in Males and Females.

(A-H) The plots show the increase in body weight for −/− (purple line) versus +/+ (green line) adult male (A, C, E, G) and female (B, D, F, H) mice on a high fat diet over 16 weeks. Data for Fto-Irx::hibE1 (A,B), Fto-Irx::hibE2 (C,D), Fto-Irx::hibE3 (E,F), and Fto-Irx::hibE4 (G,H) −/− versus +/+ control mice are shown (n shown, bottom right). *p<0.05, **p<0.01, ***p<0.001. Likelihood ratio test of regression models. Mean ± SEM.

Fig. 5.. Alterations to Naturalistic Foraging in Fto-Irx::hibE3^−/− Mice with Age.

(A) The schematic shows the foraging assay design, including the home cage attached to the arena by a tunnel. The arena has 4 potential food patches (Pots1-4) and one has food (Pot2, red) in the 30-min naïve Exploration phase (red pot). In the 30-min Foraging phase 4 hours later, the food is moved from Pot2 and buried in Pot4 (green), such that mice express memory response behaviors of the familiar environment and former food location (Pot2). The diagrams show the key locations in the assay, including zones not involving a Pot or the home (Not-At-Pot, NAP locations). (B) Foraging behaviors are tracked and segmented into discrete round-trip excursions from the home and then decomposed by 52 measures of different behavioral components. (C) The plots show Fto-Irx::hibE3^−/− (orange) and ^+/+ control mice (blue) foraging patterns from the cumulative time spent at each location (seconds) in the Exploration (green) and Foraging (purple) phases for male + female mice, including young adult (8–12-week-old) and (12-month-old) aged adult cohorts (see titles). Bonferroni corrected p-values across the 4 conditions shown (p.Bonf). Bars show mean ± SEM. * p.BH<0.05. TTP, total time at Pot (1-4). (D and E) The schematic depicts how different round trip foraging excursions are grouped into stereotypic foraging excursion types, called modules, using an unsupervised machine learning approach (C). The Venn diagram shows the numbers of foraging modules uncovered from the 156 mice and 7815 excursions analyzed in the Exploration and Foraging phases. The PHATE map shows 2-D embeddings of each excursion colored according to foraging module classification (D). The results show that excursions classified to the same module are closely clustered (see 3-D embeddings in Movie S2). (F) Results for likelihood ratio test comparing two Poisson regression models assessing the interaction between genotype, age, sex, and module on foraging excursions performed by the 156 tested male, female, young, aged, −/− and +/+ mice. Model 1 included additive effects of Genotype and Module, controlling for sex and age differences, whereas Model 2 included their interaction (Module × Genotype). Bonferroni correction for multiple testing in the Exploration and Foraging phases. Df, Model 1 vs. 2 changes in degrees of freedom; n, number of excursions. (G) Post-hoc estimated marginal means analysis of the Foraging phase data identified specific modules where genotype had a significant effect with interactions further modulated by age. The plot illustrates the difference in mean expression between homozygous (−/−; HOM) and wild-type (+/+; WT) mice for significantly affected modules during the Foraging phase, stratified by young and aged adults (BH corrected p < 0.05). The HOM–WT difference in estimated marginal mean expression is represented by dots, with error bars denoting ± standard error (SE). Only modules that show significant differential expression between HOM and WT mice are displayed. *p.adjust<0.05; **p.adjust<0.01. (H) The PHATE map shows the 2-D embeddings for the foraging excursions for module 11 (red) and module 46 (green) that show significant differential expression between ^−/− versus ^+/+ mice in aged adults in the Foraging phase (G). The purple traces show foraging excursion X-Y-Z movement sequences representative of each affected module shaded by movement velocity (purple, see legend). The inset barplot shows the time spent at Pot4 for Module 11 versus 46 excursions expressed by aged mice pooled for genotype and sex (t-test, n=16-21). *p<0.05.