Identification of a functional hypoxia-responsive element that regulates the expression of the egl nine homologue 3 (egln3/phd3) gene - PubMed (original) (raw)

Identification of a functional hypoxia-responsive element that regulates the expression of the egl nine homologue 3 (egln3/phd3) gene

Nuria Pescador et al. Biochem J. 2005.

Abstract

Low oxygen levels induce an adaptive response in cells through the activation of HIFs (hypoxia-inducible factors). These transcription factors are mainly regulated by a group of proline hydroxylases that, in the presence of oxygen, target HIF for degradation. The expression of two such enzymes, EGLN1 [EGL nine homologous protein 1, where EGL stands for egg laying defective (Caenorhabditis elegans gene)] and EGLN3, is induced by hypoxia through a negative feedback loop, and we have demonstrated recently that hypoxic induction of EGLN expression is HIF-dependent. In the present study, we have identified an HRE (hypoxia response element) in the region of the EGLN3 gene using a combination of bioinformatics and biological approaches. Initially, we isolated a number of HRE consensus sequences in a region of 40 kb around the human EGLN3 gene and studied their evolutionary conservation. Subsequently, we examined the functionality of the conserved HRE sequences in reporter and chromatin precipitation assays. One of the HREs, located within a conserved region of the first intron of the EGLN3 gene 12 kb downstream of the transcription initiation site, bound HIF in vivo. Furthermore, this sequence was able to drive reporter gene expression under conditions of hypoxia in an HRE-dependent manner. Indeed, we were able to demonstrate that HIF was necessary and sufficient to induce gene expression from this enhancer sequence.

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Figures

Figure 1

Figure 1. The EGLN3 promoter region is not induced by hypoxia

(A) Diagram of the human EGLN3 promoter region, in which the numbers indicate the nucleotide positions relative to the transcription initiation site. Black boxes represent the first EGLN3 exon comprising residues +1 to +686. The small black box corresponds to the 5′-UTR (from +1 to +329) and the large one to the coding sequence (from +329 to +686). CpG, location of a CpG-rich region according to the UCSD Genome Browser (

http://genome.ucsc.edu

). Open boxes indicate the localization of putative HRE core sequences. The regions indicated, −3410 to +169 and −1465 to +169, in the EGLN3 genomic fragment were cloned into pGL3 basic vectors upstream of the firefly luciferase gene to generate pGL3-E3P3.5 and pGL3-E3P1.5 respectively. (B) HeLa cells were transfected with the fragments indicated from EGLN3 or from the VEGF-A promoter cloned into the pGL3 basic reporter plasmid. Upper panel: diagram of the pGL3-E3P3.5 and pGL3-E3P1.5 constructs. Lower panel: after transfection, the cells were cultured under normoxic conditions (Nx) or in an atmosphere of 1% oxygen (Hx) for 24 h before analysing the luciferase activity. The mean results from ‘_n_’ independent experiments, each performed in duplicate (solid bar) are shown. Each type of symbol represents data from an individual experiment. In order to compare between different experiments, the ratio of firefly and Renilla luciferase activities for each sample was normalized to the ratio obtained for the control (empty vector under normoxia). ***, statistically significant differences (P<0.001) between the two indicated samples. The fold induction of hypoxia versus normoxia for each sample is shown.

Figure 2

Figure 2. Computer identification of HRE in the EGLN3 locus

(A) The data represent the scores obtained by comparing the sequences from VEGF-A, BNIP3, P4Hα, ROR4α, c-Met, PGK1, Glut-1, promoters (functional HREs; ●) or from random sequences containing a core [A/G]CGT motif (random HREs; ▲), to the position-specific frequency matrix. The horizontal line represents the mean value for each group of data and the number represents its value. The difference between their means was statistically significant (P<0.001, t test). (B) BLAST comparison of the concatenated 124 putative HRE sequences found in the human 40 kb EGLN3 genomic region versus the concatenated 129 sequences found for the mouse locus. (C) Alignment of the five HRE sequences identified in (B) that are conserved between human (Hs) and mouse (Mm). The score for each sequence, according to the position-specific frequency matrix, as well as their rank, is shown. (D) Genomic region of the EGLN3 locus (adapted from UCSD Genome Browser,

http://genome.ucsc.edu

). Boxes are open reading frames. The size of the box indicates whether it is UTR or CDS, as indicated in Figure 1. The arrow indicates the direction of transcription. The EGLN3 gene structure, including transcription start site, is based on RefSeq NM_022073. Conservation of sequences among different species is indicated by the black histogram, and the individual homology between different species and human is indicated by grey histograms. The localization of enhancers A–D in the EGLN3 genomic region is indicated by arrows. The sequence conservation of the five putative enhancers between different species is shown; the arrows above the HREs indicate their direction.

Figure 3

Figure 3. Enhancer A is induced by hypoxia

(A) Diagram of the H. sapiens EGLN3 genomic region in which the numbers indicate nucleotide positions relative to the transcription initiation site, as shown in Figure 1. The indicated regions (hatched boxes) containing putative hypoxia-responsive sequences were cloned into the pProl plasmid upstream of the rat minimal prolactin promoter. (B) HeLa cells were transfected with the fragments from the EGLN3 genomic region indicated, cloned upstream of a minimal promoter (open pointed box). A diagram of the constructs is depicted in the Figure, in which the hatched box represents any of the elements from Figure 3(A). After transfection, the cells were cultured under normoxic (Nx) conditions or in an atmosphere of 1% oxygen (Hx) for 24 h before analysing the luciferase activity. Enh. A* is a reporter construct derived from enhancer A (Enh. A) in which the core ACGT was mutated to TAGC. The data are presented as indicated in Figure 1. (C) The enhancer A sequence, or its mutated form, was cloned into pGL3-E3P3.5 (see Figure 1A) downstream of the firefly luciferase gene as depicted in the Figure (hatched box). HeLa cells were transfected with the indicated constructs and treated as in (B). The data are presented as indicated in Figure 1.

Figure 4

Figure 4. HIF1α binds to enhancer A in vivo

HeLa cells were cultured under normoxic (N) or hypoxia (H) conditions for 12 h and then fixed to cross-link proteins to DNA. Cells were lysed and their DNA fragmented by sonication. Cell lysates were then immunoprecipitated with control IgGs (IgG) or a polyclonal antiserum raised against HIF1α (1α). Co-immunoprecipitated DNA was then amplified by PCR with primers specific for enhancer A (primers 6 and 7, Table 1), enhancer D (primers 8 and 9, Table 1) or collagen proline-4 hydroxylase α promoter (P4Hα; primers 15 and 16, Table 1). Input, sample of fragmented genomic DNA before immunoprecipitation. None, PCR without template. The experiment was repeated three times yielding similar results.

Figure 5

Figure 5. HIFα is necessary and sufficient to induce enhancer A activity

(A) HIF1α-deficient Ka13 cells or HIF1α-competent 4.5 cells were transfected with the constructs indicated (see Figure 3A for details). After transfection, the cells were cultured under normoxic conditions (Nx) or in an atmosphere of 1% oxygen (Hx) for 24 h before analysing the luciferase activity. Results are represented as indicated in Figure 1. (B) HeLa cells were transfected with the indicated constructs derived from enhancer A (see Figure 3A for details) together with a plasmid encoding a stable HIF construct (HIF PP). After transfection, the cells were cultured under normoxic conditions for 48 h before analysing the luciferase activity. Results are represented as indicated in Figure 1.

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