©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Two Novel Regulatory Elements within the 5`-Untranslated Region of the Human -Globin Gene (*)

Persis J. Amrolia (1)(§), John M. Cunningham (1) (3), Paul Ney (2), Arthur W. Nienhuis (1), Stephen M. Jane (1) (4)

From the (1) Division of Experimental Hematology and the (2) Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, the (3) Department of Pediatrics, University of Tennessee, Memphis, Tennessee 38101, and the (4) Bone Marrow Research Laboratory, Royal Melbourne Hospital, Parkville Victoria, 3050, Australia

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Interaction between the stage selector element (SSE) in the proximal -globin promoter and hypersensitivity site 2 in the locus control region partly mediates the competitive silencing of the -globin promoter in the fetal developmental stage. We have now demonstrated that a second SSE-like element in the 5`-untranslated region of the -gene also contributes to this competitive silencing of the -gene. Utilizing transient transfection assays in the fetal erythroid cell line, K562, we have shown that the core enhancer of hypersensitivity site 2 can preferentially interact with the proximal -promoter in the absence of the SSE, completely silencing a linked -promoter. Mutation of a 20-base pair sequence of the -gene 5`-untranslated region (UTR) led to derepression of -promoter activity. A marked activation of -promoter activity was also observed with this mutation, suggesting the presence of a repressor. Fine mutagenesis dissected these activities to different regions of the 5`-UTR. The stage selector activity was localized to a region centered on nucleotides +13 to +15. Electromobility shift assays utilizing this sequence demonstrated binding of a fetal and erythroid-specific protein. The repressor activity of the 5`-UTR was localized to tandem GATA-like sites, which appear to bind a complex of two proteins, one of which is the erythroid transcription factor GATA-1. These results indicate that the 5`-UTR of the -gene contains sequences that may be important for its transcriptional and developmental regulation.


INTRODUCTION

The normal developmental program of the human -globin locus is characterized by two switches in gene expression: from embryonic () to fetal ( and ) beginning in the 5th week of gestation and from fetal to adult ( and ) during the perinatal period. This developmental program is governed by a diverse array of regulatory mechanisms (1) . Cis-acting elements within or immediately flanking the genes themselves can confer tissue and temporal specificity in transgenic mice, but the levels of expression are low (2, 3, 4) . High level expression is restored when the genes are linked to the locus control region (LCR),() a regulatory region 6-20 kilobases 5` of the -globin gene that is characterized by four erythroid-specific DNase 1 hypersensitivity sites (5, 6). This enhanced expression is achieved without disruption of the temporal regulation of the - and -genes, which appear to be autonomously silenced (7, 8) . In contrast, the increase in -gene expression mediated by the LCR is accompanied by expression throughout development. Restoration of -gene expression confined to the adult developmental stage is only achieved when the -gene is linked in the normal configuration (9, 10, 11) . These results suggest that competition between the genes for LCR sequences is essential for appropriate regulation of the -gene.

The concept of competition between globin gene promoters for a shared enhancer as a mechanism of developmental regulation was originally proposed for the chicken globin locus. In this system, silencing of the embryonic -globin gene in the adult stage of erythropoiesis is mediated by a sequence in the -promoter, the stage selector element (SSE) (12) . An erythroid and developmentally specific protein, NF-E4, appears to facilitate the preferential interaction between the chick -promoter and enhancer by binding to both regions and establishing a protein bridge that brings these two elements into apposition (13) . In the human -globin cluster, we have identified an analogous SSE in the proximal -promoter. This sequence fosters a preferential interaction between the -promoter and hypersensitivity site 2 (HS2) of the LCR in the fetal stage of erythropoiesis, thus silencing a linked -promoter (14, 15) . As with the chicken SSE, the ability of this element to confer a competitive advantage to its promoter is dependent on the binding of a developmentally specific protein, the stage selector protein (SSP). The SSP is a heteromeric complex consisting of a ubiquitous transcription factor, CP2, and a fetal/erythroid-specific partner protein (16) . CP2 has recently been demonstrated to represent the human homologue of chicken NF-E4 (16) .

Although numerous protein footprints have been demonstrated in HS2, the site of interaction with the SSE is unknown. HS2 contains a core enhancer element that binds the transcription factors NF-E2/AP-1, LCRF-1, and NRF-2 (17, 18, 19) . Additional cis-acting elements located 5` and 3` to the core enhancer have also been defined. These sites, known as co-enhancers, bind the transcriptional regulators Sp1, GATA-1, USF, and YY1 and contribute to high level expression in stable transfectants and transgenic mice (20, 21) . In the studies delineated here, we sought to determine whether the core enhancer of HS2 was sufficient in isolation for the preferential interaction between HS2 and the -promoter. Our results define a novel SSE-like element in the 5`-untranslated region (5`-UTR) of the -globin gene, which appears to preferentially interact with this enhancer in the presence of a developmentally specific protein and thus contribute to the competitive silencing of the -gene in the fetal stage of erythropoiesis. We have, in addition, identified a separate negative regulatory element in this region.


MATERIALS AND METHODS

DNA Construction

pUC007, a pUC-based plasmid (17), was used as the vector in all constructs. Constructs containing the -globin promoter (-385 to +38) subcloned 5` of the coding sequence of the chloramphenicol acetyltransferase reporter gene (CAT) and the -promoter (-260 to +35 or -35 to +35) subcloned 5` of the firefly luciferase gene (LUC or -35LUC) have been previously described (14, 17) . HS2 was subcloned as a 734-bp HindIII-BglII fragment into the HindIII-BamHI fragment of pUC007. The XhoI-AatII fragments of CAT and LUC were subcloned 3` of HS2 into a SalI-AatII fragment (HS2CAT, HS2LUC). A synthetic HS2 (HS2) containing the tandem NF-E2/AP-1 sites flanked by neutral DNA was constructed by PCR. The HS2 retains the spacing between the core enhancer and the adjacent promoter but lacks all the previously reported co-enhancer footprints. Neutral sequences were designed to be devoid of consensus sequences for known transactivators of the LCR. Initially, a 163-bp XhoI-HindIII PCR product of the pBR322 backbone and a 189-bp BglII-SalI PCR product of the pBR322 ampicillin resistance gene were subcloned 5` and 3` to the 46-bp enhancer core in pUC007 (22) . An additional 370-bp BamHI-SalI PCR product of the pBR322 tetracycline resistance gene was subcloned 3` to retain the natural spacing. The entire HS2 was released as a SalI-AatII fragment and subcloned 5` of either CAT or LUC (HS2CAT, HS2LUC) using the XhoI/SalI-AatII strategy outlined above. Dual promoter constructs linking the - and -260-promoter (HS2CATLUC, HS2CATLUC) or the - and -35-promoters (HS2CAT-35LUC, HS2CAT-35LUC) with their respective reporter genes were obtained using the same strategy.

Oligonucleotides and Site-directed Mutagenesis

Oligonucleotides were synthesized on an Applied Biosystems synthesizer model 380B using phosphoramidite chemistry and purified on Sephadex G-25 columns (Pharmacia Biotech Inc.). Prior to use in gel mobility shift assays, complementary strands were annealed in 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 10 mM MgCl by heating to 95 °C for 5 min and slow cooling to room temperature. PCR site-directed mutagenesis was performed as described (23) . Briefly, mutants were made by PCR in 2 halves incorporating a PstI restriction site at the 3`-end of one and the 5`-end of the other. Products were cleaved with PstI, ligated with T4 DNA ligase, and the full-length mutant amplified by PCR using the external primers. Once subcloned into plasmids, the sequence was verified by the chain termination method with Sequenase (U. S. Biochemical Corp.).

Cell Lines and Nuclear Extracts

Human erythroleukemia (K562) cells, murine erythroleukemia (MEL), and HeLa cells were grown in improved minimal essential medium (Biofluids) with 10% fetal calf serum and 50 µg/ml gentamicin. Nuclear extracts were prepared as described (14) .

DNA Transfection and Transient Assays

Plasmid DNA was prepared over double cesium chloride gradients. 3.5 10 K562 cells were mixed with equimolar amounts of the test plasmids in the presence of carrier plasmid (pUC 9) to a total of 50 µg. Electroporation was performed with a Bio-Rad gene pulser apparatus at 960 microfarads and 0.2 kV. After transfection, cells were grown in the presence or absence of 20 µM hemin, and each sample was split in two and harvested after 48 h. Cell lysates from one half were prepared by repeated freeze-thaw cycles and assayed for CAT activity as described (24) . The remaining half was lysed with Triton X-100, and luciferase activity was measured on a Monolight 2001 luminometer (Analytical Luminescence Laboratories). The assay was linear from 10 to 10 light units; samples above or below this were reassayed with appropriate dilution. CAT activities were standardized for lysate protein concentration. Luciferase lysates had equivalent protein levels. In our previous studies, we had demonstrated that inclusion of an internal control reporter plasmid was unsuitable in dual promoter constructs (14) . Standard single promoter constructs were used as experimental controls, and constructs were only compared within the same series of experiments. Results shown represent at least six different transfections with two independent plasmid preparations in induced cells. Similar trends were observed in separate transfections of uninduced cells. Statistics were calculated using Student's t test; values of p < 0.05 were considered significant.

RNase Protection Assay

80 µg of total RNA was prepared from K562 cells 48 h after electroporation using RNAzol B (Tel-Test, Friendswood, TX) according to the manufacturer's instructions. Probes were constructed by subcloning XhoI-BamHI PCR fragments containing the -35-wild-type or mutant promoters and the first 243 bp of the luciferase coding sequence into the XhoI-BglII fragment of pSP73. Sequence integrity was verified by dideoxy sequencing. High specific activity radiolabeled transcripts were generated with SP6 RNA polymerase. The predicted length of protected fragments from correctly initiated transcripts was 284 bp. RNase protection assays were performed as described (25) , and samples were electrophoresed on an 8% sequencing gel.

Gel Mobility Shift Assay

Assays were performed with 10 cpm of probe added to a 20-µl reaction containing varying amounts of nuclear extract, 500 ng of poly[d(GC)], 6 mM MgCl, 16.5 mM KCl, and 100 µg of bovine serum albumin (26, 27) . For competition assays, a 150-fold molar excess of unlabeled double-stranded oligonucleotide was added with the probe. In antibody studies, 3 µl of pre-immune serum or rat or rabbit anti-mouse GATA-1 antibody (kindly provided by Merlin Crossley and Stuart Orkin, HHMI, Childrens' Hospital, Boston) were preincubated for 10 min with the binding reaction prior to addition of the probe. After incubation at 4 °C for 10 min and 25 °C for 20 min, samples were electrophoresed on a 4% non-denaturing polyacrylamide gel in 0.5 Tris-borate-EDTA buffer for 90 min at 10 V/cm.


RESULTS

The Preferential Interaction between the Core Enhancer of HS2 and the -Promoter Is Not Dependent on the SSE

The erythroid-specific enhancer in HS2 has been mapped to a 46-bp fragment consisting of tandem NF-E2/AP-1 binding sites (17, 22) . In our initial experiments, we sought to determine whether this minimal enhancer was sufficient to preferentially interact with the -promoter when in competition with the -promoter in a fetal erythroid environment. A 734-bp HindIII-BglII fragment of HS2 and a synthetic HS2 (HS2) containing the 46-bp enhancer flanked by neutral DNA to retain the spacing between the enhancer and adjacent promoter but ablating all reported protein binding sites were compared. Initially, each HS2 fragment was linked to the -378 -promoter with a CAT reporter gene or the -260 -promoter with a luciferase reporter gene and transfected into K562 cells. With the -promoter, reporter gene activity was 4-fold higher in the context of HS2 than HS2 (data not shown). This presumably reflects the effect of positive regulatory elements in HS2 outside the core, which we refer to as co-enhancers. By contrast with the -promoter, reporter gene activity was equivalent with HS2 and HS2 in K562 cells (Fig. 1, constructs1 and 4). To determine if the core enhancer was sufficient for the competitive advantage of the -promoter in the fetal environment, the -378 -promoter/CAT reporter gene and -260 -promoter/luciferase reporter gene were linked in competition for HS2 or HS2 (HS2CATLUC, HS2CATLUC) and transfected into K562 cells. Constructs linking the HS2 fragments to the -promoter/CAT reporter hybrid served as controls. As seen in Fig. 1, -promoter activity in the dual promoter constructs was diminished 10-fold compared with the controls with both HS2 and HS2 (p < 0.05), indicating that the minimal enhancer was sufficient to allow preferential interaction of HS2 with the -promoter (constructs2 and 5). The reduction in -promoter activity observed in construct 5 by comparison with construct 2 is consistent with the loss of the co-enhancer effect.


Figure 1: The minimal -promoter can silence a linked -promoter in competition for the HS2 core enhancer. A, diagrammatic representation of the constructs used for transfections into K562 cells induced with hemin. Solidboxes represent either the 734-bp HindIII-BglII fragment of HS2 (constructs1-3) or the HS2 core enhancer flanked by neutral DNA to retain equivalent spacing (constructs4-6). The hatchedboxes represent the -385 -promoter linked to the CAT gene, and the openboxes represent either the -260 (constructs2 and 5) or the -35 (constructs3 and 6) -promoter linked to the luciferase reporter gene. B, reporter gene activity of these constructs. Hatchedbars represent CAT conversion, standardized per microgram of protein; openbars represent luciferase activity (lysates had equivalent protein concentration). Values shown represent the mean ± S.E. of at least six separate transfections with two different plasmid preparations. Statistical significance was assessed using Student's t test.



To determine if the interaction between the -promoter and the core enhancer was dependent on the SSE, we truncated the -promoter to -35 relative to the transcriptional start site and examined the effect in dual promoter constructs in the presence of HS2 and HS2. The residual -promoter contained the 35 nucleotides 5` to the cap site and an additional 35 nucleotides of 5`-UTR. In the context of the wild-type HS2, truncation of the -promoter resulted in a marked decrease in its activity, presumably due to loss of upstream regulatory elements (Fig. 1, construct3). Loss of the SSE in this setting resulted in derepression of the linked -promoter (p < 0.01) (Fig. 1, construct3). However, the level of -promoter activity observed in this construct was significantly less than the activity seen when the -promoter was linked to the same HS2 fragment in isolation (Fig. 1, construct1), suggesting that residual competitive sequences may be present. This was confirmed with the construct linking HS2 to the CAT/-35LUC cassette (Fig. 1, construct6). In this setting, the -35-promoter totally retained its competitive advantage, and the linked -promoter was suppressed. These findings led us to postulate the existence of alternative stage selector-like elements that interact specifically with the HS2 core enhancer within the proximal -promoter.

Localization of an Alternate Stage Selector Element in the 5`-UTR of the -Globin Gene

To further localize the sequences in the -35-promoter responsible for its competitive advantage in attracting the core HS2 enhancer, scanning mutagenesis of this region was performed. The ability of mutants to compete with a linked -promoter for interaction with HS2 was tested in K562 cells. As seen in Fig. 2, construct2, mutation of the -TATA box (-35) totally abolished -activity. Despite this, no derepression of the linked -promoter was seen, implying that the competitive advantage of the -35-promoter was independent of its transcriptional activity. Mutation of the sequences between the TATA box and the CAP site decreased -35-promoter activity slightly but had no effect on repression of a linked -promoter (data not shown). In contrast, mutation of the 5`-UTR (-35, Fig. 2, construct3) led to a 3-fold derepression of -promoter activity (p < 0.05), suggesting that this area contained a competitive element capable of preferentially attracting the HS2 core enhancer. In addition to the derepression of the -promoter, the 20-bp 5`-UTR mutant also resulted in a concomitant 7-fold increase in -promoter activity (p < 0.05), suggesting that this mutation might also have disrupted a separate negative regulatory element. To further evaluate this repressor activity, the wild-type and mutant -35-promoters were linked to the HindIII-BglII fragment of HS2 as single promoter constructs and transfected into K562 cells and the murine adult erythroid cell line, MEL. In both cell lines, the wild-type promoter was less active than the mutant, and a comparable degree of repression was observed in each, indicating that this effect was not developmentally specific (data not shown).


Figure 2: Mutation of the 5`-untranslated region of the minimal -promoter activates - and derepresses linked -promoter activity. A, diagrammatic representation of constructs. In construct1, the wild-type minimal -promoter (-35 to +35) has been linked to the -385 -promoter in competition for the HS2 core enhancer. Constructs2 and 3 are identical except for mutations of the -TATA box and 5`-untranslated region between +8 and +27, respectively. B, reporter gene activity of corresponding constructs. Hatchedbars represent CAT conversion, and openbars represent luciferase activity.



To evaluate the contribution of the 5`-UTR SSE-like element to the suppression of a linked -promoter in the context of larger -promoter and HS2 fragments, the 20-bp 5`-UTR mutation was made in the -260-promoter, and the ability of this mutant to silence a linked -promoter in competition for the 734-bp HindIII-BglII HS2 enhancer was tested. As shown in Fig. 3 (construct2), with the larger HS2, loss of the UTR SSE resulted in a significant derepression of -promoter activity (p < 0.05) despite the presence of an intact -promoter SSE. As previously shown, loss of the -promoter SSE by truncation to -35 (construct3) resulted in a significant derepression of linked -activity (p < 0.05). In the absence of the -promoter SSE, mutation of 5`-UTR (construct4) led to a more marked derepression of -promoter activity than that seen with construct 3 (p < 0.05). This suggests that the two SSE regions function cooperatively. As with the core enhancer, mutation of the 5`-UTR also led to activation of -35-promoter activity (p < 0.05, construct4). This effect was less pronounced in the setting of the -260-promoter (construct2), suggesting that upstream elements in the -promoter can compensate for the repressor activity of this region.


Figure 3: Mutation of the 5`-untranslated region in the -260 -promoter also derepresses linked -promoter activity in the context of a larger HS2 fragment. A, diagrammatic representation of constructs. In construct1 and 3, the wild-type -260 or -35-promoters have been linked to the -385 -promoter in competition for the 734-bp HindIII-BglII fragment of HS2. Constructs2 and 4 are identical to these, respectively, except for mutation of the -5`-untranslated region between +8 and +27. B, reporter gene activity of corresponding constructs. Hatchedbars represent CAT conversion, and openbars represent luciferase activity.



To dissect the sequences in the 5`-UTR responsible for the SSE and repressor functions, a series of smaller mutants spanning this area were made and tested for their ability to compete with a linked -promoter for the HS2 enhancer. As shown in Fig. 4 (constructs1 and 2), mutation of the sequence from +13 to +15 (-35) resulted in a significant derepression of linked -promoter activity without reduction in -activity (p < 0.01). No other mutant demonstrated any -promoter derepression (data not shown). This suggested that the nucleotides from +13 to +15 may represent the important contact bases for a protein mediating the SSE-like effect. It should be noted that this mutation introduced a different sequence between +13 and +15 than the 20-bp mutant, making it unlikely that the effect is specific to the mutant sequences. In contrast, mutation of the 5`-UTR between +24 and +29 (Fig. 4, construct3) had no effect on linked -promoter activity but led to a 5-fold increase in -promoter activity (p < 0.05). These nucleotides are centered on a tandem inverted binding motif for the erythroid transcription factor, GATA-1. These results suggested that the -globin 5`-UTR contains two distinct regulatory elements, one analogous to the SSE and another that functions as a repressor.


Figure 4: A stage selector-like element and a repressor sequence are localized to different regions of the -5`-untranslated region. A, diagrammatic representation of the constructs used. In construct1, the -35-promoter has been linked to the -385 -promoter in competition for the HS2 core enhancer. Constructs2 and 3 are identical to construct1 except for mutations of the 5`-untranslated region between +13 and +15 or +24 and +29, respectively. The wild-type -5`-UTR is shown under construct1, and mutated sequences in constructs2 and 3 are shown in lowercase. B, reporter gene activity of corresponding constructs. Hatchedbars represent CAT conversion, and openbars represent luciferase activity.



Functional Mutations in the 5`-UTR Do Not Alter Transcription Initiation

Previous studies have shown that deletions 3` to the TATA box in the rabbit -globin promoter and 5`-UTR altered the transcription initiation site (28, 29) . To ensure that the effects we had observed with the UTR mutants were not attributable to aberrant initiation, RNase protection analysis was performed. RNA was prepared from K562 cells transfected with single promoter constructs containing the -260, -35, and -35 promoters. The full HS2 enhancer was used to increase transcript levels. Fig. 5shows a similar pattern of one major and two minor bands clustered around the mRNA CAP site for all three promoters. The upper major band corresponds to the correctly initiated full-length 284-bp mRNA transcript. The minor smaller bands are consistent with the previously reported minor transcripts and probably represent microheterogeneity in -globin transcription initiation sites (22, 28) . No new downstream initiation site was introduced with the 5`-UTR mutation. Transcript levels with the repressor mutant were noted to be increased in comparison to the wild-type -35-promoter (Fig. 5, lanes4 and 6).


Figure 5: Mutation of the -5`-untranslated region does not alter transcription initiation. Constructs linking the wild-type -260 or -35-promoters (lanes3 and 4) or the mutant -35-promoter (lane6) were electroporated into K562 cells and RNA prepared 48 h post-transfection. RNase protection analysis was performed on 80 µg of total RNA or 10 µg of yeast RNA using 35 -wild-type (WT) or mutant (M2) RNA probes. Probe integrity was confirmed by hybridizing to yeast RNA in the absence of RNase (lanes2 and 5). The arrow indicates the correctly initiated 284-bp protected transcript.



Analysis of Proteins Binding to the -Promoter 5`-UTR

To ascertain whether the functional sequences defined in the experiments above represented protein binding sites, we performed electromobility shift assays (EMSA) using the wild-type -gene from -5 to +27 as probe (UTR 1). As shown in Fig. 6 , three retarded bands were observed with crude K562 nuclear extract (lane1). Competition with excess non-radiolabeled probe ablated binding activity, confirming the specificity of these interactions (data not shown). A mutant probe (UTR 1M), which altered only the 3 bp from +13 to +15, the site of the putative stage selector-like element, failed to bind the uppermost of these complexes (Fig. 6, lane2). To evaluate the tissue specificity of this complex, wild-type and mutant probes were used to assay nuclear extracts from adult erythroid (MEL) and non-erythroid (HeLa) cell lines (Fig. 6, lanes3-6). Binding activity of the uppermost complex was absent in these extracts, suggesting that this complex is both fetal and erythroid specific. Neither a highly purified preparation of SSP or recombinant CP2 bound to the wild-type UTR 1 probe (data not shown).


Figure 6: The alternate stage selector element binds a protein that is fetally and erythroid restricted. A, sequences of the upstream -5`-untranslated region probe wild-type (UTR 1) and mutant (UTR 1M) probes used in electrophoretic mobility shift assays. The mutation at the site of the stage selector-like element is underlined. B, EMSA of upstream 5`-UTR probes with crude K562, MEL, and HeLa nuclear extracts.



To evaluate protein binding to the repressor sequence in the 5`-UTR, a probe spanning nucleotides +10 to +42 (UTR 2) was used in the EMSA with extract derived from K562, MEL, and HeLa cells. As seen in Fig. 7, two retarded species were observed with all extracts. However, the broad lower complex contained a distinct erythroid-specific component in MEL and K562 extract (lanes1 and 2). Mutation of the tandem inverted GATA motifs with activity in functional assays (UTR 2M) abolished binding of both the upper ubiquitous band and the erythroid-specific band (Fig. 7, lane9). To determine whether the erythroid-specific band was due to binding of GATA-1, an excess of an unlabeled probe consisting of 6 repeats of the GATA motif or anti-mouse GATA-1 antibodies were added to the reaction mixture. As seen in Fig. 7 , both the unlabeled competitor (lane4) and anti-mouse GATA-1 antibody (lane6) significantly reduced the binding of the erythroid complex without altering the ubiquitous band. Preimmune sera nonspecifically diminished both bands to a minor degree (lane 5). The diminution in GATA-1 binding observed with this antisera was comparable with that observed by other investigators.() To further validate that the erythroid complex consisted of GATA-1, nuclear extract from COS cells transfected with a GATA-1 expression vector and extract from untransfected COS cells was studied with the UTR 2 probe. As seen in lanes7 and 8, the GATA-1 containing extract produced a single retarded band, which comigrated with the erythroid-specific band seen with K562 extract. No complex was observed with untransfected COS extract. These results suggest that the repressor element of the 5`-UTR binds two proteins, one of which is GATA-1.


Figure 7: The repressor element of the -5`-untranslated region binds two proteins, one of which is GATA-1. A, sequences of the downstream -5`-UTR wild-type probe (UTR 2), a similar probe with a mutation at the site of the repressor element (UTR 2M), and a probe consisting of 6 GATA repeats used as a non-radiolabeled competitor(GATA). B, EMSA of wild-type and mutant downstream 5`-UTR probes with crude K562, MEL, and HeLa nuclear extracts (lanes1-3 and 9) and extracts from untransfected COS cells (lane7) or COS cells transfected with a GATA-1 expression vector (lane8). Competition experiments were performed with K562 extract preincubated with non-radiolabeled GATA probe competitor (lane4), pre-immune serum (lane5), or anti-mouse GATA-1 antibodies (lane6).




DISCUSSION

Although the promoter elements of the human globin genes have been exhaustively studied, little is known about the role of the untranslated leader sequences of these genes. We now report the presence of two novel elements in the 5`-UTR of the -gene, which appear to have differing transcriptional activities. The first element, localized to a region centered on nucleotide +14, functions as an alternate stage selector by directing preferential interaction of the erythroid enhancer of HS2 to the -gene in a fetal erythroid environment. The second element represses -promoter activity in both fetal and adult stages of erythropoiesis. Both of these effects appear to be mediated by specific DNA binding proteins.

We have previously shown that the competitive advantage of the -promoter in the fetal stage of erythropoiesis is mediated in part by the SSE in the proximal -promoter (14) . We now demonstrate that a second element in the 5`-UTR (SSE-2) also contributes to the preferential interaction of HS2 with the -promoter. SSE-2 is distinct from the -promoter SSE in that its function is unrelated to the binding of the SSP but correlates with the binding of a second fetal/erythroid-specific protein, which we refer to as the UTR stage selector protein. Phylogenetic footprint analysis suggests that the binding site for this protein is conserved in species with a distinct fetal stage of -gene expression (30) . This observation is analogous to the phylogenetic footprint data observed with the SSE, suggesting that both factors may be integral for the competitive silencing of the -promoter in the fetal stage of erythropoiesis. Examination of the binding motif of the UTR stage selector protein reveals no obvious similarities with known consensus sequences, and thus the identity of this factor is yet to be determined.

Our studies lead us to propose a model whereby the 2 stage selector elements function by stabilizing the interaction between looping upstream regulatory sequences of the LCR and the transcriptional machinery of the -promoter. SSE-2 appears to stabilize the interaction with the core enhancer. This effect may be mediated by an interaction between UTR stage selector protein and the enhancer protein rather than an effect on the transcription initiation complex, since capture of the core enhancer can occur in the absence of transcription (Fig. 2, construct2). We have recently demonstrated that the SSE in the proximal -promoter may function by recruiting co-enhancers outside the HS2 core.() In the presence of large fragments of HS2, the 2 stage selector elements may function additively (Fig. 3). In this context, loss of the proximal promoter SSE destabilizes the loop, and preferential interaction with the -promoter is lost (14) . However, our studies with the HS2 core enhancer suggest that loss of the SSE in this setting induces less instability and compensation by the 5`-UTR SSE occurs (Fig. 1). The observation that even when both stage selector elements are removed from the -promoter, linked expression does not achieve the levels seen with the single -promoter construct. This suggests that loss of the SSEs negates the competitive advantage of the -promoter, and the two promoters now compete equally for HS2.

The co-incident derepression of -promoter activity with both the 20-bp and smaller UTR mutants suggests that a repressor element also exists in the 5`-UTR region. In contrast to the effect of SSE-2, which must be at the level of transcription since it affects a separate gene transcript, the repressor effect could be either transcriptional or secondary to a translational block or altered mRNA stability. However, we have observed that the fold increase in -promoter activity observed with the mutant was mirrored by a similar increase in mRNA levels (Fig. 5) and that the magnitude of this effect was independent of assay time. Additional support for a transcriptional mechanism comes from the observation that binding of the erythroid transcription factor GATA-1 and a second ubiquitous factor was integral for repressor activity. Although we have no evidence to differentiate which of these factors is mediating this effect, GATA-1 has previously been demonstrated to transcriptionally repress -gene expression when binding to the non-canonical (C/T)AAG motif at -117 in the -promoter (31) .

The function of the 5`-UTR has been demonstrated in viral and eukaryotic genes as centering predominantly on translational control (32-35). However, transcriptional activity of this region has also been demonstrated (36) . Sequences downstream of the HIV-1 and HIV-2 promoters have been shown to influence basal transcription secondary to binding of the transcription factor LBP-1 (37) . In eukaryotes, the Drosophila heat shock gene, hsp22, is transcriptionally regulated by a small region between the cap site and nucleotide +14 (38) . Similarly, the human gastrin gene appears to be transcriptionally responsive to the leader sequence (39) . The role of the 5`-UTR in globin gene regulation remains virtually unexplored. The transcriptionally active sequences reported here suggest that regulation of the -gene and the competitive balance between the - and -genes may be influenced by this region and the proteins that bind to it. Although the relative importance of these elements in the setting of the entire cluster remains to be elucidated, the previous identification of both the human and chicken stage selector elements in similar assays provides precedent for their usefulness.


FOOTNOTES

*
Supported in part by Cancer Center Support CORE Grant P30 CA 21765, NHLBI, National Institutes of Health, Program Project Grant P01 HL 53749-01, American Lebanese Syrian Associated Charities, the Wellcome Trust, and NHMRC Project Grant 940573. 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.

§
To whom correspondence should be addressed: Division of Experimental Hematology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38101. Tel.: 901-495-2734; Fax: 901-495-2176.

The abbreviations used are: LCR, locus control region; SSE, stage selector element; UTR, untranslated region; PCR, polymerase chain reaction; HS2, hypersensitivity site 2; SSP, stage selector protein; CAT, chloramphenicol acetyltransferase; bp, base pair(s); EMSA, electromobility shift assays.

M. Crossley, personal communication.

P. J. Amrolia and S. M. Jane, unpublished observations.


ACKNOWLEDGEMENTS

We thank Merlin Crossley and Stuart Orkin for the gift of anti-GATA-1 antibody and nuclear extract from COS cells overexpressing GATA-1.


REFERENCES
  1. Stamatoyannopoulos, G., and Nienhuis, A. W.(1994) in The Molecular Basis of Blood Diseases (Stamatoyannopoulos, G., Nienhuis, A. W., Majerus, P. J., and Varmus, H., eds) pp. 107-156, WB Saunders, Philadelphia
  2. Chada, K., Magram, J., and Costantini, F.(1986) Nature 319, 685-689 [Medline] [Order article via Infotrieve]
  3. Kollias, G., Wrighton, N., Hurst, J., and Grosveld, F.(1986) Cell 46, 89-94 [Medline] [Order article via Infotrieve]
  4. Townes, T. M., Lingrel, J. B, Chen, H. Y., Brinster, R. L., and Palmiter, R. D.(1985) EMBO J. 4, 1715-1723 [Abstract]
  5. Grosveld, F., van Assendelft, G. B., Greaves, D. R., and Kollias, G. (1987) Cell 51, 975-985 [Medline] [Order article via Infotrieve]
  6. Talbot, D., Collis, P., Antoniou, M., Vidal, M., Grosveld, F., and Greaves, D. R.(1989) Nature 338, 352-355 [CrossRef][Medline] [Order article via Infotrieve]
  7. Dillon, N., and Grosveld, F.(1991) Nature 350, 252-254 [CrossRef][Medline] [Order article via Infotrieve]
  8. Raich, N., Enver, T., Nakamoto, B., Josephson, B., Papayannopoulou, T., and Stamatoyannopoulos, G.(1990) Science 250, 1147-1149 [Medline] [Order article via Infotrieve]
  9. Enver, T., Raich, N., Ebens, A. J., Papayannopoulou, T., Costantini, F., and Stamatoyannopoulos, G.(1990) Nature 344, 309-313 [CrossRef][Medline] [Order article via Infotrieve]
  10. Behringer, R. R., Ryan, T. M., Palmiter, R. D., Brinster, R. L., and Townes, T. M.(1990) Genes & Dev. 4, 380-389
  11. Hanscombe, O., Whyatt, D., Fraser, P., Yannoutsos, N., Greaves, D., Dillon, N., and Grosveld, F.(1991) Genes & Dev. 5, 1387-1394
  12. Choi, O. R. B., and Engel, J. D.(1988) Cell 55, 17-26 [Medline] [Order article via Infotrieve]
  13. Gallarda, J. L., Foley, K. P., Yang, Z., and Engel, J. D.(1989) Genes & Dev. 3, 1845-1859
  14. Jane, S. M., Ney, P. A., Vanin, E. F., Gumucio, D. L., and Nienhuis, A. W.(1992) EMBO J. 11, 2961-2969 [Abstract]
  15. Jane, S. M., Gumucio, D. L., Ney, P. A., Cunningham, J. M., and Nienhuis, A. W.(1993) Mol. Cell. Biol. 13, 3272-3281 [Abstract]
  16. Jane, S. M., Nienhuis, A. W., and Cunningham, J. M.(1995) EMBO J. 14, 97-105 [Abstract]
  17. Ney, P. A., Sorrentino, B. P., McDonagh, K. T., and Nienhuis, A. W. (1990) Genes & Dev. 4, 993-1006
  18. Chan, J. Y., Han, X. L., and Kan, Y. W.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11371-11375 [Abstract]
  19. Moi, P., Chan, K., Asunis, I., Cao, A., and Kan, Y. W.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9926-9930 [Abstract/Free Full Text]
  20. Talbot, D., Philipsen, S., Fraser, P., and Grosveld, F.(1990) EMBO J. 9, 2169-2177 [Abstract]
  21. Ellis, J., Talbot, D., Dillon, N., and Grosveld, F.(1993) EMBO J. 12, 127-134 [Abstract]
  22. Sorrentino, B., Ney, P., Bodine, D., and Nienhuis, A. W.(1990) Nucleic Acids Res. 18, 2721-2731 [Abstract]
  23. Gustin, K. E., and Burk, R. D.(1993) BioTechniques 14, 22-23 [Medline] [Order article via Infotrieve]
  24. Gorman, C. M., Moffat, L. F., and Howard, B. H.(1982) Mol. Cell. Biol. 2, 1044-1051 [Medline] [Order article via Infotrieve]
  25. Calzone, F. J., Britten, R. J., and Davidson, E. H.(1987) Methods Enzymol. 152, 611-632 [Medline] [Order article via Infotrieve]
  26. Fried, M., and Crothers, D. M.(1981) Nucleic Acids Res. 9, 6505-6525 [Abstract]
  27. Strauss, F., and Varshavsky, A.(1984) Cell 37, 889-901 [Medline] [Order article via Infotrieve]
  28. Grosveld, G. C., Shewmaker, C. K., Jat, P., and Flavell, R. A.(1981) Cell 25, 215-226 [Medline] [Order article via Infotrieve]
  29. Grosveld, G. C., De Boer, E., Shewmaker, C. K., and Flavell, R. A. (1982) Nature 295, 120-126 [Medline] [Order article via Infotrieve]
  30. Gumucio, D. L., Heilstedt-Williamson, H., Gray, T. A., Tarle, S. A., Shelton, D. A., Tagle, D. A., Slightom, J. L., Goodman, M., and Collins, F. S.(1992) Mol. Cell. Biol. 12, 4919-4929 [Abstract]
  31. Berry, M., Grosveld, F., and Dillon, N.(1992) Nature 358, 499-502 [CrossRef][Medline] [Order article via Infotrieve]
  32. Rao, C. D., Pech, M., Robbins, K. C., and Aaronson, S. A.(1988) Mol. Cell. Biol. 8, 284-292 [Medline] [Order article via Infotrieve]
  33. Prats, A. C., Vagner, S., Prats, H., and Amalric, F.(1992) Mol. Cell. Biol. 12, 4796-4805 [Abstract]
  34. Dix, D. J., Lin, P. N., Kimata, Y., and Theil, E. C.(1992) Biochemistry 31, 2818-2822 [Medline] [Order article via Infotrieve]
  35. Geballe, A. P., Spaete, R. R., and Mocarski, E. S.(1986) Cell 46, 865-872 [Medline] [Order article via Infotrieve]
  36. Mansour, S. L., Grodzicker, T., and Tjian, R.(1986) Mol. Cell. Biol. 6,2684-2694 [Medline] [Order article via Infotrieve]
  37. Jones, K. A., Luciw, P. A., and Duchange, N.(1988) Genes & Dev. 2, 1101-1114
  38. Hultmark, D., Klemenz, R., and Gehring, W. J.(1986) Cell 44, 429-438 [Medline] [Order article via Infotrieve]
  39. Theill, L. E., Wiborg, O., and Vuust, J.(1987) Mol. Cell. Biol. 7, 4329-4336 [Medline] [Order article via Infotrieve]

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