Hypoxic regulation of lactate dehydrogenase A. Interaction between hypoxia-inducible factor 1 and cAMP response elements.

The oxygen-regulated control system responsible for the induction of erythropoietin (Epo) by hypoxia is present in most (if not all) cells and operates on other genes, including those involved in energy metabolism. To understand the organization of cis-acting sequences that are responsible for oxygen-regulated gene expression, we have studied the 5′ flanking region of the mouse gene encoding the hypoxically inducible enzyme lactate dehydrogenase A (LDH). Deletional and mutational analysis of the function of mouse LDH-reporter fusion gene constructs in transient transfection assays defined three domains, between −41 and −84 base pairs upstream of the transcription initiation site, which were crucial for oxygen-regulated expression. The most important of these, although not capable of driving hypoxic induction in isolation, had the consensus of a hypoxia-inducible factor 1 (HIF-1) site, and cross-competed for the binding of HIF-1 with functionally active Epo and phosphoglycerate kinase-1 sequences. The second domain was positioned close to the HIF-1 site, in an analogous position to one of the critical regions in the Epo 3′ hypoxic enhancer. The third domain had the motif of a cAMP response element (CRE). Activation of cAMP by forskolin had no effect on the level of LDH mRNA in normoxia, but produced a magnified response to hypoxia that was dependent upon the integrity of the CRE, indicating an interaction between inducible factors binding the HIF-1 and CRE sites.

The regulation of gene expression by oxygen is an important feature of many biological processes. In energy metabolism for instance, mRNA levels for a number of glycolytic and gluconeogenic enzymes are subject to transcriptional regulation by oxygen in a coordinate and reciprocal manner (1)(2)(3)(4)(5)(6)(7)(8)(9). Other genes that are regulated by oxygen include vascular growth factors (10), transcription factors (11), glucose transporters (12), tyrosine hydroxylase (the rate-limiting enzyme for dopamine synthesis) (13), and erythropoietin (Epo), 1 a hormone that regulates erythropoiesis in accordance with the oxygen carrying capacity of the blood (14).
Recent studies of Epo gene regulation have provided impor-tant insights into the mechanism of the cellular response to hypoxia. First, they have led to the proposal of a specific oxygen sensing mechanism, possibly involving a heme protein liganding with dioxygen (14) or forming part of a non-mitochrondrial electron transport system (15). Second, they have provided strong evidence of a transcriptional response to hypoxia, which operates in a wide variety of cells. The Epo 3Ј hypoxic enhancer can drive oxygen-regulated expression in many cells that do not produce the hormone (16,17), and a nuclear factor termed hypoxia-inducible factor-1 (HIF-1), which binds to a functionally critical region of the Epo enhancer, has also been shown to be widespread (18 -21), with the implication that the same control mechanism might operate on other genes. A link between the regulation of Epo and of glycolytic enzymes was established by the demonstration that these genes shared similar inducible characteristics, and that the gene encoding phosphoglycerate kinase-1 also contained a functionally critical HIF-1 binding site in its 5Ј enhancer (22). Further work has demonstrated HIF-1 sites at other glycolytic gene loci (23), but it is not yet clear whether all of these are functional, or how they cooperate with other binding sites to produce oxygen-regulated control sequences. Detailed analysis of the oxygen-regulated enhancer lying 3Ј to the Epo gene has defined at least three functional domains and has shown that cooperative interaction between these domains is required for function. Neither the Epo HIF-1 site nor the other domains in the Epo enhancer are individually capable of driving oxygen-regulated expression of a reporter, even when reiterated; cooperative interaction of at least two domains seems to be necessary for functional activity (17, 20, 24 -26). It is not known whether this is a general property of oxygen-regulated gene control elements containing HIF-1 sites, because only the Epo enhancer has been subject to detailed analysis.
To understand the organization of hypoxically inducible cisacting sequences, we have therefore performed a detailed analysis of a second hypoxically inducible sequence: that contained in the mouse lactate dehydrogenase A (LDH-A) 5Ј flanking region (22). Lactate dehydrogenase catalyses the interconversion of pyruvate and lactate. In anaerobic cells conversion of pyruvate to lactate regenerates NAD ϩ and is essential for continued glycolytic flux. The LDH-A gene encodes the M subunit of the enyzme; the isoform that is adapted for the anaerobic function. The M 4 homotetramer has a low K m for pyruvate and a high V max , and is the predominant form expressed in glycolytically active skeletal muscle and in many tissue culture cell lines (27). Previous work has established that expression of both LDH-A mRNA and protein are inducible by hypoxia (3,9,22); they are also inducible by cAMP (1,2,9), raising the interesting issue as to how these inducible stimuli might interact.
A series of mutations in the LDH-A 5Ј flanking region, scanning a region of 56 bp that was indicated as being functionally important by deletional studies, define three domains that are crucial for oxygen-regulated function. One of these is a HIF-1 binding site and is absolutely required for inducible activity. The two other domains lie on either side of this motif. One, which lies 5Ј to the HIF-1 site, occupies a position similar to that of a critical cooperative site in the Epo enhancer. The other lies 3Ј to the HIF-1 site and has the characteristic motif of a cyclic AMP response element (5Ј-TGACGTCA-3Ј) (28). Functional cooperativity is shown between these sites; concatamers that contain the HIF-1 site alone are not capable of driving oxygen-regulated expression of a reporter through a heterologous promoter, whereas concatamers containing the HIF-1 site in conjunction with either of the other sites are, and the combination of all three sites together confers the greatest level of hypoxic inducibility. Moreover, forskolin, which modulates activity of the cAMP system by activation of protein kinase A, is shown to increase the magnitude of hypoxic induction of the LDH-A gene by interacting with the CRE.

EXPERIMENTAL PROCEDURES
Cell Lines and Culture Conditions-All experiments were performed on HeLa (human cervical carcinoma) cells grown in minimum essential medium with Earle's salts supplemented with 10% fetal calf serum, glutamine (2 mM), penicillin (50 IU/ml), and streptomycin sulfate (50 g/ml). In studies of endogenous gene expression plates were used when 70% confluent. Hypoxic stimulation (1% O 2 , 5% CO 2 , 94%N 2 in most experiments; in some 0.5%, 3% and 5% O 2 , each with 5% CO 2 and N 2 to balance) was provided by incubation for 14 -16 h in a Napco 7100 incubator.
Transient Transfection and RNA Analysis-In all experiments the test plasmid (20 -40 g), encoding human growth hormone as a reporter, was transfected by electroporation in combination with a control plasmid (20 -40 g), which expressed human ␣ 1 -globin. Transfected cells were split equally and incubated in parallel under normoxic (20% O 2 , 5% CO 2 , 75%N 2 ) or hypoxic conditions as described above. RNA was extracted, analyzed by RNase protection, and quantitated as described previously (29). For assay of material from transiently transfected cells, double hybridization with probes that protected 120 bp of GH mRNA and 132 bp of ␣-globin mRNA was performed on 3-5 g of RNA. For assay of endogenous gene expression, 25 g of RNA was hybridized with a cRNA probe that protected 92 bp of human LDH-A mRNA transcribed from exons 2 and 3. A small quantity of antisense RNA generated by in vitro transcription of a fragment of the rat endothelin 1 gene was added to each of these samples, together with a sense probe that protected 333 bp of that transcript. Estimation of the recovery of the endothelin hybridization product served to control for the effects of variation in sample processing and gel loading between specimens.
Deletional and Mutational Analysis-The plasmid LDHGH formed the basis for deletional and mutational analysis of the mouse LDH-A promoter (22). This contained 233 bp of mouse LDH-A promoter and adjacent 5Ј sequence, extending from position Ϫ186 to 47 (where 1 is the transcription initiation site), cloned into the BamHI site in the 5Ј untranslated region of the growth hormone (GH) reporter. Progressive 5Ј truncations of the LDH element in this construct were made by PCR using appropriate oligonucleotides (Fig. 1). The region that was defined as critical for oxygen-regulated control of the reporter was then examined using a series of 14 consecutive 4-bp mutations of the LDHGH plasmid in which purines were substituted for their non-complementary pyrimidines, and vice versa (Fig. 2). These were made using the pALTER-based Altered Sites in vitro mutagenesis system as directed by the manufacturers (Promega, Southampton, United Kingdom). The nucleotide sequence of the LDH element in all plasmids was confirmed by dideoxy sequencing.
Functional Testing of Concatamerized Elements-Since mutational analysis defined three domains that were functionally important in driving oxygen-regulated expression of the GH reporter, constructs were made to determine whether these domains, concatamerized either singly or in combination with one another, were capable of conferring the ability to respond to hypoxia on a minimal heterologous promoter (Fig. 3). The three largest elements (Fig. 3, panel B: A-C) were made from LDHGH by PCR using appropriate oligonucleotides with 5Ј ends that could be digested with XbaI (5Ј-GCTCTAGA-(LDH sequence)-3Ј). The three smallest elements (Fig. 3, panel B: D-F) were synthesized as oligonucleotides, which, when phosphorylated and annealed, produced elements with 5Ј XbaI-and 3Ј SpeI-compatible ends (top strand: 5Ј-CTAGA-(LDH sequence)-A-3Ј; bottom strand: 5Ј-CTAGT-(LDH se-quence)-T-3Ј). The elements were each then cloned into an XbaI site located 10 bp 5Ј to the herpes simplex thymidine kinase TATTAA box of the reporter plasmid, which had low basal activity and was not itself inducible by hypoxia (22). The nucleotide sequence of dimers or trimers of each element was confirmed by dideoxy sequencing.
Nuclear Extract and Electrophoretic Mobility Shift Assay-A modification of the protocol of Dignam et al. (30) was used to prepare nuclear extract. Oligonucleotides used as probes or competitors (Table I) were purified by polyacrylamide gel electrophoresis. Labeling of the 5Ј nucleotide of probes was performed with [␥-32 P]ATP (3000 Ci/mmol) using phage T 4 polynucleotide kinase. Labeled oligonucleotides were annealed with a 4-fold molar excess of the complementary strand. Unlabeled oligonucleotides were annealed in molar equivalent quantities. Binding reaction mixtures (20 l) contained 50 mM KCl, 1 mM MgCl 2 , 0.5 mM EDTA, 5 mM dithiothreitol, 5% (v/v) glycerol, and 0.15-0.30 g of sonicated poly(dI-dC). Nuclear extract (5 g) was incubated with this mixture for 5 min at room temperature, before probe (approximately 0.1 ng) and specific competitors were added. Incubation was continued for another 10 min. Reactions were electrophoresed (12.5 V/cm) at 4°C using 5% polyacrylamide in 0.3 ϫ TBE (30 mM Tris, 30 mM boric acid, 0.06 mM EDTA, pH 7.3, at 20°C).
Modulation of Cyclic AMP-The mutational analysis and the functional testing of concatamerized elements both suggested that one of the functional domains important for oxygen-regulated expression had the characteristic sequence motif of a CRE (5Ј-TGACGTCA-3Ј) (28). The effect of agents that modulate activity of the cAMP system on oxygenregulated expression of the endogenous LDH gene and of transiently transfected constructs was therefore examined. Two agents were used: H8 (N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide), an inhibitor of protein kinase A; and forskolin, an activator of the cAMP-protein kinase A system. For assay of endogenous LDH expression, H8 (15 M) or forskolin (50 M) were applied to cells incubated in normoxia, and 8 h later the plates were transferred into the hypoxic incubator, where they remained for 16 h. Controls were exposed to the agents for 24 h of normoxic incubation. In transient transfection experiments, cells were incubated with the same concentrations of H8 or forskolin for 8 h before they were harvested for electroporation, and the chemicals were applied again immediately after the cells had been plated out following transfection. Control transfections were performed in parallel using cells that were not exposed to either agent. RESULTS We and others have previously reported that LDH-A mRNA is inducible by exposure to 1% O 2 (5,9,22). In this study we found that it was inducible by hypoxia over a range of oxygen concentration from 5% to 0.5%. In comparison with paired normoxic control plates we found induction as follows: 5% O 2 , 1.85 Ϯ 0.16-fold (mean Ϯ S.E., n ϭ 5); 3% O 2 , 2.03 Ϯ 0.41 (n ϭ 4); 1% O 2 , 1.67 Ϯ 0.26 (n ϭ 4); 0.5% O 2 , 2.00 Ϯ 0.10 (n ϭ 5). For comparison with previously published work, all experiments were performed with the 1% O 2 stimulus (5,9,16,17,22,29).
The effect of a series of truncations of the 5Ј end of the LDH sequence on the ability to drive expression of the reporter in normoxia and hypoxia are shown in Fig. 1. Deletion of 16 bp between L3 and L3.2 caused a reduction of only 16 Ϯ 6% (n ϭ 4) in the level of normoxic expression, but a fall in the ratio of hypoxic/normoxic expression from 7.9 Ϯ 1.9 to 1.5 Ϯ 0.3, thus defining the 5Ј extent of the oxygen-regulated control element. The effect of a series of 14 consecutive 4 bp mutations, the most 5Ј of which begins 8 bp 5Ј to the first truncation shown to ablate oxygen-regulated function (L3.2), is shown in Fig. 2. Three domains are important, defined by mutations (M) 1, M4-M5, and M10-M11, of which the one affected by M4-M5 seems to be the most significant, since these mutations completely abrogated the ability to respond to hypoxia.
The capacity of the three domains to drive hypoxic expression through a heterologous minimal promoter, when concatamerized either singly or in combination with one another, is shown in Fig. 3. Neither a dimer nor a trimer containing solely the domain defined by M4-M5 could confer oxygen-regulated expression. By contrast, dimers containing both the M4-M5 domain and either of the other domains were capable of oxygen-regulated action, although one construct containing both the M4-M5 and M10-M11 domains was not, for reasons that are unknown. A dimer containing all three domains conferred the greatest level of hypoxic inducibility.
The domain defined by M4-M5 has the motif of a HIF-1 site (5Ј-(C/G/T)ACGT(G/C)C(G/T)-3Ј). For analysis of the ability of this element to bind hypoxically inducible nuclear factors, a double-stranded oligonucleotide based on the domain was in-cubated with nuclear extract from normoxic and hypoxic HeLa cells. Figs. 4 and 5 show a pattern of binding to this oligonucleotide that closely resembles that seen using oligonucleotides derived from HIF-1 binding sites in the Epo 3Ј enhancer and the PGK 5Ј flanking sequence. They also demonstrate that there is cross-competition for binding of hypoxia-inducible factor(s) between these sequences, and that this is lost when the HIF-1 binding site of either the Epo or PGK oligonucleotide is mutated.
The domain defined by M10-M11 has the motif of a CRE. To test for functional involvement of cAMP in basal normoxic and hypoxically induced expression of LDH-A, the effects of the protein kinase inhibitor H8 and the protein kinase A activator forskolin were examined. The results are shown in Fig. 6. Neither agent altered the level of normoxic expression of the endogenous gene. Both M4 and M10 produced a lower level of normoxic expression than did the wild type LDHGH construct. The level of normoxic expression of all three constructs was reduced in the presence of forskolin, whereas H8 was without effect. Forskolin induced a substantial increase in the ratio of hypoxic/normoxic expression of both the endogenous gene (control 1.40 Ϯ 0.09, forskolin 3.80 Ϯ 0.89, n ϭ 6, p Ͻ 0.05) and the reporter after transfection with wild type LDHGH (control 6.7 Ϯ 1.4, forskolin 17.1 Ϯ 1.5, n ϭ 3, p Ͻ 0.05). As expected, the M4 construct was not capable of driving oxygen-regulated expression and did not become so in the presence of either H8 or forskolin. Construct M10 did exhibit hypoxic inducibility, but in contrast with wild type LDHGH, the magnitude of this inducibility was not altered by forskolin (ratio of hypoxic/normoxic expression:control 5.2 Ϯ 1.5, forskolin 6.4 Ϯ 0.6, n ϭ 3, p not significant), providing functional evidence that the ability of cAMP to magnify the hypoxic induction of LDH is modulated by the CRE motif placed between Ϫ48 and Ϫ41 bp upstream of the transcription initiation site. DISCUSSION Functional similarities between the oxygen-dependent regulation of genes encoding glycolytic enzymes and the regulation of the Epo gene have led to the analysis of glycolytic gene control sequences for sites that bind HIF-1, a nuclear factor that is critical for inducible operation of the Epo 3Ј enhancer (22,23). Such a site was demonstrated by deletional analysis to be crucial for activity of the element driving oxygen-regulated expression in the phosphoglycerate kinase 5Ј flanking sequence (22), providing firm evidence linking the hypoxic induction of glycolytic enzymes and Epo to a common cellular oxygen sensing mechanism. Other potential HIF-1 sites have been defined by sequence analysis of glycolytic gene loci. For several of these, HIF-1 binding activity has been shown, and for some the functional activity of concatamerized sequences has been dem-onstrated on heterologous reporters (23). However, no detailed analysis of the organization of an hypoxically regulated enhancer or promoter has been made in this group of genes, although there have been many analyses of their regulatory sequences in normoxic cells. In this paper we present such an analysis for the LDH-A promoter in HeLa cells, a cell line that demonstrates hypoxically inducible expression of the LDH-A gene and that provides a suitable basis for transfection studies. Whereas progressive deletion from Ϫ186 bp to Ϫ63 bp had only modest effects on normoxic promoter function, a sharp reduction in hypoxic activity resulted when sequences between Ϫ88 bp and Ϫ80 bp were deleted, thus defining a hypoxically inducible promoter of approximately 90 bp. Mutational analysis was focused on this region, and defined three domains within a 52-bp sequence lying 5Ј to the "TATA" box: M1 (Ϫ84/Ϫ81), M4-M5 (Ϫ72/Ϫ65), and M10-M11 (Ϫ48/Ϫ41), which were necessary for hypoxically inducible function.
The M4-M5 site corresponds to the HIF-1 binding element recently reported in independent experiments during the course of this work (23). The current analysis shows that mutation of this site completely abolishes oxygen-regulated expression, indicating that it is functional. Mutations at the other two sites, M1 and M10-M11, severely reduced but did not entirely abrogate the response to hypoxia. Thus, as with the Epo 3Ј enhancer, the HIF-1 site is absolutely necessary, but not sufficient, for inducible activity (17, 20, 24 -26). One significant difference between the function of the HIF-1 site in the LDH-A  Table I promoter and that in the Epo enhancer relates to its basal activity. Whereas the latter has little activity in normoxic cells (17), the former has strong constitutive action, and mutation at the HIF-1 site has a substantial effect on this.

. Electrophoretic mobility shift assay showing that an oligonucleotide from the LDH-A 5 flanking region (LDH) binds to an hypoxia-inducible nuclear factor, and that it cross-competes for the binding of such factors with oligonucleotides from the PGK and Epo hypoxic enhancers (for oligonucleotide sequences, see
Either HIF-1 or the constitutive complexes that bind to the HIF-1 site in the LDH-A promoter (see Figs. 4 and 5) could be responsible.
Mutation M1 had no action on constitutive promoter function but severely reduced inducible promoter function. It is interesting that there is a striking similarity between the position of this domain with respect to the HIF-1 site in LDH, and the arrangement of functional domains in the Epo 3Ј enhancer (17, 20, 24 -26). Table II shows an alignment of the LDH-A promoter (in reverse orientation) with the sequences of the mouse and human Epo enhancers. It can be seen that in each case mutational analysis has defined a functionally critical domain in the same position, between 7 and 11 bp 3Ј to the HIF-1 site. There is sequence homology between this region in mouse LDH-A and mouse Epo, with 3 of 4 bp identical, but not between the mouse sequences and that of human Epo. The nature of the factor that binds to this site is unknown. In LDH-A, but not Epo, the sequence that is mutated in M1 is part of a Myo-D left binding site (5Ј-CACGTG-3Ј) (31). However, the consensus extends into the mutants M0 and M2, neither of which affected function. Nevertheless, it is possible that in muscle cells Myo-D could affect the inducible operation of the LDH promoter by binding at this site.
The third domain (M10-M11) involved in hypoxically inducible function of the LDH-A promoter has the characteristic motif of a cyclic AMP response element (5Ј-TGACGTCA-3Ј) (28). Mutation of this site reduced, rather than abolished, the magnitude of induction by hypoxia, and in keeping with previous analyses it was also found to contribute to constitutive promoter function (1,2,32).
The minimal requirements for hypoxically inducible function were determined by assay of concatamerized subsequences placed adjacent to a minimal promoter. None of the constructs that omitted the HIF-1 site showed any inducible activity. However, the HIF-1 element alone was unable to support inducible expression, even as a multimer. By contrast, constructs that contained the HIF-1 site, accompanied by either the upstream domain or the CRE, could convey inducible function, indicating that cooperative interactions necessary for hypoxic function could take place with factors binding either of these sites. A requirement for cooperation between HIF-1 and other binding sites is consistent with analysis of the Epo enhancer (17, 20, 24 -26). However, in our analysis of the mouse phosphoglycerate kinase-1 enhancer, we found that a multimerized 18-bp sequence containing the HIF-1 site was sufficient to confer hypoxic inducibility on a heterologous promoter (22). Semenza and colleagues (23) have recently demonstrated this property using a dimer of 24 bp of sequence from the human phosphoglycerate kinase gene, and with a trimer of a 26-bp oligonucleotide derived from mouse phosphofructokinase L sequence. It is not clear whether the HIF-1 complexes at these sites are in some way different from those in Epo and LDH-A in being able to drive oxygen-regulated expression on their own, or whether the elements studied contain other cooperative domains.
The finding that the CRE was one of the functional domains in the LDH-A promoter required for the response to hypoxia was clearly of interest in relation to the ability of cAMP to stimulate LDH-A expression in some cells (1,2,32). In HeLa cells we found that neither the protein kinase A inhibitor H8 nor the c-AMP agonist forskolin produced significant changes in LDH-A mRNA in normoxia. By contrast, hypoxic induction of the endogenous gene was much magnified in the presence of forskolin. The transfected wild type LDHGH construct also had this property, but the M10 promoter mutated construct did not, demonstrating that the interaction occurred within the LDH-A promoter, and was dependent on an intact CRE. The M4 mutated promoter was completely unresponsive to hypoxia in the presence of forskolin, indicating that the HIF-1 site remained absolutely necessary for hypoxic function.
Although we did not measure protein kinase A activity, the observation that forskolin did not augment the response to hypoxia in the CRE-mutated construct strongly suggests that this action was modulated by the adenylyl cyclase/protein kinase A/CRE-binding protein system, rather than through one of forskolin's other effects (33). We do not know which of the many CRE binding species are involved in this interaction; however, the finding that while forskolin augmented the hypoxic response, H8 did not reduce it, may indicate that both constitutive and inducible CRE species are involved. The small reduction in expression of all LDHGH plasmids in normoxic cells treated with forskolin, an effect not seen on expression of the endogenous gene, was not dependent on the CRE and is unexplained. One possibility is that sequence(s) excluded from the promoter construct may act on the endogenous gene to negatively regulate the effect that forskolin had on normoxic expression of that construct.
Overall, these results indicate the potential for complex interaction between the HIF-1 site and the CRE in the LDH-A promoter, the most marked being that stimulation of the CRE leads to a magnified hypoxic response. The precise nature of this interaction may vary between cell types, since in a study of vascular smooth muscle cells Marti and colleagues (9) found that forskolin induced LDH in normoxic cells, but did not produce enhancement of the response to hypoxia, and H8 reduced the induction of LDH by hypoxia. Agonists and antagonists of the cAMP system have been reported to affect a variety of cellular responses to hypoxia, including expression of the Epo gene (34). Since cAMP levels can be increased by hypoxia, it has been proposed that this second messenger is directly involved in the cellular signaling system that responds to this stimulus (9,34). The current experiments demonstrate a different type of involvement of the cAMP system in hypoxic gene regulation, where factors binding to two distinct cis-acting sequences, the HIF-1 site and the CRE, interact.
In summary, we have performed a detailed analysis of the mouse LDH-A promoter, demonstrating that oxygen-regulated function depends upon cooperation between three domains: a HIF-1 binding site, which is absolutely necessary, but not by itself sufficient, for hypoxically inducible function; a domain adjacent to this, located in the same position relative to the HIF-1 site as a site critical for function of the Epo enhancer; and a CRE. These findings indicate both that HIF-1 is critical to the mechanism of hypoxic induction of LDH-A and demonstrate that the mechanism requires cooperation of HIF-1 with neighboring sites. The interactions of stimuli acting at the HIF-1 site and the CRE illustrate how different characteristics of an inducible response can be generated from interactions between specific binding sites.

TABLE II
Comparison of LDH and Epo sequences Sequence of part of the oxygen-regulated control element in the mouse (m)LDH 5Ј-flanking region is shown, with mutants that ablate (M4 and M5) or severely impair (M1) function underlined. Sequences from the mouse and human (h) Epo 3Ј enhancers are aligned with respect to their HIF-1 sites (boxed area). Regions of these enhancers demonstrated to be critical for function are underlined. In both cases a functionally significant domain is located at an equivalent position to that occupied by M1 in LDH: m6 of Pugh et al. in mEpo (17); m7 of Semenza and Wang in hEpo (25). Bases that are identical in mLDH and mEpo are shown in bold type.