ENH5 is active in adipose tissue and is conserved in sequence and function in mouse adipocytes.

(A) The genomic structure of the human EPAS1 locus (chr2: 46524547-46613836, hg19). The locus is zoomed in at the ENH5 region (gray highlight: chr2: 46578867-46579856, hg19).Chromatin state tracks from the NIH Roadmap Epigenomics Mapping Consortium are shown for adipose, lung, fetal kidney, and fetal brain tissue. (B) Luciferase assay results of ENH5 low- and high-altitude alleles in 3T3-L1 cells in hypoxia (1% O2, 48 hours) and normoxia. The average ± SEM of 6 technical replicates (n=6) is shown. (C) The human EPAS1 sequence is highly conserved in mice. The gray highlight indicates ENH5. (D) Luciferase assay results of mouse ENH5 in 3T3-L1 cells in hypoxia (1% O2, 48 hours) and normoxia. The average ± SEM of 6 technical replicates (n=6) is shown. (E) The genomic structure of the mouse EPAS1 locus (chr17: 86753864-86833410, mm10). The locus is zoomed in at the ENH5 region (chr17: 86801737-86802738, mm10). The cut sites denote the target sites of the CRISPR gRNAs used to generate the homozygous ENH5 KO mouse line that has been previously established9. Data were analyzed using a one-tailed t-test. * p < 0.05, ** p < 0.01, and *** p< 0.001

ENH5 does not impact adipose biology or metabolic response in vivo.

(A) 8 week-old male and female WT and ENH5 KO mice were placed on a 12-week high-fat diet (HFD) (n = 9 male WT mice (WTM), n = 10 male ENH5 KO mice (KOM), n = 12 female WT mice (WTF) and n = 14 female ENH5 KO mice (KOF). Weight was measured weekly from weening (week 3) until the end of the HFD. (B) Abdominal fat percentage of mice before and after the HFD obtained from DEXA scans. (C) Intraperitoneal glucose tolerance tests (IPGTT) in WT and ENH5 KO mice before and after the HFD. Bar graphs represent area under curve (AUC). (D) Sections of subcutaneous fat from WT and ENH5 KO mice after the HFD. Bar graphs show cell count and cell size for 4 mice per genotype per sex. Data are presented as average ± SEM. Data were analyzed using two-tailed t-test. * p < 0.05 and ** p < 0.01.

ENH5 KO more strongly downregulates expression of key genes involved in thermogenesis response upon CL316243 treatment compared to WT.

(A) Experimental design showing preadipocytes being isolated from the subcutaneous fat pads of male WT (n = 6) and ENH5 KO mice (n = 6). Preadipocytes were then differentiated into mature adipocytes in vitro and stimulated with CL316243 for 4 hours. An interaction analysis for genotype by treatment was conducted to identify differentially expressed genes between WT and ENH5 KO response to treatment. (B) GSEA showing the top 10 enriched gene sets more strongly downregulated in ENH5 KO response than WT response to CL316243 treatment. The GSEA enrichment plot shows the enrichment score for the aerobic respiration and respiratory electron transport gene set. The red line indicates the running enrichment score (y-axis) while going down the ranked gene list (x-axis). The horizontal bars indicate the position of genes from the given gene set in the ranked gene list, where those at the top of the list are more upregulated in ENH5 KO response to CL316243 treatment compared to WT response while those at the bottom of the list are more downregulated in ENH5 KO response. Adipogenesis Burton 5 (MSigDB: M1564) and Adipogenesis Burton 6 (MSigDB: M1678) represent adipogenesis signaling pathways at early and late differentiation, respectively. (C) Heatmaps of the genes making up the aerobic respiration and respiratory electron transport gene set. Genes are clustered based on the electron complex chain component they are associated with. Diagram in panel A created with BioRender.

ENH5 KO more strongly downregulates expression of key genes involved in thermogenesis response under hypoxic conditions compared to WT.

(A) Experimental design showing preadipocytes being isolated from the subcutaneous fat pads of male WT (n = 5) and ENH5 KO mice (n = 4). Preadipocytes were then differentiated in vitro to mature adipocytes, then cultured in hypoxia (1% O2) for 48 hours. An interaction analysis for genotype by treatment was conducted to identify differentially expressed genes between WT and ENH5 KO response to hypoxia. (B) GSEA showing the top 10 enriched gene sets more strongly downregulated in ENH5 KO response than WT response to hypoxia is shown. The GSEA enrichment plot shows the enrichment score for the aerobic respiration and respiratory electron transport gene set. The red line indicates the running enrichment score (y-axis) while going down the ranked gene list (x-axis). The horizontal bars indicate the position of genes from the given gene set in the ranked gene list, where those at the top of the list are more upregulated in ENH5 KO response to hypoxia compared to WT response while those at the bottom of the list are more downregulated in ENH5 KO response. Adipogenesis Burton 6 (MSigDB: M1678) represents adipogenesis signaling pathways at late differentiation. (C) Heatmaps of the genes making up the aerobic respiration and respiratory electron transport gene set. Genes are clustered based on the electron complex chain component they are associated with. Diagram in panel A created with BioRender.

WT and ENH5 KO response to CL316243 treatment.

(A) Top 10 enriched pathways from WikiPathways for WT and ENH5 KO genes downregulated in response to CL316243. n= 6 for male WT mice and n = 6 for male ENH5 KO mice. (B) GSEA enrichment plot showing the enrichment score for the adipogenesis gene set. The red line indicates the running enrichment score (y-axis) while going down the ranked gene list (x-axis). The horizontal bars indicate the position of genes from the given gene set in the ranked gene list, where those at the top of the list are more upregulated in ENH5 KO response to CL316243 treatment compared to WT response while those at the bottom of the list are more downregulated in ENH5 KO response. Adipogenesis Burton 6 (MSigDB: M1678) represents adipogenesis signaling pathways at late differentiation. (C) Heatmaps of the genes making up the adipogenesis gene set. Genes are clustered based on different roles of adipogenesis they are associated with.

WT and ENH5 KO response to hypoxia.

(A) Biological processes enrichment using gene ontology analysis of WT and ENH5 KO genes upregulated in response to hypoxia. n = 5 for male WT mice and n = 4 for male ENH5 KO mice. (B) Top 10 enriched pathways from WikiPathways for WT and ENH5 KO genes downregulated in response to hypoxia. (C) GSEA enrichment plot showing the enrichment score for the adipogenesis gene set. The red line indicates the running enrichment score (y-axis) while going down the ranked gene list (x-axis). The horizontal bars indicate the position of genes from the given gene set in the ranked gene list, where those at the top of the list are more upregulated in ENH5 KO response to hypoxia compared to WT response while those at the bottom of the list are more downregulated in ENH5 KO response. Adipogenesis Burton 6 (MSigDB: M1678) represents adipogenesis signaling pathways at late differentiation. (D) Heatmaps of the genes making up the adipogenesis gene set. Genes are clustered based on different roles of adipogenesis they are associated with.

Joint hypoxia and thermogenic stimulation does not have an additive effect on transcriptional regulation in ENH5 KO adipocytes.

(A) Experimental design showing preadipocytes being isolated from the subcutaneous fat pads of male WT (n = 6) and ENH5 KO mice (n = 6). Preadipocytes were differentiated in vitro to mature adipocytes, then cultured in hypoxia (1% O2) for 48 hours followed by a 4 hour CL316243 treatment while in hypoxia. An interaction analysis for genotype by treatment was conducted to identify differentially expressed genes between WT and ENH5 KO response to thermogenesis in hypoxia. (B) Top 10 enriched pathways from WikiPathways for WT and ENH5 KO genes downregulated in response to hypoxia + CL316243. (C) GSEA showing the top 10 enriched gene sets more strongly downregulated in ENH5 KO response than WT response to the joint treatment is shown. The GSEA enrichment plot shows the enrichment score for the serum and rapamycin sensitive genes gene set. The red line indicates the running enrichment score (y-axis) while going down the ranked gene list (x-axis). The horizontal bars indicate the position of genes from the given gene set in the ranked gene list, where those at the top of the list are upregulated in ENH5 KO response to thermogenesis in hypoxia compared to WT response while those at the bottom of the list are downregulated in ENH5 KO response. (D) Box plot of the change in expression of Pdk2 in ENH5 KO and WT adipocytes in response to all three experimental conditions. Dotted horizontal line separates hypoxia + CL16243 treatment to indicate that this experiment was conducted in a hypoxic background. Diagram in panel A created with BioRender.