Genetic analysis of pathways regulated by the von Hippel-Lindau tumor suppressor in Caenorhabditis elegans

Abstract

The von Hippel-Lindau (VHL) tumor suppressor functions as a ubiquitin ligase that mediates proteolytic inactivation of hydroxylated alpha subunits of hypoxia-inducible factor (HIF). Although studies of VHL-defective renal carcinoma cells suggest the existence of other VHL tumor suppressor pathways, dysregulation of the HIF transcriptional cascade has extensive effects that make it difficult to distinguish whether, and to what extent, observed abnormalities in these cells represent effects on pathways that are distinct from HIF. Here, we report on a genetic analysis of HIF-dependent and -independent effects of VHL inactivation by studying gene expression patterns in Caenorhabditis elegans. We show tight conservation of the HIF-1/VHL-1/EGL-9 hydroxylase pathway. However, persisting differential gene expression in hif-1 versus hif-1; vhl-1 double mutant worms clearly distinguished HIF-1-independent effects of VHL-1 inactivation. Genomic clustering, predicted functional similarities, and a common pattern of dysregulation in both vhl-1 worms and a set of mutants (dpy-18, let-268, gon-1, mig-17, and unc-6), with different defects in extracellular matrix formation, suggest that dysregulation of these genes reflects a discrete HIF-1-independent function of VHL-1 that is connected with extracellular matrix function.

Figure 1. HIF-1–Dependent Effects of VHL-1 Inactivation

Representative RNase protection assays of genes that were differentially expressed in the vhl-1 versus wild-type microarray in (A) mixed-stage and (B) synchronized populations of worm. All genes are regulated by the VHL-1/HIF-1/EGL-9 pathway. (C) Regulation of nhr-57 mRNA in egl-9; vhl-1 worms and by egl-9 RNAi and DIP in vhl-1 worms. For RNAi experiments controls were L4440 vector alone (−) and C17G10.1, an irrelevant putative dioxygenase. F21C3.5 is a constitutively expressed gene used to control for RNA integrity. RNase protection assays were performed using worms cultured under normoxic conditions, unless otherwise indicated.

Figure 2. HIF-1–Independent Effects of VHL-1 Inactivation

RNase protection assays of genes that were differentially expressed in the hif-1; vhl-1 versus hif-1 microarrays in (A) mixed-stage and (B) synchronized populations of worm. The results confirm the existence of VHL-1–dependent, HIF-1–independent effects on gene expression.

Figure 3. Chromosomal Clustering of VHL-1–Dependent (HIF-1–Independent) Genes

(A) Chromosomal localization of VHL-1–dependent, HIF-1–independent genes. The positions of the genes from Table 4 are indicated by vertical ticks along the C. elegans chromosomes (shown to scale). Where two such genes are too close to be clearly resolved, the tick is marked by an asterisk. The single significant spatial clustering of VHL-1–dependent, HIF-1–independent genes is indicated by a red rectangle. The histogram under each chromosome shows the gene density (deeper bar, greater density) calculated as a sliding window of 100,000 bp moving with 10,000-bp increments along each chromosome. Dark blue indicates total annotated gene density, and light blue indicates the density of genes from the microarray that passed preliminary quality control. (B) Organization of the VHL-1–regulated (HIF-1–independent) gene cluster from Chromosome V. The relative positions and sizes of gene transcription units are shown to scale, with genes transcribed left to right above the horizontal line and right to left below the line. Names in black indicate genes that passed all selection criteria to be considered upregulated in hif-1; vhl-1 versus hif-1 worms (see Table 4). Genes with a mean >2.0-fold upregulation are indicated by green boxes, 1.5- to 2-fold are yellow, and <1.5-fold are red. Genes for which no data were obtained are shown as light grey.

Figure 4. Responses of VHL-1–Dependent, HIF-1–Independent Genes to egl-9 Inactivation, Hypoxia, and 2-Oxoglutarate Dioxygenase Inhibitors

RNase protection assays showing regulation of VHL-1–dependent, HIF-1–independent genes by (A) EGL-9 and hypoxia and (B) pharmacological inhibitors of 2-oxoglutarate dioxygenases: DIP and DMOG. None of the genes is regulated by EGL-9, but two genes (C01B4.7 and C01B4.8) show modest induction by hypoxia, DIP, and DMOG.

Figure 5. Sensitivity of VHL-1–Regulated Genes to Defects in Extracellular Matrix-Associated Proteins

RNase protection assays showing altered expression of VHL-1–regulated genes that are HIF-1 independent (upper six panels) and HIF-1 dependent (F22B5.4) in worms bearing mutations affecting (A) procollagen prolyl and lysyl hydroxylases and (B) other extracellular matrix-associated proteins. A common pattern of upregulation is observed in hif-1; vhl-1, vhl-1, dpy-18, let-268, gon-1, mig-17, and unc-6 worms but not other mutants. This contrasts with the HIF-1–dependent gene F22B5.4, which is upregulated in vhl-1 worms but none of the other mutants. (C) RNase protection assay for C01B4.9 illustrating DPY-18–mediated changes in expression that are independent of HIF-1.