Oxygen‐regulated erythropoietin gene expression is dependent on a CpG methylation‐free hypoxia‐inducible factor‐1 DNA‐binding site

Abstract

The hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in the expression of oxygen-regulated genes such as that for erythropoietin. Following exposure to low oxygen partial pressure (hypoxia), HIF-1 binds to an hypoxia-response element located 3′ to the erythropoietin gene and confers activation of erythropoietin expression. The conserved core HIF-1 binding site (HBS) of the erythropoietin 3′ enhancer (CGTG) contains a CpG dinucleotide known to be a potential target of cytosine methylation. We found that methylation of the HBS abolishes HIF-1 DNA binding as well as hypoxic reporter gene activation, suggesting that a methylation-free HBS is mandatory for HIF-1 function. The in vivo methylation pattern of the erythropoietin 3′ HBS in various human cell lines and mouse organs was assessed by genomic Southern blotting using a methylation-sensitive restriction enzyme. Whereas this site was essentially methylation-free in the erythropoietin-producing cell line Hep3B, a direct correlation between erythropoietin protein expression and the degree of erythropoietin 3′ HBS methylation was found in different HepG2 sublines. However, the finding that this site is partially methylation-free in human cell lines and mouse tissues that do not express erythropoietin suggests that there might be a general selective pressure to keep this site methylation-free, independent of erythropoietin expression.

Ischemia dysregulates DNA methyltransferases andp16INK4amethylation in human colorectal cancer cells

“…47 Thus the promoter region of EPO is a target for ischemia-mediated hypomethylation as an adaptive mechanism in ischemic tissue. There are several other genes involved in adaptation to ischemia which utilize the HIF1 pathway, such as vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS).…”

“…There are several other genes involved in adaptation to ischemia which utilize the HIF1 pathway, such as vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). 47 We have demonstrated that the tumor microenvironment may be an important modulator of DNMT expression and the effects of ischemia on global methylation and expression changes in human colorectal cancer are currently being investigated. The impact of agents which modulate the tumor (1:250; Imgenex) over night at 4°C followed by goat anti-mouse Cy3 (1:200; Jackson Immunoresearch, West Grove, PA, USA) for 1 h at 22°C.…”

Epigenetics: The epicenter of the hypoxic response

“…This is evident in the erythropoietin and the class III beta-tubulin genes, where it has been shown that hypoxia-induced expression is dependent upon the tissuespecific methylation status of a HRE in the 3'-UTR. [17][18][19] As global changes in DNA methylation can occur under chronic hypoxic conditions, it is possible that this may impact on the methylation status of HRE sites, 1,20 and thus shape the HIF-dependent transcriptional profile according to the intensity and duration of the hypoxic insult. Gene specific DNA hypomethylation may reveal previously inaccessible HIF binding sites, thus exposing new active regions for HIF or other hypoxia-responsive transcription factors.…”

Genome-wide identification and annotation of HIF-1α binding sites in two cell lines using massively parallel sequencing

“…These results indicated that only a part of HRE motifs had an open nucleosome structure suitable for subsequent transcriptional activation, although HRE motifs were present throughout the human genomic sequences. Previous HIF-1a ChIP-chip studies have reported that less than 1% of the HRE motifs within the genomic sequences that are located in regions used for the microarrays are bound by HIF-1a (Mole et al 2009;Wenger et al 1998Wenger et al , 2005. Epigenetic regulation of the human genome plays an essential role in specifying the binding sites of HIF-1a in addition to the consensus binding sequences.…”