Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans
Data files
Feb 25, 2024 version files 95.78 KB
Abstract
Dedicated genetic pathways regulate cysteine homeostasis. For example, high levels of cysteine activate cysteine dioxygenase, a key enzyme in cysteine catabolism in most animal and many fungal species. The mechanism by which cysteine dioxygenase is regulated is largely unknown. In an unbiased genetic screen for mutations that activate cysteine dioxygenase (cdo-1) in the nematode C. elegans, we isolated loss-of-function mutations in rhy-1 and egl-9, which encode proteins that negatively regulate the stability or activity of the oxygen-sensing hypoxia-inducible transcription factor (hif-1). EGL-9 and HIF-1 are core members of the conserved eukaryotic hypoxia response. However, we demonstrate that the mechanism of HIF-1-mediated induction of cdo-1 is largely independent of EGL-9 prolyl hydroxylase activity and the von Hippel-Lindau E3 ubiquitin ligase, the classical hypoxia signaling pathway components. We demonstrate that C. elegans cdo-1 is transcriptionally activated by high levels of cysteine and hif-1. hif-1-dependent activation of cdo-1 occurs downstream of an H2S-sensing pathway that includes rhy-1, cysl-1, and egl-9. cdo-1 transcription is primarily activated in the hypodermis where it is also sufficient to drive sulfur amino acid metabolism. Thus, the regulation of cdo-1 by hif-1 reveals a negative feedback loop that maintains cysteine homeostasis. High levels of cysteine stimulate the production of an H2S signal. H2S then acts through the rhy-1/cysl-1/egl-9 signaling pathway to increase HIF-1-mediated transcription of cdo-1, promoting the degradation of cysteine via CDO-1.
README: Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans
https://doi.org/10.5061/dryad.kd51c5bdk
All data is from fluorescence microscopy, C. elegans growth assays, or C. elegans survival assays.
Description of the data and file structure
Figures 1E, 2B, 2C, 3B, 3C, and 5D represent Pcdo-1::GFP expression in C. elegans. Data are lists of mean GFP fluorescent values in a defined region of interest. Quantification was performed in FIJI and values are reported in arbitrary units. Genotypes and experimental conditions are displayed when appropriate.
Figure 4- figure supplements 1B and 2B represent CDO-1::GFP expression in C. elegans. Data are lists of mean GFP fluorescent values in a defined region of interest. Quantification was performed in FIJI and values are reported in arbitrary units. Genotypes and experimental conditions are displayed when appropriate.
Figures 4C, 4D, and Figure 1- figure supplement 1 represent the quantification of animal length in microns as measured from the tip of head to the end of tail after a set amount of time post-L1 stage of development. Measurements were performed in FIJI. Genotypes and experimental conditions are displayed when appropriate.
Figure 3D, 3E, and 3F represent the percentage of animals surviving overnight exposure to 0 (3D), 100 (3E), or 1,000 (3F) micromolar supplemental cysteine. Genotypes and experimental conditions are displayed when appropriate.
Table 1 data represent the number of days required for 5 L4-staged animals to starve the E. coli food source under standard C. elegans culture conditions. Genotypes and experimental conditions are displayed when appropriate.
Complete experimental details are available in the methods section of the associated manuscript: Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans.
Methods
All data is from fluorescence microscopy, C. elegans growth assays, or C. elegans survival assays.