A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery

<p>Abstract</p> <p>Background</p> <p>Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand (hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest.</p> <p>Results</p> <p>Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the β domain of VHL and a hydroxyproline-containing HIFα peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFα peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system.</p> <p>Conclusion</p> <p>The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes.</p

Analysis of HIF-prolyl hydroxylases binding to substrates

Hypoxia inducible transcription factors (HIF) are mainly regulated by a group of proline hydroxylases (EGLNs) that, in the presence of oxygen, target HIF for degradation. HIFα contains two independent oxygen degradation domains (N-ODD and C-ODD) that are substrates for these enzymes. In this work, we employed the yeast two-hybrid assay to study the sequence determinants required for the binding of EGLN1 and 3 to HIF1α in a cellular context. Our results demonstrate that, while EGLN1 is able to recognize both ODDs within full length HIF1α protein, EGLN3 only binds to CODD. The analysis of the residue substitutions within CODD uncovered novel critical determinants for EGLN1 and 3 binding. In addition, our results show that both enzymes have a very similar, albeit not identical, residue preference at specific positions in their substrate sequences. © 2006 Elsevier Inc. All rights reserved.This work was supported by grants from Fondo de Investigaciones Sanitarias (FIS01/0264 to L.P.), from Ministerio de Ciencia y Tecnologı´a (SAF2002-02344 and SAF2005-00180 to L.P. and SAF2004-00824 to M.O.L.), and from Red Cardiovascular (RECAVA).Peer Reviewe

Acute Vhl Gene Inactivation Induces Cardiac HIF-Dependent Erythropoietin Gene Expression

Von Hippel Lindau (Vhl) gene inactivation results in embryonic lethality. The consequences of its inactivation in adult mice, and of the ensuing activation of the hypoxia-inducible factors (HIFs), have been explored mainly in a tissue-specific manner. This mid-gestation lethality can be also circumvented by using a floxed Vhl allele in combination with an ubiquous tamoxifen-inducible recombinase Cre-ER T2. Here, we characterize a widespread reduction in Vhl gene expression in Vhl floxed-UBC-Cre-ER T2 adult mice after dietary tamoxifen administration, a convenient route of administration that has yet to be fully characterized for global gene inactivation. Vhl gene inactivation rapidly resulted in a marked splenomegaly and skin erythema, accompanied by renal and hepatic induction of the erythropoietin (Epo) gene, indicative of the in vivo activation of the oxygen sensing HIF pathway. We show that acute Vhl gene inactivation also induced Epo gene expression in the heart, revealing cardiac tissue to be an extra-renal source of EPO. Indeed, primary cardiomyocytes and HL-1 cardiac cells both induce Epo gene expression when exposed to low O2 tension in a HIF-dependent manner. Thus, as well as demonstrating the potential of dietary tamoxifen administration for gene inactivation studies in UBC-Cre-ER T2 mouse lines

Identification of a region on hypoxia-inducible-factor prolyl 4-hydroxylases that determines their specificity for the oxygen degradation domains

HIFs [hypoxia-inducible (transcription) factors] are essential for the induction of an adaptive gene expression programme under low oxygen partial pressure. The activity of these transcription factors is mainly determined by the stability of the HIFα subunit, which is regulated, in an oxygen-dependent manner, by a family of three prolyl 4-hydroxylases [EGLN1–EGLN3 (EGL nine homologues 1–3)]. HIFα contains two, N- and C-terminal, independent ODDs (oxygen-dependent degradation domains), namely NODD and CODD, that, upon hydroxylation by the EGLNs, target HIFα for proteasomal degradation. In vitro studies indicate that each EGLN shows a differential preference for ODDs, However, the sequence determinants for such specificity are unknown. In the present study we showed that whereas EGLN1 and EGLN2 acted upon any of these ODDs to regulate HIF1α protein levels and activity in vivo, EGLN3 only acted on the CODD. With the aim of identifying the region within EGLNs responsible for their differential substrate preference, we investigated the activity and binding pattern of different EGLN deletions and chimaeric constructs generated by domain swapping between EGLN1 and EGLN3. These studies revealed a region of 97 residues that was sufficient to confer the characteristic substrate binding observed for each EGLN. Within this region, we identified the minimal sequence (EGLN1 residues 236–252) involved in substrate discrimination. Importantly, mapping of these sequences on the EGLN1 tertiary structure indicates that substrate specificity is determined by a region relatively remote from the catalytic site

<i>Erythropoietin</i> and <i>glucose transporter-1</i> gene expression in isolated primary cardiomyocytes in response to hypoxia.

<p>Isolated rat cardiomyocyte cultures were subjected to basal normoxic conditions and/or hypoxia (1% O₂) for 24 hours. <i>Epo</i> (A) and <i>Glut1</i> (B) expression was then analyzed by RT-PCR and normalized to that of <i>Hprt</i>. The data from four independent experiments are expressed as the change relative to the normoxic values. Statistical significance was assessed using a two-tailed paired t-test (*, p<0.05).</p

Gross appearance of tamoxifen-fed Vhl^floxed-Cre-ERT2 mice.

<p>(A) Vhl^wt-UBC-Cre-ER^T2 (n = 3), Vhl^floxed (n = 9) and Vhl^floxed-UBC-Cre-ER^T2 (n = 10) mice were administered tamoxifen as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022589#pone-0022589-g001" target="_blank">Figure 1</a> and the spleen/body weight ratio was then determined. Statistical significance was assessed using a two-tailed Student's t-test (*, p<0.05; **, p<0.01). Representative images of spleens (B), snouts (C) and paws (D) of Vhl^floxed-UBC-Cre-ER^T2 and control Vhl^floxed mice are shown.</p

<i>Vhl</i> and <i>Hif1α</i> gene expression in tamoxifen-fed Vhl^floxed-UBC-Cre-ER^T2 and Hif1α^floxed-UBC-Cre-ER^T2 mice.

<p>(A) Vhl^wt-UBC-Cre-ER^T2 (n = 3), Vhl^floxed (n = 6) and Vhl^floxed-UBC-Cre-ER^T2 (n = 6) mice were placed on a tamoxifen diet for ten days followed by ten additional days on a normal diet. Gene expression was assessed by RT-PCR in the tissues indicated, the expression of the <i>Vhl</i> gene was normalized to that of <i>Hprt</i> and it was expressed as the change relative to Vhl^floxed mice. (B) Tamoxifen intake was measured over the 10 days of tamoxifen administration in Vhl^wt-UBC-Cre-ER^T2 (n = 3), Vhl^floxed (n = 6) and Vhl^floxed-UBC-Cre-ER^T2 (n = 6) mice. (C,E) Hif1α^wt-UBC-Cre-ER^T2 (n = 4), Hif1α^floxed (n = 3) and Hif1α^floxed-UBC-Cre-ER^T2 (n = 5) mice were administered tamoxifen as indicated above. <i>Hif1α</i> (C) or <i>Vhl</i> (E) gene expression was normalized to that of <i>Hprt</i> and expressed as the change relative to Hif1α^floxed mice. (D) Tamoxifen intake in Hif1α^wt-UBC-Cre-ER^T2 (n = 4), Hif1α^floxed (n = 3) and Hif1α^floxed-UBC-Cre-ER^T2 (n = 5) mice was measured as in B. Total intake per day was expressed relative to the body weight at the end of the tamoxifen treatment and the values represent the mean ± SEM. Statistical significance was assessed using a two-tailed Student's t-test, (*, p<0.05; **, p<0.01) when comparing Vhl^wt-UBC-Cre-ER^T2 or Hif1α^wt-UBC-Cre-ER^T2 with Vhl^floxed-UBC-Cre-ER^T2 or Hif1α^floxed-UBC-Cre-ER^T2 respectively; (^##, p<0.01) when comparing Vhl^floxed or Hif1α^floxed with Vhl^floxed-UBC-Cre-ER^T2 or Hif1α^floxed-UBC-Cre-ER^T2 respectively.</p

<i>Erythropoietin</i> and <i>glucose transporter-1</i> gene expression in HL-1 cardiomyocyte cell line in response to activation of the oxygen-sensing HIF pathway.

<p>(A,B,C) HL-1 cells were transfected with a siRNA for <i>Hif1α</i> (siHIF1α) or a scrambled siRNA control (siSCR) and 24 hours after transfection, the cells were exposed to normoxic or hypoxic (1% O₂) conditions. The expression of <i>Epo</i>, <i>Glut1</i> and <i>Hif1α</i> was measured as described above and the data from three independent experiments are expressed as the change relative to the normoxic values. Statistical significance was assessed using a two-tailed Student's t-test (*, p<0.05).</p

Body weight during and after tamoxifen diet administration in Vhl^floxed-UBC-Cre-ER^T2 and control mice.

<p>Body weight of Vhl^wt-UBC-Cre-ER^T2 (n = 3), Vhl^floxed-UBC-Cre-ER^T2 (n = 6) control Vhl^floxed (n = 6) mice was measured before tamoxifen treatment (TFX 0d), at the end of 10 days on a tamoxifen diet (TFX 10d) and one day after returning to a normal diet (N +1d). Statistical significance was assessed using a two-tailed Student's t-test, (*, p<0.05; **, p<0.01; ns, no significant differences).</p