©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Use of a Marked Erythropoietin Gene for Investigation of Its Cis-acting Elements (*)

Vincent Ho (§) , Anthony Acquaviva(§)(¶) , Elia Duh (**) , H. Franklin Bunn (§§)

From the (1) Division of Hematology-Oncology, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To examine the function of conserved noncoding regions in the erythropoietin (Epo) gene, we have prepared clones and pools of Hep3B cells stably transfected with a marked 4.1-kilobase Epo gene and deletions thereof. The marked transcripts had single base substitutions at three sites in the coding portion of Exon 5, enabling them to be distinguished from endogenous Epo mRNA by ribonuclease protection and competitive polymerase chain reaction. The basal expression and hypoxic induction of the marked Epo gene that had no deletions were indistinguishable from that of the endogenous Epo gene. Likewise, deletion of conserved intervening sequence 1 had minimal effect on hypoxic induction. In contrast, a 3`-deletion that included the conserved 3`-enhancer element resulted in a substantial, but not complete, suppression of hypoxic induction while a 3`-deletion downstream of the enhancer resulted in enhancement. A 188-base pair deletion of a conserved 3`-untranslated region in Exon 5 had minimal effect on hypoxic induction. However, the truncated Epo mRNA had a markedly prolonged half-life (15 h) in comparison to the endogenous Epo mRNA (2.0 h) or the marked full-length Epo mRNA (2.1 h). Further deletions in the 3`-UTR showed that a relatively small region of approximately 50 bases is responsible for the relatively rapid turnover of Epo mRNA. These experiments provide information on cis-acting elements of the Epo gene that cannot be obtained from conventional reporter gene transfection experiments.


INTRODUCTION

Erythropoietin is a 30.4-kDa glycoprotein that regulates red cell mass (1) . Reduction in oxygen tension markedly increases the production of this hormone in the kidney and liver as well as in two human hepatoma cell lines, Hep3B and HepG2 (2, 3) . Hypoxia causes about a 100-fold enhancement in Epo() mRNA expression in both the murine kidney (4, 5, 6) and in Hep3B cells (7) , an induction due primarily to enhanced transcription (8, 9) . In addition, the Epo gene may also be regulated at the level of mRNA stability (9, 10) , although the evidence for this is indirect and therefore less convincing than recently published studies on another hypoxia inducible gene, tyrosine hydroxylase (11, 12) .

A logical and often fruitful approach in identifying important cis-acting elements in a gene is to test highly conserved sites in the 5`-flank as well as in noncoding regions downstream. As shown in Fig. 1, segments of strong homology in the noncoding portions of the human and mouse Epo genes include: a stretch upstream of the transcriptional start site; two segments within the first intron; all of the untranslated region in the 5th exon (excluding a B1 repeat insertion in the mouse gene); and, 130 bp downstream of the polyadenylation site, a segment extending 371 bp (13, 14, 15) . Transfection experiments into Hep3B and HepG2 cells utilizing some of these conserved elements linked to reporter genes have proven useful in determining their contribution to hypoxic induction. For example, the region of homology downstream from the termination of transcription contains a 40-bp enhancer which plays a critical role in hypoxic induction (16, 17, 18, 19, 20, 21) . This 3`-enhancer interacts with a hypoxia inducible transcription factor, HIF-1 (20, 22, 23) , and, 14 bp downstream, with the nuclear receptor HNF-4 (24) . Functional analysis of the Epo promotor indicates that 118 bp upstream of the start site are required for both basal transcription as well as for hypoxic induction (19) . It is likely that the full response requires interaction and perhaps cooperativity between the enhancer and promotor elements.


Figure 1: Diagrams of the wild type human Epo gene, the marked full-length ( mE-FL) and some of the marked deletion constructs used in this study. The five exons are shown in rectangles; the coding regions are shaded. Regions of sequence homology between human and mouse Epo genes are shown at the top. Black, >75% homology; shaded, 50-75% homology. MP, minimal promotor; 3`UTR, = 3`-untranslated region; A, the tandem repeat polyadenylation sequence; IVS1, first intervening sequence. The following restriction sites are shown: AccI ( Acc) in the wild type gene was mutated to HindIII ( Hind) in the marked genes; E, EspI, S, SacI; A, ApaI, H, HphI; Eco, EcoRI.



Much less information is available about the possible functional roles of other regions of homology in the Epo gene. Segments in the first intron and in the 3`-untranslated region may impact on mRNA processing and stability. As such, they cannot be tested by conventional reporter gene analyses. To address this question, we have devised a strategy that has enabled us to investigate cis elements of the Epo gene in their native context. We have used the Epo gene as its own reporter by marking it with innocuous point mutations in the coding region, thereby enabling its transcript to be readily distinguished from endogenous Epo mRNA. We prepared stable transfectants expressing this marked Epo gene and deletions thereof. Analyses of the expression of the marked Epo gene, in comparison to the endogenous gene has provided useful new information on functionally important cis elements.


MATERIALS AND METHODS

Constructs A genomic clone of human Epo in pUC18 was kindly provided by Genetics Institute, Cambridge, MA. The Epo gene insert is a 4100-bp HindIII- EcoRI fragment (GenBankfile HUMERPA (accession no. M11319)) (25) extending from 385 bp upstream of the transcriptional start site to 762 bp downstream of the polyadenylation signal. In this report, as in HUMERPA, the numbering of bases begins with the 5`-A of the HindIII site.

The Epo gene in this construct was ``marked'' by site-directed mutagenesis. Three base substitutions in the coding region of Exon 5 (2691:AG; 2694: CT; 2706: CT) obliterated an AccI site and created a HindIII site 13 bp upstream of it. As demonstrated below, these changes enabled the marked transcript to be distinguished from the endogenous transcript by means of both ribonuclease protection analysis (RPA) and competitive PCR. These three substitutions were at third bases of codons thereby preserving Epo's native amino acid sequence. This precaution would be important in the unlikely event that the expression of steady state mRNA levels is influenced by the protein product, analogous to the regulation of tubulin gene expression (26) . We have designated this construct mE-FL ( marked Epo gene with full- length sequence) in order to distinguish it from constructs having deletions ( e.g. mEIVS1 = marked Epo gene with deletion of IVS1).

To facilitate preparation of deletion constructs the upstream HindIII site in HUMERPA was replaced by a unique BamHI site. Since the Epo gene has a weak promoter (19) , it was necessary to prevent cryptic promoter activity from site(s) upstream of the vector. Therefore a 625-bp NdeI- BamHI fragment containing a tandem repeat transcriptional stop sequence from pXP2 ( Ain Fig. 1) was inserted into the NdeI and BamHI sites upstream of the marked Epo gene. Deletions in the mE-FL were generally made by digestions at selected restriction sites followed by filling in overhangs and blunt end ligation. Several of the constructs involving non-unique restriction sites required partial digestions. The IVS1 construct was prepared by substituting an EclXI- Asp718 fragment of Epo cDNA for an EclXI- Asp718 fragment of Epo genomic DNA.

Constructs expressing wild type and marked Epo riboprobes were prepared by inserting a PvuII- NcoI Epo fragment (2650-2890) from wild type Epo or from the marked Epo (mE-FL) into pBluescript KS(+). This fragment of Epo genomic DNA extended from the 5`-end of Exon 5 into its noncoding portion of Exon 5, thereby including the three sites in the coding region of Exon 5 that were ``marked'' by substitution. Because the construct was linearized in the polylinker, 15 bp downstream of the Epo insert, the full length riboprobe could be readily distinguished from protected fragments. Transfections Stable transfections were performed by electroporation. 10 µg of pSVNeo (expressing the neomycin resistance gene driven by the strong promoter of SV40 large T) and 300 µg of supercoiled marked Epo constructs were co-transfected into 3 10Hep3B cells with a voltage of 250 and a capacitance of 1,080 µF. After incubation for 48-72 h in standard medium, 0.4 mg/ml G418 was added. Previous dose-response experiments indicated that this was an appropriate dose to select neomycin-resistant cells. Approximately 3 weeks were required for the outgrowth of neomycin-resistant colonies. For most experiments, colonies on a 10-cm plate were pooled in order to avoid bias owing to the site of integration of the Epo gene. The number of colonies per pool ranged from 5 to greater than 50. For a few experiments, individual colonies were isolated and expanded by trypsinization in a 1-cm cylindrical ring which was fixed to the culture plate by autoclaved vacuum grease. In each transfection procedure, a plate of cells was mock transfected and selected in the same concentration of G418 to assure that no breakthrough colonies occurred. Stably transfected and nontransfected Hep3B cells were split into equal aliquots grown to approximately 70% confluence in 15 cm plates and incubated for 12 h in either 1 or 21% Oas described above. Following trypsin treatment to release cells from the plates, DNA and RNA samples were prepared by standard protocols. Analysis

Ribonuclease Protection

Ribonuclease protection was employed to provide quantitation of absolute levels of marked and endogenous Epo mRNAs. For most of the experiments (and all the results shown in Figs. 3-5), the marked riboprobe was used (see section above under ``Constructs''). In the few experiments in which wild type riboprobe was used, the results were fully consistent. The riboprobe plasmid was linearized at a site distal to the Epo insert and radiolabeled antisense cRNA was prepared by incubating 1 µg of DNA in the presence of T7 RNA polymerase and [P]CTP. An aliquot of labeled riboprobe containing 2 10cpm was mixed with 20 µg of RNA sample incubated overnight at 55 °C and then digested with RNase T, and RNase A for 45 min. Samples were then loaded onto a 6% polyacrylamide gel and electrophoresed at 1700 V (80-100 watts) for approximately 2 h. Autoradiograms were prepared by exposure to X-Omat film. Endogenous and marked Epo mRNA was quantitated by scanning the dried gel with a PhosphorImager (Molecular Dynamics Inc.), and the respective bands were quantitated by ImageQuant software.

Competitive PCR

Competitive PCR was used to assay both the number of copies of marked Epo constructs integrated into stably transfected cells and the expression of marked Epo mRNA, relative to endogenous Epo mRNA. The locations of the PCR primers are shown in Fig. 2 A. Different upstream primers were used for amplification of genomic DNA versus cDNA. For the former, a 24-base sequence in Intron 4 was used (5`3`): AGGAGCGGACACTTCTGCTTGCCC. For amplification of cDNA the upstream primer consisted of 28 bases of sequence at the 3`-end of Exon 4 (ACTCTGCTTCGGGCTCTGGGAGCCCAG) followed by 3 bases of sequence at the 5`-end of Exon 5 (AAG). The use of this primer prevented any amplification of genomic DNA or transfected plasmid DNA. The same downstream primer was used for both amplifications: AAGCAATGTTGGTGAGGGAGGTGGTGGAT. cDNA was prepared from 1-µg samples of RNA by incubation in 1 RT buffer (7) , 4 units reverse transcriptase and 2 µg random oligonucleotide primers. PCR reactions were carried out as described previously (7, 28) . One aliquot of the PCR products was digested with AccI, which cleaves only the endogenous genomic DNA and cDNA fragments. Another aliquot was digested with HindIII which cleaves only the marked fragments. In keeping with the principle of competitive PCR, the endogenous and marked DNAs are co-amplified with equal efficiencies (28) . Therefore, the ratio of their amplification products will be identical to the ratio in the sample prior to amplification, irrespective of number of cycles or incubation conditions. Accordingly, competitive PCR gives an accurate measurement of the ratio of marked to endogenous genomic DNA and the ratio of marked to endogenous mRNA. The ethidium bromide stained PCR gels (shown in Fig. 2 B) were analyzed by a Molecular Dynamics densitometer utilizing ImageQuant software and also by visual inspection. The reliability of our PCR analysis was considerably enhanced by digestion of each specimen with two enzymes ( AccI and HindIII), thereby providing an internal check. The fractions of fragments formed by digestion with either enzyme was predictably less than the fraction of parent (pre-amplification) Epo DNA having that restriction site, owing to the inability of the enzyme to cut heteroduplexes.() Accordingly, the ratio of marked to endogenous product was calculated using the binomial distribution to correct for heteroduplex formation.


Figure 2: Analysis of genomic DNA and reverse transcribed RNA by PCR. A, PCR amplification of marked ( M) and endogenous ( E) Epo genes and cDNAs. The dark rectangles represent exon 4 ( left) and exon 5 ( right) separated by intron 4. In exon 5, the AccI site (2702) of wild type Epo and the HindIII site (2689) of marked Epo are shown. The upstream primer used to amplify genomic DNA ( dotted arrow) hybridizes to the 5`-end of intron 4. The upstream primer use to amplify cDNA contains 28 bases of sequence at the 3`-end of exon 4 and 3 bases of sequence at the 5`-end of exon 5. The same downstream primer was used for amplification of both genomic DNA and cDNA. B, ethidium bromide-stained gel showing amplification products of reverse transcribed RNA from cells stably transfected with marked Epo genes. Following digestion with AccI ( top) and HindIII ( bottom), fragments of expected size were obtained. Lower left-hand lane, size markers ( HaeIII-digested X174 DNA). Two left-hand lanes, mE-FL3, the marked ``full-length'' gene having 400 bp upstream of the promotor. Middle two lanes, m5`E-FL1, a construct having 5 kb of Epo flanking sequence upsteam of the promotor; this construct failed to be incorporated into the genome and therefore produced no transcripts. Two right-hand lanes, mBgS-1, having a deletion in the 3`-UTR.




RESULTS

Fig. 1 depicts the marked wild type and mutant Epo constructs used in our experiments. Hep3B cells were transfected with these constructs as described under ``Materials and Methods.'' Initially, all of these constructs were transiently transfected into Hep3B cells both as calcium phosphate precipitates and by electroporation, under conditions which we have previously shown to give maximum transfection efficiency (19, 27) . Unfortunately, analysis of Epo mRNA by reverse transcription and competitive PCR revealed that the expression of the marked gene in both normoxic and hypoxic cells was less than 10% that of the endogenous gene (data not shown). This low level of expression, presumably a reflection of the inherent weakness of the Epo promotor (19, 27) , precluded our use of transient transfections.

To assure a higher level of expression of the marked Epo gene, we prepared stably transfected cell lines. In most experiments, neomycin resistant colonies were pooled in order to avoid bias owing to variability in integration site and its possible effect on basal expression and hypoxic induction of the marked gene. The average number of copies of the marked gene per cell in a given pool varied considerably from 0.2 to 13 with a mean of 3.1 ± 0.8 (S.E., n = 18) for all of the cell pools. We observed no significant correlation between the copy number of the marked gene and its expression. Subconfluent adherent cells were incubated for 12 h either at 21% Oor at 1% O. The expression of the endogenous and the marked Epo genes was analyzed by RPA as well as by competitive PCR.

The validity of our experimental design required demonstration that the expression of the transfected full-length Epo gene (mE-FL) was comparable to that of the endogenous gene both constitutively (21% O) and following hypoxic challenge (1% O). Fig. 3shows RPA gels for RNA samples from five independent pools transfected with mE-FL containing from 5 to greater than 50 colonies per pool. In each of the five pools incubated at 21% O, there was barely detectable expression of both the marked Epo gene and, as expected, the endogenous gene. Moreover, in each of the five pools, the expression of the marked gene was substantially enhanced by hypoxic challenge, as shown in the hypoxia lanes in Fig. 3. These RPA results were generally in good agreement with the competitive PCR data. In three of the five pools tested, the hypoxic induction of the marked gene was the same as that of the endogenous with ratios of M/E 1% to M/E 21% that were close to unity (). In the other two pools (mE-FL7 and mE-FL10) hypoxic induction of the marked gene was less than that of the endogenous gene, probably because a few colonies in each pool constitutively overexpressed Epo at 21% O, thereby increasing the M/E ratio. Nevertheless, as shown by RPA in Fig. 3 , even in these two pools, the expression of the marked Epo gene undergoes robust enhancement following exposure to hypoxia. Taken together, the RPA and competitive PCR results show that the marked wild type Epo gene underwent normal or nearly normal hypoxic induction when stably transfected into Hep3B cells.


Figure 3: Ribonuclease protection analysis of RNA from pools of cells stably transfected with the 4.1-kb ``full-length'' marked Epo gene (mE-FL). The antisense riboprobe had the sequence of the marked Epo gene (intron 4 and exon 5) and therefore protected a larger (242-base) fragment of marked Epo mRNA in comparison to endogenous wild type (180-base fragment). Hep3B, nontransfected Hep3B cells expressing only the endogenous Epo mRNA. Y, yeast RNA; N, normoxia (21% O); H, hypoxia (1% O).



We then tested marked Epo genes in which putative regulatory elements were deleted. As shown in Fig. 4 , the marked IVS1 Epo gene, in which exons 1 and 2 were fused, thereby eliminating intron 1, appears to have nearly normal basal expression and hypoxic induction. The competitive PCR data in also suggests normal induction although there is more variability among the three IVS pools than among the other sets of data. In contrast, deletion downstream of the ApaI site in the 3`-flank (AE), thereby eliminating the above-mentioned 40-bp enhancer, resulted in a marked decrease in hypoxic induction of the marked gene in two independent cell pools. The RPA result is supported by the competitive PCR analysis () showing that the ratio of expression of the marked to endogenous Epo mRNA fell markedly when the cells were challenged by hypoxia. Nevertheless, as shown in Fig. 4 , significant hypoxic induction was retained, indicating that the 3`-enhancer is not solely responsible for hypoxic up-regulation of Epo gene expression. In a construct having a deletion further downstream, thereby preserving the 3`-enhancer, hypoxic induction was enhanced above normal, as shown both by RPA (Fig. 4) as well as by competitive PCR (). This result suggests the presence of cis elements in the 3`-flank between 3550 and 4100 that impact negatively on hypoxic induction.


Figure 4: Ribonuclease protection analysis of RNA from pools of cells stably transfected with: mE-IVS1 (deletion of first intervening sequence); mEAE (3`-deletion that includes the 3`-enhancer); mEHE (3`-deletion downstream of the 3`-enhancer); and mEES (deletion of highly conserved segment within the 3`-UTR). As in Fig. 3, the marked Epo riboprobe was used. The protected fragment of the marked Epo mRNA from mEES was 22 bases shorter because of the deletion in the 3`-UTR. N, normoxia (21% O); H, hypoxia (1% O).



The function of the highly conserved 3`-untranslated region was examined by means of a series of marked deletion mutants. RPA analysis (Fig. 4) shows near normal induction of the mutant ES (EspSac) having the largest deletion in the 3`-UTR (2873-3061). However, both the RPA and the competitive PCR analyses () showed that at 21% Othe basal expression of the marked ES gene was somewhat higher than that of the endogenous gene.() This result, observed in five out of six separate cell pools, could be due either to increased basal transcription of the marked gene or enhanced mRNA stability. In order to distinguish between these possibilities, the in vivo decay of the marked Epo mRNA was compared to that of the endogenous Epo mRNA by inducing high level expression with a 12 h hypoxic challenge, followed by rapid replacement with 21% O. RNA samples were prepared at 0, 1, 2, 4, and 8 h. The radioactivity of the protected bands in the RPA gels was measured by a PhosphorImager. As shown in Fig. 5 , the half-life of the endogenous Epo mRNA was 2.0 ± 0.1 h ( n = 4), a value very close to those that we previously obtained by Northern blot analysis (1.5 h) (9) and by competitive PCR (2.0 h) (7) . As shown in Fig. 5A, the marked wild type Epo mRNA (mWT) had a half-life of 2.1 ± 0.1 h ( n = 5), a value indistinguishable from that of the endogenous mRNA. This result was fully anticipated since the two mRNAs differ only by 3 bases in the coding portion of exon 5, a region not expected to affect mRNA stability. In contrast, as shown in Fig. 5, B and C, the large (187 bases) deletion in the conserved 3`-UTR (ES) resulted in the expression of a truncated mRNA with markedly prolonged projected half-life of 15 ± 3 h ( n = 5). The large difference between the stability of the marked wild type mRNA compared to the ES mRNA that was observed with cell pools was also observed in single clonal colonies of mE-FL and ES. In order to further delineate the site that confers relative instability on Epo mRNA, a set of smaller deletions were tested. The half lives of these mutant Epo mRNAs are shown in Fig. 5 C. BsS, a 58-base deletion at the 3`-end of ES, had a normal half-life (2.1 h), while that of EBg, an 84-bp deletion at the 5`-end of ES, was nearly normal (3.2 h).() These results taken together suggests that the major contributor of Epo mRNA's relative instability lies in the 46-base middle region (Fig. 5 C). In support of this conclusion the mutant BgS mRNA, which has a deletion that includes this 46-base segment, had a prolonged half-life (5.8 h). Thus, these experiments testing deletions in the 3`-UTR suggest that a relatively small stretch is responsible for its relatively rapid degradation and short half-life.


Figure 5: Stability of endogenous and marked Epo mRNA. High levels of Epo mRNA were induced by overnight exposure to 1% O. At time 0, the cells were rapidly exposed to 21% Oand the levels of Epo mRNA were analyzed by ribonuclease protection and quantitated by PhosphorImaging. A, decay of endogenous Epo mRNA ( squares), marked Epo mRNA expressed by cells stably transfected with full-length Epo gene, mE-FL ( circles) and marked truncated Epo mRNA expressed by cells stably transfected with mEES ( triangles). B, ribonuclese protection analysis of RNA from cells stably transfected with the marked gene having a deletion in the 3`-UTR (mEES1). C, restriction sites in 3`-UTR used for preparation of deletion constructs. The brackets depicted above show the tin hours for each of the four deletions that were tested. Restriction sites: Esp, 2874; Bgl, 2957; Bsm, 3003; Sac, 3061.




DISCUSSION

The most commonly employed strategy for studying the function of cis-acting elements in a gene is to transfect into an appropriate cell line a construct in which a given element is attached to a reporter gene, not normally expressed by that cell. Although this approach is highly adaptable and informative, it suffers from obvious drawbacks. First, gene fragments artificially inserted into reporter gene constructs may give misleading and unreliable results with respect to tissue and developmental specificity as well as response to specific cellular signals. Second, such experiments provide no information about post-transcriptional gene regulation. To circumvent these problems we have studied the Epo gene in its most native possible context by marking the Epo gene and using the marked transcript as the reporter. Baseline experiments using the ``full-length'' (FL) 4.1-kb marked Epo gene provide evidence that this approach is valid. As shown in Figs. 3 and 5 A and , basal expression, hypoxic induction and stability of the mEpo-FL mRNA were indistinguishable from that of the endogenous Epo gene. These results offer satisfactory assurance that 385 bp of upstream flank and 762 bp of downstream flank are sufficient for the stably integrated gene to function normally in Hep3B cells. Moreover, marking the gene by 3-bp substitutions in the coding region of Exon 5 did not significantly affect expression, hypoxic induction or mRNA stability. Finally, the fact that reproducible results were obtained with replicate pools as well as clonal lines of stable transfected cells indicates that integration site did not impact significantly on the function of the marked gene. Thus, our demonstration that the expression of mE-FL faithfully mimics that of the endogenous Epo gene indicates that experiments testing deletions of this construct can be interpreted with confidence.

As shown in Fig. 4and , we observed a marked diminution in hypoxic induction of the marked Epo mRNA when the transfected construct contained a deletion that included the 3`-enhancer (mEAE). This experiment demonstrates that this enhancer is functional in the context of the intact Epo gene. Nevertheless, even in the absence of this downstream element, the Epo gene still responded substantially to hypoxic challenge. This observation poses a challenge to conclusions in the recent literature that Epo's 3`-enhancer is both necessary and sufficient for response to hypoxia in Hep3B cells (18, 20) . The induction that we observed in the absence of this 3`-enhancer is consistent with reporter gene experiments demonstrating that Epo's promotor per se can confer a significant induction by hypoxia (19, 24) .

During the past decade, there has been growing appreciation of the importance of sequences in the 3`-untranslated region as determinants of mRNA stability. Examples of special biologic importance include the AU rich motif that is shared among the 3`-UTRs of a number of transcription factor and cytokine mRNAs (29, 30, 31) and the iron regulatory stem loop motif in the mRNA of the transferrin receptor (32, 33) . The 3`-UTR of Epo's mRNA lacks any known sequence homology except perhaps to the 3`-UTR of the tyrosine hydroxylase gene (12) . We have sought to determine whether Epo gene expression is affected by post-transcriptional regulation. Nuclear run-on experiments in Hep3B cells have demonstrated at least a 10-fold increase in Epo gene transcription in response to hypoxia and cobalt (9) . Because the level of Epo gene expression in unstimulated cells is so low, the quantitation of the signal was imprecise. Therefore it is possible that the induction of transcription is considerably greater than 10-fold. In order to assess whether hypoxia also affected Epo mRNA stability, an actinomycin D chase experiment was performed (9) . Surprisingly, inhibition of de novo transcription by actinomycin D increased the half-life of Epo mRNA by a factor of four. Cycloheximide, an inhibitor of protein synthesis, also resulted in a marked prolongation in the half-life of Epo mRNA. Electrophoretic mobility gel shift experiments on extracts from normoxic and hypoxic Hep3B cells demonstrated 70- and 135-kDa cytosolic proteins which bound specifically to a portion of Epo mRNA in the 3`-untranslated region immediately downstream of termination of translation (approximately 2664-2884) (10) . This region includes a 28-base pyrimidine rich stretch that is homologous to a region in the 3`-UTR of tyrosine hydroxylase mRNA that bound preferentially to cytosolic extracts from hypoxic cells (12) . In contrast, the Epo mRNA bound equally well to protein in normoxic and hypoxic extracts (10) . Furthermore, there was no apparent correlation between the tissue distribution of these proteins and the ability of cells to express Epo (10) . Taken together, these results suggest that the half-life of Epo mRNA is reduced by the specific binding of proteins to the 3`-UTR. However, there is no evidence thus far that these proteins are either stimulus-specific or tissue-specific. Because the stability of Epo mRNA is affected by ongoing transcription and protein synthesis, it is difficult to design and execute experiments that address this problem.

In this study, we demonstrate that a conserved 46 base sequence in the Epo 3`-UTR (2957-3003) is required for the relatively short half-life of Epo mRNA. Deletions in this region which prolonged the tof the marked mRNA generally gave rise to enhanced basal (21%) expression of marked Epo mRNA as assayed by PCR. (See and Fig. 2B, bottom panel, showing increased ratios of marked to endogenous mRNAs from cells exposed to 21% O). Despite the semiquantitative nature of these PCR analyses, they provide information that could not be obtained from ribonuclease protection assays, owing to the weakness of the RPA signals from 21% Osamples. This 46-base segment is at least 67 bases downstream of the protein binding region described above. It is possible that the upstream binding of proteins mediates ribonuclease cleavage at the downstream site. It will be of considerable interest to ascertain whether the same cytosolic proteins bind to the mRNAs of Epo and tyrosine hydroxylase and perhaps to mRNAs of other oxygen sensitive genes.

  
Table: Stable transfections with marked Epo gene: analysis by competitive PCR

The first column shows the marked Epo construct tested. The second column shows the number of pools tested. The next two columns show ratios of marked to endogenous reverse transcribed Epo mRNA from cells exposed to 21% O, and from cells exposed to 1% O. The right hand column (1%/21%) shows the hypoxic induction of the marked Epo gene relative to the endogenous. 1%/21% = [M/E]/[M/E]. Since the expression of the endogenous gene (E/E) varies only slightly among the pools, the data in this 1%/21% column reflects the induction of the marked Epo gene relative to the endogenous; a value of 1 means that the marked gene is equally as inducible as the endogenous. This table shows mean values ± S.E.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant RO1-DK41234. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Contributed equally to this work and therefore should both be considered first authors.

Current address: Tufts University School of Medicine, Boston, MA 02111.

**
Current address: Joslin Diabetes Center, Harvard Medical School, Boston, MA 02115.

§§
To whom correspondence should be addressed: Hematology-Oncology Division, LMRC-2, Brigham and Women's Hospital, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-732-5841; Fax: 617-739-0748.

The abbreviations used are: Epo, erythropoietin; RPA, ribonuclease protection analysis; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s).

After 50 cycles of amplification, heteroduplex formation is maximal and the digested products follow a binomial distribution (a+ 2ab + b). Thus, if the endogenous (E) and marked (M) Epo transcripts were present in equivalent amounts, the final PCR products would be 25% EE, 50% EM, and 25% MM. AccI would digest only EE and HindIII would digest only MM.

As shown in Table I the mean M/E for the mEES construct at 21% Owas 2.1 ± 0.4 (S.E.), compared to a mean M/E of 1.24 ± 0.31 for the mE-FL construct ( p = 0.05).

The basal (21% O) expression of these two deletion mutants (EBg and BsS) relative to endogenous Epo was low (0.4 and 0.4, respectively) compared to values of ES and BgS which have long half-lives.


ACKNOWLEDGEMENTS

We appreciate helpful discussions with Kerry Blanchard, Debby Galson, and Mark Goldberg.


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