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Multiple Regulatory Elements in the 5′-Flanking Sequence of the Human ε-Globin Gene

1998; Elsevier BV; Volume: 273; Issue: 17 Linguagem: Inglês

10.1074/jbc.273.17.10202

1083-351X

Jin Li, Constance Tom Noguchi, Webb Miller, Ross C. Hardison, Alan N. Schechter,

RNA modifications and cancer

We have previously reported, on the basis of transfection experiments, the existence of a silencer element in the 5′-flanking region of the human embryonic (ε) globin gene, located at −270 base pairs 5′ to the cap site, which provides negative regulation for this gene. Experiments in transgenic mice suggest the physiological importance of this ε-globin silencer, but also suggest that down-regulation of ε-globin gene expression may involve other negative elements flanking the ε-globin gene. We have now extended the analysis of ε-globin gene regulation to include the flanking region spanning up to 6 kilobase pairs 5′ to the locus control region using reporter gene constructs with deletion mutations and transient transfection assays. We have identified and characterized other strong negative regulatory regions, as well as several positive regions that affect transcription activation. The negative regulatory regions at −3 kilobase pairs (εNRA-I and εNRA-II), flanked by a positive control element, has a strong effect on the ε-globin promoter both in erythroid K562 and nonerythroid HeLa cells and contains several binding sites for transcription factor GATA-1, as evidenced from DNA-protein binding assays. The GATA-1 sites within εNRA-II are directly needed for negative control. Both εNRA-I and εNRA-II are active on a heterologous promoter and hence appear to act as transcription silencers. Another negative control region located at −1.7 kilobase pairs (εNRB) does not exhibit general silencer activity as εNRB does not affect transcription activity when used in conjunction with an ε-globin minimal promoter. The negative effect of εNRB is erythroid specific, but not stage-specific as it can repress transcription activity in both K562 erythroid cells as well as in primary cultures of adult erythroid cells. Phylogenetic DNA sequence comparisons with other primate and other mammalian species show unusual degree of flanking sequence homology for the ε-globin gene, including in several of the regions identified in these functional and DNA-protein binding analyses, providing alternate evidence for their potential importance. We suggest that the down-regulation of ε-globin gene expression as development progresses involves complex, cooperative interactions of these negative regulatory elements, εNRA-I/εNRA-II, εNRB, the ε-globin silencer and probably other negative and positive elements in the 5′-flanking region of the ε-globin gene. We have previously reported, on the basis of transfection experiments, the existence of a silencer element in the 5′-flanking region of the human embryonic (ε) globin gene, located at −270 base pairs 5′ to the cap site, which provides negative regulation for this gene. Experiments in transgenic mice suggest the physiological importance of this ε-globin silencer, but also suggest that down-regulation of ε-globin gene expression may involve other negative elements flanking the ε-globin gene. We have now extended the analysis of ε-globin gene regulation to include the flanking region spanning up to 6 kilobase pairs 5′ to the locus control region using reporter gene constructs with deletion mutations and transient transfection assays. We have identified and characterized other strong negative regulatory regions, as well as several positive regions that affect transcription activation. The negative regulatory regions at −3 kilobase pairs (εNRA-I and εNRA-II), flanked by a positive control element, has a strong effect on the ε-globin promoter both in erythroid K562 and nonerythroid HeLa cells and contains several binding sites for transcription factor GATA-1, as evidenced from DNA-protein binding assays. The GATA-1 sites within εNRA-II are directly needed for negative control. Both εNRA-I and εNRA-II are active on a heterologous promoter and hence appear to act as transcription silencers. Another negative control region located at −1.7 kilobase pairs (εNRB) does not exhibit general silencer activity as εNRB does not affect transcription activity when used in conjunction with an ε-globin minimal promoter. The negative effect of εNRB is erythroid specific, but not stage-specific as it can repress transcription activity in both K562 erythroid cells as well as in primary cultures of adult erythroid cells. Phylogenetic DNA sequence comparisons with other primate and other mammalian species show unusual degree of flanking sequence homology for the ε-globin gene, including in several of the regions identified in these functional and DNA-protein binding analyses, providing alternate evidence for their potential importance. We suggest that the down-regulation of ε-globin gene expression as development progresses involves complex, cooperative interactions of these negative regulatory elements, εNRA-I/εNRA-II, εNRB, the ε-globin silencer and probably other negative and positive elements in the 5′-flanking region of the ε-globin gene. The expression of the individual genes of the human β-globin cluster is regulated in both a developmental and a tissue-dependent manner. The developmental “switches” in expression follow the sequential arrangement of the globin genes, beginning at the 5′ region of the gene cluster and including the five active ε, Gγ, Aγ, δ, and β-globin genes (1Stamatoyannopoulos G. Nienhuis A.W. Stamatoyannopoulos G. Nienhuis A.W. Majerus P.W. Varmus H. The Molecular Basis of Blood Diseases. W. B. Saunders, Philadelphia1994: 107-156Google Scholar). The effort to understand the mechanism of hemoglobin switching has focused on localizing the cis-acting DNA sequence elements which are involved in regulating globin gene expression, and identifying and characterizing the transcription factors or proteins that bind to those DNA motifs or related proteins (2Felsenfeld G. Boyes J. Chung J. Clark D. Studitsky V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9384-9388Crossref PubMed Scopus (157) Google Scholar, 3Shivdasani R.A. Orkin S.H. Blood. 1996; 87: 4025-4029Crossref PubMed Google Scholar). Each globin gene and its immediate flanking region appear to contain sufficient information for developmentally correct expression as suggested by transgenic mouse experiments (4Chada K. Magram K. Costantini F. Nature. 1986; 319: 685-689Crossref PubMed Scopus (114) Google Scholar, 5Kollias G. Wrighton N. Hurst J. Grosveld F. Cell. 1986; 46: 89-94Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 6Magram J. Chada K. Costatini F. Nature. 1985; 315: 338-340Crossref PubMed Scopus (126) Google Scholar, 7Shih D.M. Wall R.J. Shapiro S.G. J. Biol. Chem. 1993; 268: 3066-3071Abstract Full Text PDF PubMed Google Scholar). Phylogenetic footprinting has been used to identify evolutionarily conserved regions and other potential protein binding sites in the globin gene cluster (8Gumucio D.L. Shelton D.A. Bailey W.J. Slightom J.L. Goodman M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6018-6022Crossref PubMed Scopus (83) Google Scholar, 9Hardison R. Chao K.M. Adamkiewicz M. Price D. Jackson J. Zeigler T. Stojanovic N. Miller W. DNA Seq. 1993; 4: 163-176Crossref PubMed Scopus (24) Google Scholar, 10Slightom J.L. Bock J.H. Tagle D.A. Gumucio D.L. Goodman M.S., N. Jackson J. Miller W. Hardison R. Genomics. 1997; 39: 90-94Crossref PubMed Scopus (18) Google Scholar). Located at the distal 5′ region of the β-globin cluster immediately upstream of the embryonic ε-globin gene are the DNase I hypersensitive sites (HS 1 to HS 5) 1The abbreviations used are: HS, hypersensitivity site; LCR, locus control region; GS, gene silencer; hAEC, human adult erythroid cell; NR, negative regulatory region; PR, positive regulatory region; bp, base pair(s); kb, kilobase pair(s). of the locus control region (LCR) (6–13 kb 5′) that are important in controlling transcription and replication of the β-globin cluster. The proposed role of the LCR in developmental regulation is controversial. Studies in transgenic mouse show that linkage of the LCR to individual globin gene results in much higher expression in vivo, and an apparent alteration in the developmental specificity of the γ- and β-globin genes, depending on proximity and arrangement of the transgene (11Behringer R.R. Ryan T.M. Palmiter R.D. Brinster R.L. Towns T.M. Genes Dev. 1990; 4: 380-389Crossref PubMed Scopus (192) Google Scholar, 12Enver T. Ebens A.J. Forrester W.C. Stamatoyannopoulos G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7033-7037Crossref PubMed Scopus (65) Google Scholar, 13Enver T. Raich N. Ebens A.J. Papayannopoulou T. Costantini F. Stamatoyannopoulos G. Nature. 1990; 344: 309-313Crossref PubMed Scopus (238) Google Scholar). In contrast, developmental specificity of expression of human ε-globin gene appears to be more autonomous and does not require a particular arrangement with respect to the fetal γ- or adult β-globin genes. DNA constructs lacking the LCR show developmental switching of globin genes in transgenic mice showing the LCR is expendable for developmental regulation, at least in this assay. We have previously identified an ε-globin gene silencer (εGS), using reporter gene transfection assays, in vitrotranscription and DNA-protein binding assays, located in the region between −300 bp and −250 bp 5′ to the ε-globin gene cap site (14Cao S.X. Gutman P.D. Dave H.P. Schechter A.N. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5306-5309Crossref PubMed Scopus (100) Google Scholar, 15Peters B. Merezhinskaya N. Diffley J.F. Noguchi C.T. J. Biol. Chem. 1993; 268: 3430-3437Abstract Full Text PDF PubMed Google Scholar, 16Wada K.Y. Peters B. Noguchi C.T. J. Biol. Chem. 1992; 267: 11532-11538Abstract Full Text PDF PubMed Google Scholar). The potential biological significance of the silencing activity of εGS was supported by in vivo studies using transgenic mice (7Shih D.M. Wall R.J. Shapiro S.G. J. Biol. Chem. 1993; 268: 3066-3071Abstract Full Text PDF PubMed Google Scholar, 17Raich N. Clegg C.H. Grofti J. Romeo P.H. Stamatoyannopoulos G. EMBO J. 1995; 14: 801-809Crossref PubMed Scopus (122) Google Scholar, 18Raich N. Papayannopoulou T. Stamatoyannopoulos G. Enver T. Blood. 1992; 79: 861-864Crossref PubMed Google Scholar). Additional studies have revealed other cis-acting regulatory elements further 5′ to the ε-globin gene (9Hardison R. Chao K.M. Adamkiewicz M. Price D. Jackson J. Zeigler T. Stojanovic N. Miller W. DNA Seq. 1993; 4: 163-176Crossref PubMed Scopus (24) Google Scholar,20Fibach E. Manor D. Oppenheim A. Rachmilewitz E.A. Blood. 1989; 73: 100-103Crossref PubMed Google Scholar, 21Huang X. Hardison R.C. Miller W. Comput. Appl. Biosci. 1990; 6: 373-381PubMed Google Scholar), including a positive regulatory element, located at −700 bp, and a negative regulatory element located at about −400 bp. In general, the 5′ region of the ε-globin gene provides much of the activity for developmental regulation of the ε-globin gene expression as evidenced from transgenic mouse studies (7Shih D.M. Wall R.J. Shapiro S.G. J. Biol. Chem. 1993; 268: 3066-3071Abstract Full Text PDF PubMed Google Scholar). However, the expression of limited levels of the human ε-gene (5–10% of the mouse εy or β) with constructs in which the silencer has been mutated (18Raich N. Papayannopoulou T. Stamatoyannopoulos G. Enver T. Blood. 1992; 79: 861-864Crossref PubMed Google Scholar) 2B. Peters, unpublished data. suggests that other important negative regulatory elements may exist around the ε-globin gene. In the present study, we have investigated the functional role of the ε-globin gene 5′-flanking region up to −6 kb, which includes HS 1, and have identified several functionally important cis-elements that markedly affect expression driven by the ε-globin promoter. Construction of serially deleted mutants enabled us to systematically study the positive and negative cis-acting elements involved in ε-globin control. We observed multiple regulatory sequences in this region and focused on several strong negative elements located in the regions around −1.7 and −3.0 kb. In all cases, the negative elements are flanked by positive regulatory regions. These elements contain several DNA-protein binding motifs, including the erythroid specific transcription factor GATA-1. DNA sequences in the regulatory region located at −1.7 kb are conserved in all mammals examined, whereas the DNA sequences located at −3.0 kb are present only in the prosimian primate orangutan, galago, and human. Our data suggest that in addition to the εGS and the stage-specific positive element located more proximal to the ε-promoter, expression of the ε-globin gene including specifically its down-regulation during development involves multiple positive and negative elements. An ε-globin promoter/reporter gene construct was made by linking human ε-globin gene containing 5′ sequences from the promoter +46 to −6073 bp 5′ of the cap site, to a luciferase reporter gene (LUC)-coding plasmid pGL-Basic (Promega), generating a parent construct pε6073 that includes DNase I HS 1 at about −5 kb. A series of 5′-deletion mutants were made by linearizing pε6073 with SacI and SpeI followed by exonuclease III digestion, at 1-min intervals. The ends of the deleted mutants were filled in with the Klenow fragment of DNA polymerase I and self-ligated. A second set of 5′ series of deletions was made from pε3028 to generate smaller deletion mutants. The 5′ ends of the deletion mutants were determined by dideoxy sequencing. The human erythroleukemia K562 and HeLa cells were grown in RPMI 1640 or AMEM medium (Biofluid, Rockville, MD), respectively, supplemented with 10% fetal bovine serum,l-glutamine and penicillin/streptomycin. Primary human adult erythroid cells (hAEC), were grown in a two-phase liquid culture system as described previously (20Fibach E. Manor D. Oppenheim A. Rachmilewitz E.A. Blood. 1989; 73: 100-103Crossref PubMed Google Scholar). Briefly, mononuclear cells from the peripheral blood of normal donors, isolated on a Ficoll-Hypaque gradient, were grown in α-minimal essential medium with 10% fetal calf serum and 10% conditioned medium collected from 5637 human bladder carcinoma cells (phase I). After 7 days the cells were washed and recultured in liquid medium supplemented with 1 unit/ml recombinant erythropoietin (phase II). Both K562 and HeLa cells were transfected by electroporation with Gene Pulser (Bio-Rad) at 250 V (220 V for HeLa) and 960 μF with a plasmid DNA amount ranging from 10 to 40 μg. Transfections with hAEC were carried out after 10–11 days of incubation by combining phase II cultured cells from different donors. Transfected cells were collected and lysed after 48 h of incubation, and 20 μl of the cell lysate were used to determine luciferase activity analyzed with a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA), in which the substrate d-luciferin was automatically injected. The results are expressed as the average of at least three experiments with the activity of luciferase normalized to the amount of protein used in each experiment. A construct containing the LUC reporter gene under control of the SV40 promoter was used separately as the positive control to establish a value for promoter activity of 1.0. DNA probes were made by labeling sense primers with [γ-32P]dATP followed by polymerase chain reaction amplification to generate DNA fragments. The probes range from −3198 to −2898 bp 5′ for εNRA-I/εNRA-II and from −1838 to −1588 bp 5′ for εNRB. The labeled probes were purified by SpinBind (FMC, Rockland, ME). The mixtures of probe (20,000 cpm) and nuclear extract (50–100 μg) were incubated for 30 min on ice followed by the addition of DNase I (0.25–0.5 unit) and incubation for 4 min at room temperature. Equal volumes of stop solutions containing 400 μg/ml proteinase K were added and samples incubated for 30 min at 37 °C, and 2 min at 70 °C. After phenol/chloroform extraction and ethanol precipitation the DNA samples were dissolved in loading buffer and analyzed on 6% polyacrylamide sequencing gels. Gel shift studies were carried out by annealing a pair of oligonucleotides, labeled with [γ-32P]dATP followed by SpinBind (FMC, Rockland, ME) gel purification. The reactions were carried out on ice for 30 min in a 15-μl total volume and loaded onto a 4% polyacrylamide gel. In competition experiments, an unlabeled probe or the same fragment with mutation with 12.5–100-fold molar excess was included in the reactions as indicated. Oligonucleotide sequences for gel shift are as follows with the mutated bases underlined: εNRA II-1G: 5′-CCCAG AGCTG TATCT TAATTGT; εNRA II-Δ1G: 5′ CCCAG AGCTG GCGCCTAATTGT. Pairwise alignments of the DNA sequences from the β-globin gene clusters of human, galago, rabbit, and mouse were computed using the program SIM (21Huang X. Hardison R.C. Miller W. Comput. Appl. Biosci. 1990; 6: 373-381PubMed Google Scholar) and displayed as percent identity plots (22Hardison R.C. Ocltjen J. Miller W. Genome Res. 1997; 7: 959-966Crossref PubMed Scopus (258) Google Scholar). In a percent identity plot, all the gap-free aligning segments in the region of interest are automatically plotted as a series of horizontal lines (each between the coordinates of the human sequence present in a gap-free alignment) placed along the y axis according to the percent identity in each aligning segment. Notable features in the human sequence are also placed along the x axis. The simultaneous alignment of these four DNA sequences were obtained from the Globin Gene Server (www.globin.cse.psu.edu) (23Hardison R.C. Chao K.M. Schwartz S. Stojanovic N. Ganetsky M. Miller W. Genomics. 1994; 21: 344-353Crossref PubMed Scopus (31) Google Scholar). The region encompassing εNRA in human and the homologous regions from orangutan (EMBL accession no.X05035) and galago (GenBankTM accession no. U60902) were aligned simultaneously using the program YAMA2 (24Chao K.M. Hardison R. Miller W. J. Computational Biol. 1994; 1: 271-291Crossref PubMed Scopus (42) Google Scholar). In the displays of the multiple alignments, boxes are drawn around blocks of at least six columns where each column has an identical nucleotide in at least 75% of the positions; this is equivalent to requiring invariant columns for alignments of three sequences. The human embryonic epsilon globin (ε) 5′-flanking sequence was linked to the luciferase reporter gene and tested by transient transfection in K562 cells, a human erythroleukemia cell line that expresses embryonic and fetal globin genes. As shown in Fig. 1 A, the transcription activity of ε-promoter in transfected cells measured as luciferase reporter gene activity varies greatly with different lengths of 5′-flanking sequences. A high level of activity 2.5-fold greater than the SV40 promoter was observed for the minimal ε-promoter construct pε177, as expected given the active transcription activity of the endogenous ε-globin gene in K562 cells. The εGS in the region of −300 to −250 bp (14Cao S.X. Gutman P.D. Dave H.P. Schechter A.N. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5306-5309Crossref PubMed Scopus (100) Google Scholar) and other negative elements located at −419 bp (25Trepiccio W.L. Dyer M.A. Baron M.H. Mol. Cell. Biol. 1993; 13: 7457-7458Crossref PubMed Google Scholar) contribute to the lowered reporter gene activity of pε883 when compared with that of the minimal ε-promoter construct (pε177). Extending the 5′ region to encompass HS 1, we find that the transcription activity of pε6073 is 10-fold lower than that of pε883 suggests the existence of one or more strong negative element(s) in the region from −800 to −6000 bp. We have studied the transcriptional activity profile of this region of the ε-globin gene-flanking sequences in detail by constructing a series of deletion mutants extending up to 6 kb 5′ of the human ε-globin gene linked to luciferase reporter gene. The transcriptional activities of these reporter gene constructs were tested in transient transfection assays in embryonic/fetal erythroid K562 and nonerythroid HeLa cells (Fig. 1 A). In K562 cells, transcription activity of the ε-globin gene minimal promoter was comparable with that of SV40, in contrast to HeLa cells in which the ε-globin minimal promoter activity is only 10% of that SV40. Analysis of the deletion mutants in these cells revealed several regulatory regions flanking the ε-globin gene 5′ extending from −883 bp to HS 1. A striking feature of the behavior of the reporter gene constructs is that positive regulatory regions are generally flanked by negative regulatory regions, i.e. certain constructs appear as “spikes” in the graph. The two most striking combinations of this type are a pair of positive (εPRA) and negative regions (εNRA-I/εNRA-II) located between −2.8 and −3.1 kb that are active in both K562 cells and HeLa cells and a pair of positive (εPRB) and negative (εNRB) regions located around −1.7 kb that function only in K562 cells. Another, less potent regulatory pair includes the positive regulatory region between −1995 bp and −1747 bp flanked on the 5′ side by a negative regulatory that functions in both K562 and HeLa cells. The positive region between −1084 and −1135 bp and an overall negative region between −1135 and −1460 bp are active only in K562 cells. Additional positive regulatory regions (Fig. 1 A) are localized between −2385 and −2772 bp and between −3199 and −3329 bp that increase transcription activity by about 3-fold in K562 cells, and between −3329 and −3986 bp that increases transcription activity in HeLa cells. Other negative regulatory regions that reduce transcription activity are localized between −883 and −1084 bp, −2000 and −2385 bp, and −3986 and −4442 bp, and are active in both K562 cells and HeLa cells. Extending the 5′ region from −4442 to −6073 bp further decreases reporter gene activity in K562 cells. The greatest change in transcription activity observed in these transient assays are the increases associated with the regions εPRA and εPRB, and the decreases associated with the regions εNRA-I/εNRA-II and εNRB. To further understand the negative regulation of the ε-globin gene, we have focused on the two regions that exhibited marked decrease in transcription activity in K562 cells localized at −3 kb (εNRA-I/εNRA-II) and −1.7 kb (εNRB). εNRA-I/εNRA-II are active in both K562 and HeLa cells while the activity of εNRB is absent in HeLa cells, suggesting that the negative activity of this region is erythroid-specific. A summary of the results of the deletion series are shown in Fig. 1 B (top panel), aligned with graphs of the sequence matches observed in pairwise comparisons of the human sequence with that of other mammals. In these percent identity plots, the percent identity (from 50 to 100%) for each gap-free aligning segment is plotted using the coordinates of the human sequence, and notable features such as exons and interspersed repeats are placed along the horizontal axis (22Hardison R.C. Ocltjen J. Miller W. Genome Res. 1997; 7: 959-966Crossref PubMed Scopus (258) Google Scholar). Fig. 1 B shows the percent identity plots for alignments of the human sequence with that from the prosimian primate galago, from rabbit, and from mouse as three panels, including the region from HS 1 of the LCR through the ε-globin-coding sequence. In general, almost all of the galago sequence aligns with a high similarity to the human sequence. Extensive matches are also seen for comparisons of the human sequence with rabbit and mouse, although a roughly 1.6-kb segment between HS 1 and the ε-globin gene does not match (corresponding to about −4–2.4 kb in the human). Matching sequences extending this far 5′ to the gene are not characteristic of all mammalian globin genes. For instance, the 5′-flanking region of the human β-globin gene matches with that of galago to about −3000 bp, and with mouse to about −770 (23Hardison R.C. Chao K.M. Schwartz S. Stojanovic N. Ganetsky M. Miller W. Genomics. 1994; 21: 344-353Crossref PubMed Scopus (31) Google Scholar). The regions delineated in the results of the deletion series as εNRA-I/εNRA-II and εNRB show significant regions of matching in those comparisons. Thus the simultaneous alignment of these sequences is helpful in analyzing this region in more detail, as described below. However, regions comparable to human εNRA-I/εNRA-II and εPRA are found only in orangutan and galago, and only this pairwise alignment is informative, in contrast to greater cross-species matching more proximal to the ε-globin gene itself. The tissue-specificity of εNRB was further examined by comparison of the two constructs, pε1747 and pε1707, in human adult erythroid primary cells (hAEC) as well as in the K562 and HeLa cell lines (data not shown). The decrease in transcription activity of pε1747 compared with pε1707 is erythroid-specific as observed in both K562 and hAEC cells but not in HeLa cells, suggesting the erythroid-specific property of εNRB. Protein binding to the εNRB was studied by in vitro DNase I footprinting with nuclear extracts from both K562 and HeLa cells. Two strongly protected regions were detected only with K562 nuclear extracts (Fig. 2). These footprints are located around −1752 to −1735 bp and −1718 to −1710 bp and overlap with regions that are conserved in the 5′ region of corresponding embryonic globin genes in mouse, rabbit, and galago (Fig. 2,bottom). εNRB alone, however, does not act as a true silencer. Interestingly, no significant negative activity is observed when εNRB is linked directly to the ε minimal promoter and tested in either K562 or HeLa cells, when linked to a heterologous promoter transcription activity is again reduced (Fig. 3). This suggests that εNRB alone may exhibit negative regulation depending on the promoter, but does not act as a true silencer.Figure 3Transcription effects of εNRB on the ε-minimal promoter and a heterologous promoter (SV40).Luciferase activity of the ε-minimal promoter construct with and without εNRB was measured in transfection assays in K562 and HeLa cells. An SV40 promoter construct with and without εNRB was also analyzed in K562 cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The region between −3127 and −2902 bp which is active in both K562 cells and HeLa cells, has a much stronger negative effect in the erythroid cells (Fig. 1 A), perhaps related to GATA-1 binding (Fig. 4). This region contains two negative control regions, εNRA-I (−3127 to −3071 bp) and εNRA-II (−3028 to −2902 bp), each associated with a decrease in reporter gene activity. In K562 cells, the region separating these two motifs (−3071 and −3028 bp) exhibits a modest positive effect (Fig. 1 A). The combined effect of εNRA-I and εNRA-II in the 225-bp region reduces transcription activity 20-fold when added back to construct pε2902 to create pε3127. The negative effects of εNRA-I and εNRA-II were also observed in HeLa cells with about a 13-fold increase in transcription activity comparing pε2902 with pε3127. The activity of pε3127 is 3–4-fold lower than the ε-globin minimal promoter construct, pε177. The εNRA-I and εNRA-II regions were combined with a heterologous SV40 promoter in reporter gene constructs pεNRA-I/SV40 and pεNRA-II/SV40, respectively. The activity of these reporter genes were assayed and compared with that of SV40 alone (Fig. 5). The region εNRA-I decreases SV40 transcription activity by about 50% in K562 cells and more than 60% in HeLa cells. A similar decrease in transcription activity is observed when εNRA-I is combined with the epsilon minimal promoter (pεNRA-I/ε177) (data not shown). The εNRA-II has an even greater effect on SV40 promoter activity. The decrease in SV40 promoter activity by εNRA-II is almost 20 fold in K562 cells and about 10-fold in HeLa cells. The ability of εNRA-I and εNRA-II to decrease SV40 promoter activity is consistent with the decreases observed when these subregions are examined in the series of deletion mutants for the ε-globin 5′ region (Fig. 1 A). To attempt to identify the sequence motif responsible for the negative effect of εNRA-I and εNRA-II, we carried out DNase I footprint analysis and correlated the results with aligned DNA sequences from this region. Since the sequence corresponding to εNRA is not present in mouse or rabbit, we reasoned that it would be informative to look at additional primate species. The only other primate species for which sequence data extends this far is the orangutan, and a simultaneous alignment of human, orangutan, and galago sequences is shown in Fig. 6 B. Fig. 6 A shows the DNase I footprinting assay of region εNRA. The probe was generated by a polymerase chain reaction with32P-labeled primer, and the nuclear extract from K562 cells was used in the reactions. Several regions are footprinted by DNase I digestion designated as FP1–FP5. These include a conserved progesterone receptor binding motif (FP1) and a GATA-1 binding motif (FP2). A major footprinted region (FP3) appears within the region −3071 and −3028 bp which exhibits a small positive effect on transcription activity when comparing the constructs pε3028 with pε3071 in K562 cells. This footprinted region (FP3) is included within a block of sequence that is invariant among human, orangutan, and galago. Two minor footprinted regions (denoted FP4 and FP5) are at potential GATA-1 binding motifs in εNRA-II at about −2976 and −2949 bp, respectively. An inverted AGATAG sequence appears in the region corresponding to FP4 in the galago ε-globin 5′-flanking region and the region corresponding to FP5 is only partially conserved in this comparison. Although two of the GATA1 binding sites have mismatches in galago that would be expected to decrease binding affinity, these binding sites are identical between orangutan and human. To assess the role of the G