Fig 2: HCMV-miR-US33-5p downregulates SLC3A2 by targeting EPAS1. A RNA sequencing data showing EPAS1 differential expression between the HCMV-miR-US33-5p and control groups. B, C HCMV-miR-US33-5p was transfected into HA-VSMCs. qRT-PCR (B) and Western blot (C) were used to analyze the EPAS1 expression level in HA-VSMCs. Western blot was performed three times; representative images are presented. Numbers above lanes represent the relative protein level, normalized relative to control samples. D Potential EPAS1 binding sites in SLC3A2 promoter region. E Luciferase reporter assay of the effect of EPAS1 on the SLC3A2 promoter. F ChIP assay showed that EPAS1 bound to SLC3A2 promoter. G ChIP-qPCR assay of the enrichment of EPAS1 to SLC3A2 promoter. H Potential HCMV-miR-US33-5p binding site in EPAS1 promoter. I Luciferase reporter assay of the effect of HCMV-miR-US33-5p on the EPAS1 promoter. J RIP analysis of the enrichment of Ago2 on 3′-UTR of EPAS1 and HCMV-miR-US33-5p. **P < 0.01, ***P < 0.001 (n = 3, ANOVA analysis for A and D; Student’s t-test for F)

Fig 3: BMAL1 forms an active heterodimer with HIF2a.(A) Phylogenetic tree for bHLH-PAS proteins. (B) Percent sequence identity for bHLH and PAS domains in BMAL1 and ARNT. (C,D) Detection of BMAL1 (transcripts per million, TPM) (C) and clock correlation distance (CCD) heatmaps (D) calculated from RNA sequencing data from tumors and adjacent normal tissues in cancer genome atlas projects: colorectal adenocarcinoma (COAD), lung adenocarcinoma (LUAD), breast cancer (BRCA), kidney clear cell renal cell carcinoma (KIRC), and renal papillary cancinoma (KIRP). (E,F) Dependency (CHRONOS) scores (E) and correlations thereof (F) for bHLH-PAS members in RCC cell lines from DepMap 29,30. (G) Detection of indicated proteins in whole cell extracts (input) or following immunoprecipitation of the FLAG tag from 786O cells transiently expressing the indicated plasmids. (H) Relative luciferase units detected from U2OS cells expressing HRE-Luciferase and additional indicated plasmids with (red) or without (black) exogenous stabilized HIF2α (P405A, P531A, N837A). In (C,E) boxplots depict the median and interquartile range (IQR), whiskers extend either to the minimum or maximum data point or 1.5*IQR beyond the box, whichever is shorter. Outliers (values beyond the whisker) are shown as dots in (C). In (H) bars represent mean ± s.e.m. for three experimental replicates and symbols represent the mean of n=5 measurements for each experiment. In (C,E,H) ** Padj < 0.01, *** P< 0.001, **** P < 0.0001 by two-way ANOVA with Tukey’s (C,E) or Sidak’s (H) correction for multiple comparison.

Fig 4: Identification of an acute transcriptional response to hypoxia driven by HIF1A.a Genome browser view of PRO-seq, ChIP-seq, and RNA-seq signals across the ENO1 locus for HCT116 wild-type (WT) and HIF1A−/− cells exposed to normoxia (21% O2, blue) or hypoxia (1% O2, red). b Western blot analysis of HIF1A and HIF2A levels in HCT116 wild-type cells exposed to 21% or 1% O2 for the indicated times. Images are representative of at least two replicates with similar results. Source data are provided as a Source Data file. c DESeq2 differential expression analysis of PRO-seq signal within gene body regions for HCT116 WT (blue) and HIF1A−/− (green) cells. Horizontal dashed lines indicate an FDR threshold of 10% for negative binomial Wald test; numbers and colored points indicate significant genes at this threshold. d Comparison of fold changes in PRO-seq gene body signal induced by 90 min hypoxia in HCT116 WT versus HIF1A−/− cells. Red/green points denote genes with significant up/down regulation; points representing all other genes are colored by density. e Gene set enrichment analysis (GSEA) of “Hallmark” gene sets against genes ranked by gene body fold changes induced by 90 min hypoxia in HCT116 WT cells. Red/green points denote gene sets with significant (FDR 10%) positive/negative enrichment. f Distributions of fold changes in PRO-seq signal at gene bodies and transcription start sites (TSS) and pausing index (PI) for upregulated genes in HCT116 WT (blue) and HIF1A−/− (green) cells. Horizontal spread of data points is proportional to density; boxes indicate medians and upper and lower quartiles; numbers above the HIF1A−/− plots indicate effect size estimates and p-values for two-sided Mann–Whitney U tests against the corresponding measure in WT cells. g Metagene showing typical PRO-seq signal profiles across upregulated genes in HCT116 WT (blue) and HIF1A−/− (green). Data are represented as splined linear fit lines with 95% confidence intervals in gray. h Upregulated genes ranked by WT/HIF1A−/− fold-change in normoxia, comparing the effect of HIF1A in normoxia (green/black/red points) and hypoxia (gray points). i Heatmap of gene body RPKM Z-scores for select example genes in HCT116 WT and HIF1A−/− cells in normoxia and hypoxia. See also Supplementary Figs. 1, 2 and Supplementary Data 1, 2.

Fig 5: Purified BMAL1 and HIF2a form a stable complex in vitro.(A) Heparin chromatography elution of BMAL1 and HIF2a co-expressed in insect cells. SDS-PAGE analysis shows a co-eluted stoichiometric complex of BMAL1-HIF2a. (B) Mass photometry of purified BMAL1-HIF2a complex. A minor peak centered at 91 kDa corresponds to the molecular weight of HIF2a, suggesting that it is in slight excess. The major peak, centered at 157 kDa, is consistent with the calculated molecular weight for the BMAL1-HIF2a heterodimer.