Selection and Analysis of a Mutant Cell Line Defective in the Hypoxia-inducible Factor-1 α-Subunit (HIF-1α)

Abstract

Hypoxia-inducible expression has been demonstrated for many groups of mammalian genes, and studies of transcriptional control have revealed the existence of hypoxia-responsive elements (HREs) in the cis-acting sequences of several of these genes. These sequences generally contain one or more binding sites for a heterodimeric DNA binding complex termed hypoxia-inducible factor-1 (HIF-1). To analyze this response further, Chinese hamster ovary cells were stably transfected with plasmids bearing HREs linked to genes encoding immunoselectable cell surface markers, and clones that showed reduced or absent hypoxia-inducible marker expression were selected from a mutagenized culture of cells. Analysis of these cells revealed several clones with transacting defects in HRE activation, and in one the defect was identified as a failure to express the α-subunit of HIF-1. Comparison of hypoxia-inducible gene expression in wild type, HIF-1α-defective, and HIF-1α-complemented cells revealed two types of response. For some genes (e.g. glucose transporter-1), hypoxia-inducible expression was critically dependent on HIF-1α, whereas for other genes (e.g. heme oxygenase-1) hypoxia-inducible expression appeared largely independent of the expression of HIF-1α. These experiments show the utility of mutagenesis and selection of mutant cells in the analysis of mammalian transcriptional responses to hypoxia and demonstrate the operation of HIF-1α-dependent and HIF-1α-independent pathways of hypoxia-inducible gene expression in Chinese hamster ovary cells. Hypoxia-inducible expression has been demonstrated for many groups of mammalian genes, and studies of transcriptional control have revealed the existence of hypoxia-responsive elements (HREs) in the cis-acting sequences of several of these genes. These sequences generally contain one or more binding sites for a heterodimeric DNA binding complex termed hypoxia-inducible factor-1 (HIF-1). To analyze this response further, Chinese hamster ovary cells were stably transfected with plasmids bearing HREs linked to genes encoding immunoselectable cell surface markers, and clones that showed reduced or absent hypoxia-inducible marker expression were selected from a mutagenized culture of cells. Analysis of these cells revealed several clones with transacting defects in HRE activation, and in one the defect was identified as a failure to express the α-subunit of HIF-1. Comparison of hypoxia-inducible gene expression in wild type, HIF-1α-defective, and HIF-1α-complemented cells revealed two types of response. For some genes (e.g. glucose transporter-1), hypoxia-inducible expression was critically dependent on HIF-1α, whereas for other genes (e.g. heme oxygenase-1) hypoxia-inducible expression appeared largely independent of the expression of HIF-1α. These experiments show the utility of mutagenesis and selection of mutant cells in the analysis of mammalian transcriptional responses to hypoxia and demonstrate the operation of HIF-1α-dependent and HIF-1α-independent pathways of hypoxia-inducible gene expression in Chinese hamster ovary cells. Naturally occurring genetic mutations have provided many insights into gene function. In an effort to extend this source of information, a large number of targeted or untargeted means of introducing mutations and selecting by genotype or phenotype have been devised. In somatic cells, one classical approach has been to subject a mutagenized culture to a substance whose toxicity is dependent on the activity of a set of genes involved in a signal transduction or metabolic pathway (1Hankinson O. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 373-376Crossref PubMed Scopus (159) Google Scholar, 2Laiho M. Weis F.M.B. Massague J. J. Biol. Chem. 1990; 265: 18518-18524Abstract Full Text PDF PubMed Google Scholar, 3Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). However, the number of properties that are “intrinsically selectable” in this way is limited, leading some workers to explore the possibility of introducing selectable properties by transfection of recombinant plasmids (4Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (315) Google Scholar, 5Bulla G.A. Fournier R.E.K. Somat. Cell Mol. Genet. 1992; 18: 361-370Crossref PubMed Scopus (21) Google Scholar). For instance, stable transfection of a plasmid bearing an interferon-responsive promoter linked to a bacterial guanidine phosphoribosyl transferase gene has been used to select cells from the fibrosarcoma line HT1080 that are defective in the response to interferons (4Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (315) Google Scholar). Similar promoters linked to the gene encoding the cell surface antigen CD2 have been used to select other lines defective in the interferon response using fluorescence-activated cell sorting (6Watling D. Guschin D. Müller M. Silvennoinen O. Witthuhn B.A. Quelle F.W. Rogers N.C. Schindler C. Stark G.R. Ihle J.N. Kerr I.M. Nature. 1993; 366: 166-170Crossref PubMed Scopus (421) Google Scholar). The recent recognition of a widespread transcriptional response to hypoxia (see Refs. 7Semenza G.L. Wang G.L. Mol. Cell. 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Rev. 1996; 76: 839-885Crossref PubMed Scopus (1037) Google Scholar), mediated by the activation of a heterodimeric basic-helix-loop-helix PAS 1The abbreviations used are: PAS, Per-aryl hydrocarbon receptor-ARNT-Sim; AK3, adenylate kinase-3; ARNT, aryl hydrocarbon receptor nuclear translocator desferrioxamine; EPAS-1, endothelial PAS protein-1; FACS, fluorescence-activated cell scanning; Glut-1, glucose transporter-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP78, glucose-regulated protein 78; HO-1, heme oxygenase-1; HAT, hypoxanthine-aminopterin-thymidine; HIF-1, hypoxia-inducible factor-1; HRE, hypoxia-responsive element; HPRT, hypoxanthine phosphoribosyl transferase; ICR191, 3-chloro-7-methoxy-9-(3-[chloroethyl]-amino propylamino)-acridine dihydrochloride; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); VEGF, vascular endothelial growth factor; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline. 1The abbreviations used are: PAS, Per-aryl hydrocarbon receptor-ARNT-Sim; AK3, adenylate kinase-3; ARNT, aryl hydrocarbon receptor nuclear translocator desferrioxamine; EPAS-1, endothelial PAS protein-1; FACS, fluorescence-activated cell scanning; Glut-1, glucose transporter-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP78, glucose-regulated protein 78; HO-1, heme oxygenase-1; HAT, hypoxanthine-aminopterin-thymidine; HIF-1, hypoxia-inducible factor-1; HRE, hypoxia-responsive element; HPRT, hypoxanthine phosphoribosyl transferase; ICR191, 3-chloro-7-methoxy-9-(3-[chloroethyl]-amino propylamino)-acridine dihydrochloride; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); VEGF, vascular endothelial growth factor; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline. protein complex termed hypoxia-inducible factor-1 (HIF-1) (10Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4928) Google Scholar) presents an important challenge, in terms of both defining the responsive target genes and analyzing the underlying mechanism of oxygen sensing and signal transduction. Here we describe the use of a selection strategy based on the linking of HIF-1-binding hypoxia-responsive elements (HREs) with minimal promoters coupled to genes encoding cell surface antigens. We describe the selection, from a mutagenized culture of Chinese hamster ovary (CHO-K1) cells, of cells that are deficient in HRE-dependent transactivation and the identification of one line that is functionally defective in the α-subunit of HIF-1. We demonstrate the utility of this cell line in characterizing HIF-1α-dependent and HIF-1α-independent patterns of hypoxia-inducible gene expression. Cells were grown in Ham's F-12 (Sigma) supplemented with 10% fetal calf serum (Globepharm), l-glutamine (2 mm), penicillin (50 IU/ml), and streptomycin sulfate (50 μg/ml). Transfections were performed by electroporation using a 1-millifarad capacitor array charged at 350 V. Plasmids bearing HREs linked to reporter genes or selectable markers are illustrated in Fig. 1. pHPGK-CD2 was as described previously (11Dachs G.U. Patterson A.V. Firth J.D. Ratcliffe P.J. Townsend K.M.S. Stratford I.J. Harris A.L. Nat. Med. 1997; 3: 515-520Crossref PubMed Scopus (346) Google Scholar). p(E1–25)5SV-ESel contained five copies of a 25-base pair HRE from the mouse 3′-erythropoietin enhancer coupled to the SV40 early promoter (from p(E1–25)5SV-GH (12Pugh C.W. Ebert B.L. Ebrahim O. Ratcliffe P.J. Biochim. Biophys. Acta. 1994; 1217: 297-306Crossref PubMed Scopus (39) Google Scholar)) ligated into pBluescript and linked to the complete E-selectin coding sequence and SV40 splice/poly(A) from pCDM8. contained two copies of a pair HRE from the mouse to the of the promoter into bearing a and contained and genes, linked to the SV40 early contained the gene from into stable of cells bearing two hypoxia-inducible cell surface markers was by transfection with pHPGK-CD2 and by selection in and of one with p(E1–25)5SV-ESel and and selection in was performed by of cells to 3-chloro-7-methoxy-9-(3-[chloroethyl]-amino propylamino)-acridine at for using a based on Ref. 1979; transfection for transcriptional responses were performed using the with plasmid as a control for transfection Cells from transfection were for or hypoxia or The of the HIF-1α, ARNT, or genes to the hypoxia-inducible response was by of cells with and the expression plasmid the sequence from (10Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4928) Google Scholar) into S. O. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar), or 1997; PubMed Scopus Google cells were for at oxygen and grown in or in hypoxia for a of gene expression were performed on cells were performed on of cells in with and hypoxia or were in a with or and the For genes, experiments were performed for a of or in growth For of the and the cells were grown in for a in to the To for gene expression in cells were in with or glucose for transfection of the mutant with a gene was performed by electroporation using the sequence from into and by selection in (Sigma) For fluorescence-activated cell cells were in in at at and in of with and fetal calf was performed on for using a mouse CD2 and mouse E-selectin Cells were and in in analysis For selection by A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), cells were as and in with and fetal calf at was performed with mouse CD2 at a or with mouse at The cells were in in of and were at for the was to the cells. These cells were in culture and in were as were with of a of (Sigma) in for at The were in PBS, by with of (2 serum in and at cells were cells and an hypoxanthine phosphoribosyl of selected in For of and cells were and that the cells were the cells were with and of was for The was in of a and the cells were in at for in complete and and was was using a and an was performed as described in Ref. J.D. Ebert B.L. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar using a binding from the mouse enhancer For nuclear were by and to was using a a recombinant of HIF-1α, a and was by a in PIPES, and and by an or in for For of were on a and to a The were with with using for and by for HIF-1α. were performed as described previously C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of the was performed using a and was to an control for a nuclear for Chinese hamster genes were by using based on sequences for other and into or of for Chinese hamster or mouse genes are in For genes, the of was as nuclear GAPDH, GRP78, and HO-1, and of and and HIF-1α, and and VEGF, of used in protein of endothelial growth in a activity was in cell using a and a activity in cell was as in a and in using selection genetic markers by transfection to that are functionally defective in of an transcriptional mechanism is that the transfected gene is generally stable the a selection an transfected marker gene a large of cells that have or expression of the marker bearing defects in the mechanism of gene We two to this two immunoselectable markers were to selection of cells that one or the other selection of the two and to cells by selection for markers was the to the of HREs in hypoxia-inducible gene expression in cells, were performed with plasmids bearing coupled to for cell surface marker gene expression was by and analysis The two plasmids that showed the of marker expression were for stable transfection and are illustrated in cells were transfected with pHPGK-CD2 and and clones were selected in was transfected with p(E1–25)5SV-ESel and and clones were selected in clones were for hypoxia-inducible expression of the transfected cell surface markers, and one was selected on the of the expression of both analysis of and of cells cells with CD2 and markers are by cells express marker gene as by and For the line was to In one to a of of for in in at the cell we that mutations of hypoxia-inducible cells, in were to at for described a of to J. S. D. Kerr I.M. Stark G.R. Mol. Cell. Biol. PubMed Scopus Google Scholar, John J. D. M. Kerr I.M. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), cells were to and to a two of the and The cells were in the these to and the selection Cell one was The of cells that were to HRE-dependent of gene expression was by of the cell surface markers and selection by a cells were with to CD2 or and on a with Cells that to to the were and in culture the cells to were and of was To the of selection in this experiments were performed in a marker cell a was with cells in a of of of this cell for of the was for expression by These cells a of 10% in the by in a that been was that for a to in of to for a mutant of the mutagenized cells was performed with and to a of the selection was in and was for of both CD2 and E-selectin and with cells. for one of the are in Fig. in the hypoxia expression of both markers was in a of cells. from were by and for of both surface markers by clones that showed of these markers were for defects in HRE activation by transfection with a plasmid bearing an HRE-dependent reporter In this of these clones showed of HRE activity that were the or to wild However, showed a in the hypoxia-inducible response from to of that in wild cells. For of these this was stable of showed an absent response. Fig. expression of CD2 and E-selectin in was stable with to this phenotype and has been subject to the of was in cell of was selected in of the and this phenotype was by to gene is for in of the cells and the cells were using and a gene from and an gene from the were selected in of several from two independent showed of the response encoding the heterodimeric HRE binding complex been identified at an in this (10Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4928) Google Scholar), we the defect in by transfection with encoding or cells were with and expression plasmids encoding HIF-1α, ARNT, or are in A. of HIF-1α, ARNT, a of reporter gene expression. of expression in cells were with of expression plasmid and by of transfected cells to For the activity of in the cells is These that cells were defective in some of expression or the to to hypoxia and the activity of a To this defect further, cells and wild cells were with a gene C.W. M. Ratcliffe P.J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and a reporter Similar activity was in both cell that the underlying mechanism was of and protein was and the of in cells was to reduced with wild were and the of in cells was at the of on the of the revealed a signal to the of wild In for were analysis for protein was performed on nuclear from wild and mutant cells For nuclear from cells were in and wild cells show nuclear of whereas was in cells. using a binding from the mouse demonstrated of the complex in nuclear from cells the were in the was as in wild cells. basic-helix-loop-helix PAS termed EPAS-1, has been sequence to HIF-1α, with ARNT, and DNA sequences 1997; PubMed Scopus Google M. S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, Brown M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), we in wild and mutant cells by signal was in cell in of to of was by this in from Chinese hamster We the of a expression plasmid to the defect in HRE activity in transfection in Fig. were to with expression. of to an in both and HRE activity and of hypoxia-inducible reporter gene expression. The analysis that cells a defect in expression. To explore the of this on hypoxia-inducible gene the of groups of genes was in wild cells and cells. of the genes of these genes of and showed a or by hypoxia in wild cells and were for genes selected from groups that showed responses or were of the VEGF, Glut-1, GAPDH, AK3, HO-1, and genes. were For some genes, a of hypoxia-inducible expression was in wild cells that was reduced or in or of by hypoxia was in the wild cells, and expression was in cells in a was in cells with The by hypoxia was for cells were to an of expression was in cells was in cells. For and AK3, a of hypoxia-inducible expression was in cells. In and showed responses to hypoxia that were reduced in cells. that was for the of genes at oxygen that independent pathways of hypoxia-inducible gene expression To this further, the expression of genes was to more oxygen for For genes, the of was that with oxygen for In cells, the response to hypoxia was for and gene expression. The of and genes from in that the responses were in for instance, was and with oxygen in and cells, that pathways of hypoxia-inducible expression were on these genes in cells. and genes have both been demonstrated to by O. J. Cell. PubMed Scopus Google Scholar, P.J. Jiang B.-H. J. Semenza G.L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, B.L. Firth J.D. Ratcliffe P.J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), we the response of these two genes to and other to HIF-1. are in was to a by and in cells. of these responses were in cells. In was to a by was by in cells, and these responses were in cells. responses to were the set of genes, two patterns were For Glut-1, AK3, and genes, the response to hypoxia was by whereas for this was the of glucose and heme in in and cells. of and is of cells were in hypoxia hypoxia or for glucose has been to several of these genes M. D. A. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar, D. Nature. 1997; PubMed Scopus Google Scholar), for VEGF, Glut-1, HO-1, and were of cells in glucose are in Fig. showed by as with by this response was in cells. The demonstrate that the of some genes in response to hypoxia is in cells, a for this in these To this further, cells were transfected with and with selection in were and for expression of the marker genes. expression of the cell surface markers was in a of the by for expression of E-selectin was to this of clones were by analysis that these clones of Chinese hamster clones were for Glut-1, GAPDH, and and in clones were are for one a of in of by both hypoxia and In some clones of by of an of was in cells, that of expression of target genes in cells. The of these experiments was to select mutant cells with a defective transcriptional response to we were of hypoxia-inducible genes with selectable we used stably recombinant plasmids to selectable markers of HREs linked to cell surface antigens. strategy was in the of several clones that showed reduced HRE activity as by transfection and one with absent HRE to defective in the α-subunit of the transcriptional complex HIF-1. were some to that of hypoxia-inducible activity for some of the in transfection of the responsive stably marker was that the use of selection markers, more control of the of gene expression is we an strategy to that has been described for the selection of interferon (6Watling D. Guschin D. Müller M. Silvennoinen O. Witthuhn B.A. Quelle F.W. Rogers N.C. Schindler C. Stark G.R. Ihle J.N. Kerr I.M. Nature. 1993; 366: 166-170Crossref PubMed Scopus (421) Google Scholar). The of has been used for expression of genes encoding cell surface A. Proc. Natl. Acad. Sci. U. S. 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