The HIF-1-mediated hypoxia pathway is a pathway of cellular resilience that is activated under conditions of low oxygen (hypoxia). The transcriptional changes for this pathway are mediated by the HIF-1 transcription factor, which acts in a heterodimer with AHA-1 . hif-1 encodes the HIF-1 α subunit, which is sensitive to oxygen levels, while aha-1 encodes the constitutively expressed HIF-1 β subunit. Under normoxic conditions, HIF-1 is hydroxylated by the prolyl hydroxylase EGL-9 (Epstein et al., 2001). Hydroxylated HIF-1 can then be recognized by the von Hippel-Lindau E3 ubiquitin ligase VHL-1 , resulting in ubiquitination and proteasomal degradation of HIF-1 (Bishop et al., 2004). Under hypoxic conditions (0.1% - 1% oxygen), there are insufficient levels of oxygen for EGL-9 to hydroxylate HIF-1 . As a result, HIF-1 is not hydroxylated and not marked for degradation by VHL-1 . Instead, HIF-1 is able to accumulate in the nucleus and induce changes in gene expression. HIF-1 can also escape degradation when either EGL-9 or VHL-1 are disrupted. In addition, HIF-1 degradation can be prevented by disrupting the RHY-1 (Regulator of hypoxia-inducible factor-1) transmembrane protein, which normally acts to reduce CYSL-1 levels and prevent CYSL-1 from binding to and inhibiting EGL-9 (Kruempel et al., 2020; Shen et al., 2006).

A number of previous studies have demonstrated a role for the HIF-1-mediated hypoxia response in lifespan determination. Increasing the levels or activation of HIF-1 either directly or through disruption of its negative regulators vhl-1 , egl-9 , or rhy-1 , extends lifespan, indicating that activation of the hypoxia pathway can promote longevity (Kruempel et al., 2020; Leiser et al., 2015; Mehta et al., 2009; Zhang et al., 2009). In contrast, the results of studies examining the effects of hif-1 disruption on lifespan have been more varied with some experiments showing increased, decreased, or unchanged lifespan depending on the exact conditions utilized (Leiser and Kaeberlein, 2010). HIF-1 has also been shown to contribute to the longevity of multiple long-lived mutants. HIF-1 is required for lifespan extension in the long-lived mitochondrial mutants clk-1 , isp-1 and nuo-6 (Lee et al., 2010; Wu et al., 2018), but dispensable for the enhanced longevity of daf-2 , eat-2 , glp-1 and osm-5 mutants (Lee et al., 2010; Soo et al., 2023). The HIF-1 target gene fmo-2 is required for the lifespan extension induced by dietary restriction and by mild mitochondrial dysfunction (Leiser et al., 2015; Van Raamsdonk, 2026).

Multiple previous studies have used RNA sequencing (RNA-seq) to find genes that are differentially expressed following HIF-1 activation. Although the expression of these genes is dependent on HIF-1 , the effect of HIF-1 on gene expression could either be direct through HIF-1 binding to the promoter or indirect. As a result, we refer to these genes as HIF-1 modulated genes, though at least some of these genes are direct targets of HIF-1 .

Shen et al. identified genes that are upregulated during hypoxia with p<0.05 and fold change greater than or equal to 2 and not upregulated by hypoxia in hif-1 ( ia4 ) mutants (Shen et al., 2005). Vora et al. identified genes that are upregulated in egl-9 ( sa307 ) and egl-9 ( sa307 ); hif-1 ( ia4 ):odIs131[ hif-1 ::gfp] mutants compared to wild-type and egl-9 ; hif-1 ( ia4 ) worms (Vora et al., 2022). Feng et al. identified genes that are induced by 1.6 fold more in wild-type worms compared to hif-1 ( ia4 ) worms after a 2 hour exposure to hypoxia (Feng et al., 2024b). In a separate paper, Feng et al. identified genes upregulated in vhl-1 ( ok161 ), rhy-1 ( ok1402 ), egl-9 ( sa307 ) and swan-1 ( ok267 ); vhl-1 ( ok161 ) worms and made a list of genes commonly upregulated amongst all four strains (Feng et al., 2024a). Doering et al. identified genes upregulated by hypoxia in wild-type worms and nhr-49 mutants but not in hif-1 mutants (Doering et al., 2022). We previously compared gene expression between isp-1 and isp-1 ; hif-1 mutants, as the HIF-1-mediated hypoxia pathway has been found to be activated in isp-1 mutants (Lee et al., 2010). For our study, HIF-1 modulated genes were selected as genes that are upregulated by at least 30% in isp-1 worms compared to wild-type worms and decreased by at least 30% in isp-1 ; hif-1 worms compared to isp-1 worms.

In addition to these RNA-seq experiments, two studies have used Chromatin Immunoprecipitation sequencing (ChIP-seq) to find which gene promoters are bound by HIF-1 following activation. Feng et al. performed a ChIP-Seq experiment in which the hif-1 a isoform was labelled with a HA tag in the egl-9 ( sa307 ) background and an anti-HA antibody was used for ChIP (Feng et al., 2024b). Vora et al. performed a ChIP-Seq experiment in which egl-9 ( sa307 ); hif-1 ( ia4 );odIs131[ hif-1 ::gfp] worms were compared to hif-1 ( ia4 );odIs131[ hif-1 ::gfp] worms (Vora et al., 2022).

To identify high confidence HIF-1 modulated genes, we initially compared the gene sets identified by Shen et al. (genes upregulated during hypoxia in a hif-1- dependent manner), Vora et al. (genes upregulated by egl-9 mutation in a hif-1- dependent manner) and Feng et al. (genes induced 1.6 fold more in wild-type worms than hif-1 worms after hypoxia) ( Figure 1B ). We found that there were 41 genes in at least two of these gene sets with just 8 genes found in all three. We then examined whether these 41 genes were identified in the other RNA-seq experiments and the published ChIP-Seq experiments. We found that three genes were identified in seven of the eight studies ( Extended data Table S1 ) (The ChIP-seq experiment by Feng et al. only identified one of the 41 genes). This included egl-9 , F22B5.4 and sqrd-1 . There were 8 more genes that were identified in six of the eight studies: efk-1 , phy-2 , cysl-2 , gbh-2 , ethe-1 , mce-1 , ZK550.6 , and pck-1 .

As we have previously used RNA sequencing to examine gene expression in nuo-6 and nuo-6 ; hif-1 worms, as well as wild-type and hif-1 worms (Wu et al., 2018), we next examined the expression of a selection of these genes using this RNA-seq data. We found that disruption of hif-1 significantly decreased the expression of F22B5.4 , sqrd-1 , cysl-2 , mce-1 and pck-1 , thereby providing additional support that these genes are dependent on HIF-1 ( Figure 1C ).

We recently examined gene expression in a panel of nine long-lived mutants representing multiple different pathways of lifespan extension (Rudich et al., 2026). To determine the extent to which the HIF-1-hypoxia pathway is activated in each of these strains, we examined the expression of HIF-1 modulated genes that were identified by at least four of the eight studies we examined. We found that nuo-6 and isp-1 showed the greatest activation of HIF-1 modulated genes, while osm-5 and eat-2 worms showed the least activation ( Figure 1D ). This is consistent with our observation that nuo-6 and isp-1 belong to a different longevity group than osm-5 and eat-2 worms, based on gene expression (Rudich et al., 2026). It is important to note that the expression of these HIF-1 modulated genes was examined at day 1 of adulthood. In addition to genotype, the transcriptional output of HIF-1 may vary by developmental stage, tissue, oxygen level, exposure duration and environmental stressors.

In order to facilitate other researchers quantifying the expression of HIF-1 target genes to measure activation of the HIF-1-hypoxia response, we designed primers to measure six of the high confidence HIF-1 target genes using quantitative RT-PCR (details on the binding location and amplicon size of the primers can be found in Extended data File 1 ). These genes included egl-9 , F22B5.4 , sqrd-1 , cysl-2 , mce-1 and pck-1 . To test the function of these primers, we isolated RNA from isp-1 and isp-1 ; hif-1 worms with wild-type worms as control. We normalized the expression of each gene to act- 3, as our previous RNA-seq data showed that act-3 levels are not significantly different between wild-type and isp-1 worms. As anticipated, we found that the expression of all six selected HIF-1 target genes is upregulated in isp-1 mutants and the upregulation of these genes in isp-1 worms is completely prevented by the disruption of hif-1 ( Figure 1E ). This confirms that the primers we designed can be used to effectively measure the expression levels of these HIF-1 target genes.

Overall, this work combined multiple previous RNA-seq and ChIP-seq studies to identify high confidence HIF-1 target genes that can be used to monitor the activation of the HIF-1-mediated hypoxia response. We also designed and validated qPCR primers to quantify these genes using quantitative RT-PCR.

Strains

The following strains were used: N2 (wild-type), isp-1 ( qm150 ), isp-1(qm150);hif-1(ia4) . Strains were maintained at 20°C on NGM plates seeded with OP50 E. coli bacteria.

RNA Isolation

Worms were synchronized by a 4-hour limited time egg laying, collected at the pre-fertile young adult stage, and washed 3 times with M9. Trizol was then added and the samples were frozen in liquid nitrogen and kept at –80°C until RNA isolation. To extract the RNA, the pellet was thawed and re-frozen in liquid nitrogen 3 times, after which more Trizol was added to a total volume of 250 µl. This was followed by 3 vortex cycles consisting of 30s vortexing and 30s at room temperature. Samples were then left to sit at room temperature for 15 minutes and chloroform was added at 1:5 volume of Trizol. After shaking for 15 seconds, samples were left to sit at room temperature for 3 minutes. Then, samples were centrifuged at 12,000 x g for 15 minutes at 4°C, and the upper aqueous phase was transferred to a new tube. An equal volume of isopropanol was added, and it was left sitting on ice for 1 hour. After, samples were centrifuged at 12,000 x g for 20 minutes at 4°C, the supernatant was removed, and the pellet was washed with 75% ethanol. Then, samples were centrifuged at 12,000 x g for 10 minutes at 4°C, the ethanol was removed, and 100% ethanol was added before centrifuging at 12,000 x g for 3 minutes. The ethanol was removed and the pellet was air dried for 5 minutes until no traces of ethanol were found. The pellet was then dissolved in RNAse free double distilled water. RNA was isolated from 3 biological replicates.

Quantitative PCR

The samples underwent DNase I treatment to remove genomic DNA, using a DNase I, RNase-free kit (Thermo Scientific) following the manufacturer's instructions. Then, cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), according to the manufacturer's protocol. qPCR was performed using SYBR Green Master Mix (Applied Biosystems) in a Viia7 Real Time PCR System (Applied Biosystems). RNA levels were normalized to the expression of the act-3 gene. After running the RT-PCR, expression levels were calculated as 2 ^-CT (CT is the cycle threshold, which is the number of cycles at which time the fluorescence crosses a specified threshold). The expression levels for each individual gene was then divided by the expression level of the control gene act-3 . Finally, the copies/copy act-3 was divided by the average copies/copy act-3 of the wild-type samples. Three biological replicates were performed.

Primers

The sequences for the primers used for qPCR are below:

Strains utilized: