Genomic convergence in hibernating mammals elucidates the genetics of metabolic regulation in the hypothalamus

Abstract

Extreme metabolic adaptations can elucidate genetic programs that govern mammalian metabolism. Here, we used convergent evolutionary changes in hibernating lineages to define conserved cis-regulatory elements (CREs) and metabolic programs. We characterized mouse hypothalamus gene expression and chromatin dynamics across fed, fasted, and refed states and then used comparative genomics of hibernating versus nonhibernating lineages to identify cis elements with convergent changes in hibernators. Multi-omics approaches pinpointed CREs, hub genes, regulatory programs, and cell types underlying lineage divergence. Hibernators accumulated loss-of-function effects for CREs regulating hypothalamic responses, and the refeeding period after fasting served as a key phase for molecular processes with convergent evolutionary changes. This work provides a genetic framework for harnessing hibernator adaptations to understand human metabolic control.

Fig. 1.. Dynamics of metabolic states, gene expression, and chromatin accessibility across stages of the Fed-Fasted-Refed (FFR) response in the mouse hypothalamus.

(A) Schematic of the Fed-Fasted-Refed (FFR) cycle in obligate hibernators, highlighting metabolic states, processes, and seasonal cycles. Facultative hibernators show acute torpor responses to food deprivation and/or cold, rather than seasonal cycles. Homeotherms show little metabolic and physiological flexibility, though mice are facultative heterotherms capable of brief stress-induced torpor bouts. (B) Internal body temperature and respiratory exchange ratio (RER) in adult female mice across Fed (24°C, yellow), Fasted + Cold (18°C, blue), and Refed (green) states over 10 days. Red and orange dashed lines represent mean RER for dark and light cycles, respectively. Black p-values are fed vs. refed contrasts and purple p-values are Day 1 vs. 9 contrasts. Data are mean ± SEM (n = 10). See fig. S1 for further data. (C) Heatmap of RNA-Seq gene expression in the adult mouse hypothalamus for differentially expressed genes across FFR states (FDR <5%, n=3-4), showing fold change scaled by row. FA: fasted; RF: refed; 12hrs RF: refed 12 hours after 72 hours fasting; 1w RF: refed 1 week after 72 hours fasting; h: hours; w: weeks; Cold: 18°C vs. 24°C ambient temperature. (D) Circular plot showing the number of significant differentially expressed genes (FDR < 5%) at each step of the FFR cycle in the mouse hypothalamus, with bar and arrow colors indicating gene expression comparisons. (E and F) The heatmap (E) shows genes with increased expression from 72-hour fasted to 1-hour refed, and the bar plot (F) highlights KEGG pathways enriched for these DEGs (FDR < 5%). (G) Signal plots of ATAC-Seq data where the highlighted area indicates a significant peak in the 12-hr refed condition (n = 8, FDR<5%). (H) Venn diagram showing significant ATAC-Seq peaks genome-wide in the hypothalamus, with increased chromatin accessibility in refeeding (RF) compared to Fed and 72-hour fasted (FA) states (Genrich R package; FDR < 5%, n = 8).

Fig. 2.. Convergent genomic changes in hibernators affect gene regulatory mechanisms governing hypothalamic FFR responses.

(A) Schematic summary of hibernator-homeotherm comparative genomics analysis. (B) Phylogenetic tree from the 241-way mammalian alignment (241-mammalian-2020v2b.maf), showing obligate hibernators (blue), homeotherm controls (red), and background homeothermic species (black) used to define conserved regions (phastCons, FDR < 5%). Conserved regions were tested for accelerated evolution (ARs, phyloP, LRT; FDR < 5%) or deletions in hibernators and controls. (C) Images of each hibernator (blue) and control homeothermic species (red), with the number of significant accelerated regions (ARs, FDR 5%) and deletions (DELs) identified from ~1.3 million conserved regions shown below. Images and data correspond to species in the adjacent phylogenetic tree (A). (D and E) Bar plots showing the number of ARs (C) or deleted regions (D) shared by at least 2 of 4 hibernators (pHibARs, blue) or homeotherm controls (pHomeoARs, red). (F and G) H3K27ac+ PLAC-Seq identifies significant promoter regulatory contacts genome-wide in the mouse hypothalamus (FDR < 1%, total read count ≥ 12, observed/expected > 2; n = 10). (F) Heatmap showing the number of significant contacts on chromosome 18. (G) Contact plot for Spats2l (a significant FFR response DEG) illustrates how PLAC-Seq links pHibARs (light blue) and pHibDELs (dark blue) to contacted gene promoters. Contact loop height reflects the observed/expected read count. Tracks also include significant H3K27ac+ ChIP-Seq peaks (FDR < 5%, orange), FFR cycle ATAC-Seq peaks (FDR < 5%, black), gene models, and highlighted promoters (gray bars). (H) Pie charts of hypothalamus PLAC-Seq data show the proportions of pHibARs (light blue) and pHibDELs (dark blue) overlapping 10 kb windows with promoter-promoter (P-P, darker), enhancer-promoter (E-P, medium), or no promoter (No P, light) contacts. (I) Bar plot showing the odds ratio of pHibARs (blue) and pHomeoARs (red) in 10 kb regions contacting promoters of hypothalamus FFR response differentially expressed genes (DEGs) compared to background conserved regions. pHibARs show significant enrichment over pHomeoARs (Woolf test). ****p < 0.0001, **p < 0.01, *p < 0.05. (J) pHibARs and pHomeoARs were tested for enrichment in open chromatin sites (significant ATAC-Seq peaks, FDR < 5%, n = 8) relative to background conserved regions. pHibARs show significant enrichment compared to pHomeoARs (Woolf test). ****p < 0.0001; ns, not significant.

Fig. 3.. pHibARs regulate hub genes driving FFR response gene co-expression modules and ARs indicate CRE loss-of-function.

(A) Schematic summary of FFR response hub gene discovery and hibernator-homeotherm comparative analysis. (B) Bar plot showing the number of hub genes for each of the 41 FFR response gene co-expression modules in the mouse hypothalamus. (C) Bar plot showing the -log10(p-value) for pHibAR and pHomeoAR enrichment at FFR response hub genes compared to background conserved regions (LOLA, R). Red line indicates p = 0.05. (D) The Venn diagram shows the overlap between FFR response hub genes and genes regulated by pHibARs (Hypergeometric test, p = 2.7 × 10⁻⁵). (E) Comparison of the number of FFR response co-expression modules with and without Hibernation-Hub genes. (F) H3K27ac+ PLAC-Seq promoter contacts for the blue2 FFR co-expression module Hibernation-Hub gene, En1. Red contacts overlap pHibARs (light orange highlight) and blue contacts do not. The kWithin measures En1 intramodular connectivity. pHibARs are in orange, with gene models below (En1 highlighted in blue). (G) Bar plot showing significantly enriched Gene Ontology molecular function terms for hibernation-hub genes compared to all genes expressed in the adult mouse hypothalamus (FDR < 5%, ClusterProfiler R; bar colors correspond to BH-adjusted p-values). (H) Schematic summarizing potential effects of ARs on H3K27ac+ active CREs in the 13-lined ground squirrel compared to mice: (1) gain of CRE activity (new H3K27ac+ peaks in squirrels), (2) neutral effects (squirrel-mouse shared H3K27ac+ peaks with sequence divergence), or (3) loss of CRE activity (H3K27ac+ peaks present in mice but absent in squirrels). (I) Bar plots showing the odds ratio for squirrel ARs and mouse ARs overlapping H3K27ac+ peaks: squirrel-specific (blue), mouse-squirrel shared (purple), and mouse-specific (red), compared to conserved regions. Woolf test. ***p < 0.0001. (J) Plots showing H3K27ac+ ChIP-Seq results for the 13-lined ground squirrel (blue) and mouse (red) hypothalamus (normalized read counts for two replicates). Significant peaks in one or both species are highlighted.

Fig. 4.. Single-nucleus multi-omics links FFR responses and Hibernation-Hub genes to distinct hypothalamic cell types.

(A) Schematic summary of cellular multi-omics analysis of mouse hypothalamus FFR responses and the identification of hibernator-homeotherm genomic divergence in cellular FFR response regulatory mechanisms. (B and C) UMAP plots showing cell type (A) and cluster (B) classifications (Seurat and Harmony) from hypothalamus snRNA-Seq data for fed (n = 3), 72-hr fasted (n = 3), and 12-hr refed (n = 3) adult female mice, identifying 8 major cell types (A) and 42 clusters (B) from 89,063 cells. (D) The bar plot shows mean ± SEM cell counts per cluster across Fed, 72-hr fasted, and 12-hr refed conditions (n = 3), normalized by total cells per replicate. P-value is shown for cell cluster by FFR condition interaction effect with post-tests (*p < 0.05, **p < 0.01). (E) The heatmap shows the -log10 q-values for FFR co-expression module Hibernation-Hub gene (rows) enrichment among marker genes of cell clusters (columns), compared to all hypothalamic genes. Only significant modules are displayed. Red text highlighted cell clusters are significantly affected by FFR condition (see D). Cell types and clusters are color-coded by the legend. (F) UMAP heatmaps display snRNA-Seq expression for two example darkolivegreen module Hibernation-Hub genes. (G) Violin plots show snRNA-Seq expression level of darkolivegreen4 Hibernation-Hub genes across all cell clusters. Red text highlighted cell clusters are significantly affected by FFR condition (see D).

Fig. 5.. FFR-driven chromatin changes reveal hibernation-linked genetic programs across hypothalamic cell types.

(A) The bar plot shows the number of significant ATAC-Seq peaks for each hypothalamic cell type and FFR condition (Genrich, FDR < 5%, n = 3). P-values are from regression tests of FFR condition and cell-type effects on peak counts. Cell types: Endothelial (Endo), oligodendrocyte precursor cells (OPC), oligodendrocytes (Oligo), microglia (Micro), astrocytes (Astro), GABAergic neurons (GABA), glutamatergic neurons (GLUT), and unclassified neurons (Neuron). (B) Mosaic plot showing the relationship between ATAC-Seq peak numbers, cell type and FFR condition (Chi-Square p-value shown). Pearson residuals indicate relative enrichment (blue) or depletion (red) for each category. (C) Bar plots show the odds ratio for pHibARs and pHomeoARs in significant ATAC-Seq peaks across hypothalamic cell types during Fed, 72-hr fasted, and 12-hr refed conditions relative to conserved regions. LOLA R package enrichment test, ***p < 0.001, **p < 0.01. See also Fig. S15. (D) Bar plot showing the number of active CREs (significant ATAC-Seq peaks) overlapping pHibARs and/or pHibDELs in each cell type, highlighting hibernation-linked CREs. (E) UMAP of snRNA-Seq showing Hspa8 expression across cells and FFR conditions, with decreased expression in the 72-hr fasted condition. (F) Violin plots showing Hspa8 expression across cell types and FFR conditions. (G) Cell type-specific hibernation-linked regulatory programs for Hspa8 in the hypothalamus. H3K27ac+ PLAC-Seq data shows Hspa8 promoter contacts (10 kb cis-bin windows), with tracks for pHibARs (red), pHibDELs (orange), and significant snATAC-Seq peaks (blue). Peaks overlapping pHibARs are highlighted (red lines). Green tracks show snATAC-Seq peaks with significant differential accessibility between Fed, 72-hr fasted, and/or 12-hr refed conditions (q < 0.1), with PLAC-Seq bins overlapping differentially accessible regions (DARs) highlighted in green. Promoter contacts are color-coded: FFR DARs (green), pHibARs in ATAC-Seq peaks (red), pHibDELs in ATAC-Seq peaks (orange), and other ATAC-Seq peaks (blue). Gene models below, with Hspa8 promoter-associated distal contact sites highlighted in purple.