Regulation of HIF prolyl hydroxylases by hypoxia-inducible factors

Hypoxia and induction of hypoxia-inducible factors (HIF-1alpha and HIF-2alpha) is a hallmark of many tumors. Under normal oxygen tension HIF-alpha subunits are rapidly degraded through prolyl hydroxylase dependent interaction with the von Hippel-Lindau (VHL) tumor suppressor protein, a component of E3 ubuiquitin ligase complex. Using microarray analysis of VHL mutated and re-introduced cells, we found that one of the prolyl hydroxylases (PHD3) is coordinately expressed with known HIF target genes, while the other two family members (PHD1 and 2) did not respond to VHL. We further tested the regulation of these genes by HIF-1 and HIF-2 and found that siRNA targeted degradation of HIF-1alpha and HIF-2alpha results in decreased hypoxia-induced PHD3 expression. Ectopic overexpression of HIF-2alpha in two different cell lines provided a much better induction of PHD3 gene than HIF-1alpha. In contrast, we demonstrate that PHD2 is not affected by overexpression or downregulation of HIF-2alpha. However, induction of PHD2 by hypoxia has HIF-1-independent and -dependent components. Short-term hypoxia (4 h) results in induction of PHD2 independent of HIF-1, while PHD2 accumulation by prolonged hypoxia (16 h) was decreased by siRNA-mediated degradation of HIF-1alpha subunit. These data further advance our understanding of the differential role of HIF factors and putative feedback loop in HIF regulation.     

Regulation of gene expression by site-specific inversion

A site-specific inversion event is responsible for phase transition in Salmonella, as indicated by heteroduplex analysis of recombinant molecules carrying the gene coding for H2 flagellin in Salmonella. The inversion region corresponds to approximately 800 base pairs in length, and the inversion process does not appear to be dependent upon the E. coil RecA recombination pathway. Specific deletion derivatives of the cloned fragments no longer produce H2-specific flagella, effectively mapping the H2 gene within about 300 bp of the inversion region. Recombinant products of the hybrid molecules arose spontaneously, and they were used in the mapping of restriction sites within the inversion region. The restriction maps further demonstrate the extent and nature of the inversion.     

Regulation of ferrochelatase gene expression by hypoxia

Ferrochelatase (FECH), the last enzyme of the heme biosynthetic pathway, catalyzes the insertion of iron into protoporphyrin to form heme. This pathway provides heme for hemoglobin and other essential hemoproteins. The regulatory role of oxygen in the pathway has not been clearly established. In this study, we examined whether FECH gene expression is upregulated during hypoxia by a mechanism which involves the hypoxia-inducible factor 1 (HIF-1). Two HIF-1 binding motifs were identified within the -150 bp FECH minimal promoter sequence. Exposure of HEL, K562, and Hep-G2 cells to hypoxia for 18 hours resulted in a significant increase in FECH mRNA expression (p < 0.05). Hypoxia also transactivated the minimal promoter for the FECH gene in the cells. Transient co-expression of wild-type HIF-1alpha or a dominant negative HIF-1alpha with the FECH minimal promoter luciferase construct stimulated or blocked FECH promoter activity, respectively. Expression of the von Hippel-Lindau (VHL) tumor suppressor factor blocked the expression of both FECH mRNA and HIF-1alpha protein during normoxic culture of renal carcinoma cell line (RCC4). The results suggest that the FECH gene is a target for HIF-1 during hypoxia.