Proc. Natl. Acad. Sci. USA
Vol. 87, pp. 8115-8119, October 1990 Developmental Biology
Interaction of c-globin cis-acting control elements with erythroid-specific regulatory macromolecules JING WU*, G. JOAN GRINDLAYt, CLARE JOHNSON*, AND MAGGI ALLAN*t *Departments of Genetics and Medicine, College of Physicians and Surgeons of Columbia University, 630 West 168th Street, New York, NY 10032; and
tBeatson Institute, Garscube Estate, Switchback Road, Bearsden, Scotland, United Kingdom Communicated by Gary Felsenfeld, July 3, 1990
ABSTRACT We have used a competition assay to investigate the influence of erythroid-specific cellular factors on transcription from the human E-globin major cap site promoter and the minor promoter located 200 base pairs (bp) upstream from the e-globin cap site. In the human erythroid cell line K562, competition of the E-globin major cap site promoter linked to the chloramphenicol acetyltransferase (CAT) gene (EP-CAT) with the same promoter fragment linked to a neomycin resistance gene (EP-NEO) leads to a reduction in CAT activity. This indicates the specific presence of K562 cells of factor(s) which interact with the 200-bp promoter fragment (isolated from the gene body or flanking sequences) to activate transcription from the E-globin major cap site. Competition of the e-globin major promoter (as EP-CAT) with the upstream minor E-globin promoter (as EP2-NEO) also leads to a reduction in CAT activity, indicating that both promoters share erythroid-specific trans-acting factors. The reverse competition (EP2-CAT with EP-NEO) leads to an increase in CAT activity, suggesting that the existence of erythroid-specific factor(s) which repress transcription from the 200-bpupstream E-globin promoter.
major mRNA cap site (the "-200 bp promoter") (6) and the major cap site promoter in response to the erythroid environment. The experimental approach is designed to determine the nature of erythroid-specific trans-acting factors interacting with these two promoters; specifically, whether they are activators or repressors and whether the same factor(s) can interact with different e-globin DHS/promoters.
MATERIALS AND METHODS Cell Culture and Transfections. Cell lines were grown on SLM medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO). Twenty-four hours prior to transfection cells were passaged to give 50%-confluent cultures and the medium was replaced 3-4 hr prior to addition of DNA. Calcium phosphate precipitates were formed as described by Wigler et al. (12). Cells were exposed to precipitates for 14-16 hr, after which medium was replaced. Cells were transfected with DNA at a range of concentrations (0.1-40 ,ug per 75-cm2 flask) for each construct to determine the point at which maximum expression of each promoter was obtained. In each case, input DNA was made up to 40 ,ug per 75-cm2 flask by the addition of pAT153, a pBR322 derivative. Chloramphenicol Acetyltransferase (CAT) Assays. CAT assays were used as an indirect means of determining the promoter activity of DNA fragments inserted immediately upstream of the bacterial CAT gene (13). Cell lines were transiently transfected with CAT constructs as described above and CAT assays were performed exactly as described by Wu et al. (9). Ascending chromatography on SIL-G silica gel thin layer plates (Kodak) was performed in 95% chloroform/5% methanol (vol/vol). Autoradiography was overnight at room temperature. The radioactive spots were cut out and their radioactivities were measured, and percentage conversion from unacetylated to acetylated [14C]chloramphenicol was calculated. The levels of CAT activity were then plotted against levels of input DNA. RNA Preparation and Analysis. Total RNA was prepared by a variation of the method of Chirgwin et al. (14). S1 Nuclease Mapping. S1 nuclease mapping was carried out by the method of Berk and Sharp (15) as modified by Weaver and Weissman (16). Construction of Plasmids. The promoterless CAT vector pCO contains the pUC9 multiple cloning site, the CAT gene, and a 100-bp herpes simplex virus (HSV)-2 immediate early terminator element at the 3' end of the CAT gene (Fig. 2 and ref. 9). Test promoters were inserted (using linkers if necessary) into the BamHI site of pCO. To perform competition experiments, test promoters were also inserted (by means of linkers where necessary) into the HindIlI site of the promoterless vector pNEO. pNEO con-
The human e-globin gene is expressed in embryonic erythroid cells during the first trimester of gestation. Activation of the gene is accompanied by a complex process of cell divisions and differentiation in which several intermediary cell types can be identified between the "committed" erythroid stem cell stage and the terminally differentiated reticulocyte (1). The biological complexity associated with the regulation of this highly tissue-specific gene may be reflected in the finding that the E-globin gene possesses at least 10 erythroid-specific regions of DNase 1 hypersensitivity (DHS) in the 5' flanking region (2, 3). These DHS vary in their pattern of sensitivity to DNase 1 at different stages during the activation and inactivation of this gene (4, 5). Six of the E-globin DHS possess minor promoters (2, 4, 6, 7), and recently at least three of these DHS/promoters have been shown to be implicated with controlling transcription of the E-globin gene at different stages of erythroid differentiation and development (8-10). (For a map of the E-globin gene region see Fig. 1.) In some cases, regulatory function occurs as a direct result of activity of the minor upstream E-globin promoters (9, 10), and the finding that several well-documented regulatory elements also possess minor promoters (discussed in ref. 11) suggests that this type of control mechanism may be of general significance. The finding that the E-globin gene possesses multiple regulatory elements raises the question of whether multiple trans-acting factors interact with these elements or whether the same factor can interact with more than one element. In this study, we investigate the nature of the relationships between the E-globin promoter 200 bp upstream from the
Abbreviations: DHS, region of DNase 1 hypersensitivity; HSV, herpes simplex virus; CAT, chloramphenicol acetyltransferase; NEO, neomycin resistance. 4To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Proc. Natl. Acad. Sci. USA 87 (1990)
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tains the neomycin resistance (NEO) gene coding for aminoglycoside 3'-phosphotransferase (3')-II from transposon TnS and therefore carries resistance to the antibiotic G418 (20). At its 3' end is the HSV-1 TK polyadenylylation site contained within a Sma I-Pvu II fragment (T).
RESULTS Experimental Strategy. To identify cellular factors specifically involved in transcriptional regulation, we have used a competition assay described by Scholar and Gruss (21) and Mercola et al. (22). Briefly, a test promoter, covalently linked to a gene whose product is readily monitored, is cotransfected into appropriate cell lines with increasing amounts of competitor DNA. The competitor sequence is covalently linked to a gene which does not interfere with the test gene. In this study, we have used the CAT gene as test and the NEO gene as competitor. If cellular factors controlling transcription of the test promoter are limiting, then increasing amounts of competitor should lead to a decrease in CAT activity. Transcription from the Major e-Globin Cap Site Is Specifically Up-Regulated 100- to 200-Fold after Transfection into K562 Cells. To ensure a valid comparison of promoter activity in different cell lines, two parameters of the assay must be established. First, it is necessary to determine the quantity of input promoter required to attain maximum CAT activity in each cell line. Second, to control for variable HB
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transfection efficiency in different cell lines, CAT activity of the test promoter must be expressed as a function of activity of a promoter whose activity is constant within a range of cell lines. The test promoter used in this study is a 200-bp BamHI-Pvu II fragment containing the E-globin major cap site, inserted by means of BamHI linkers into the promoterless CAT vector pCO (Materials and Methods and legend to Fig. 2). The control promoter is a 200-bp Sma I-Sau3A fragment containing the HSV-2 immediate early promoter (Materials and Methods and legend to Fig. 2). Increasing amounts of the constructs EP-CAT and IE-CAT (from 0.1 to 40 jig of input DNA per 75-cm2 flask) were introduced into the embryonic human erythroid cell line K562 (23) and into the human nonerythroid cell line HeLa. Fortyeight hours later, CAT activity was measured and plotted as a function of input DNA. The maximum level of CAT activity achieved by IE-CAT was given an arbitrary value of 100 in each cell line and levels of EP-CAT are expressed relative to this. After transfection into K562 cells (Fig. 3a), IE-CAT reaches saturation at 20 ,ug of input DNA, while eP-CAT reaches saturation at 5 ,ug of input DNA. This implies that a IE-CAT
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FIG. 2. Construction of CAT and NEO plasmids containing different promoters. The line drawings indicate the constructs used in competition assays. The promoterless CAT construct pCO contains the pUC9 multiple cloning site (mcs), the bacterial CAT gene, and an HSV-2 immediate early gene terminator element (T) inserted at the 3' end of the CAT gene (9). Test promoters were inserted, using BamHI linkers, into the BamHI site of pCO. The promoters used are (i) eP, a 200-bp BamHI-Pvu II fragment containing the E-globin major promoter (see Fig. 1); (ii) EP2, a 350-bpXba I-BamHI fragment containing the E-globin -200 bp promoter (see Fig. 1); (iii) IE, a 200-bp HSV-2 immediate early promoter (9, 17); and (iv) TK, an HSV-2 thymidine kinase promoter (18). The promoterless NEO construct pNEO contains the kanamycin-neomycin resistance gene coding for aminoglycoside 3'-phosphotransferase (3')-II from transposon Tn5 and is therefore resistant to the antibiotic G418 (19, 20). The EP, EP2, IE, and TK promoters were inserted by means of HindIII linkers into the HindIII site of pNEO.
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FIG. 3. CAT activity in cell lines transfected with increasing amounts of RP-CAT and IE-CAT. Variable amounts of -P-CAT and IE-CAT (0.1-40 ,ug per 75-cm2 flask) were transfected by calcium phosphate precipitation into K562 cells (a) and HeLa cells (b). CAT activity was measured 48 hr later and was plotted as a function of input DNA. To yield a total of 40 ,ug of DNA per 75-cm2 flask, the plasmid pAT153 was used as carrier.
Developmental Biology: Wu et al. limiting levels of cellular trans-acting factors are present in K562 cells, sufficient to saturate 20 ,ug of IE-CAT and 5 ,ug of EP-CAT. In HeLa cells, IE-CAT reaches a maximum level of expression after transfection with 0.5 ,tg of DNA, while EP-CAT reaches maximum expression with 20 jig of input DNA (Fig. 3b), implying the presence of cellular factors sufficient to saturate 0.5 Ag of the IE construct and 20 ,ug of the E-globin construct. These results imply that, although the E-globin promoter can be transcribed in HeLa cells, the factor(s) mediating transcription are different from those interacting with the E-globin promoter in K562 cells. The finding that 40-fold higher input level of EP-CAT (compared with IE-CAT) are required to generate maximum promoter activity in HeLa cells suggests that the HeLa factor(s) interact only weakly with the E-globin promoter and therefore a large excess of --globin DNA is required to drive the reaction to completion. In HeLa cells, comparison of promoter activity at the linear part of the curve (up to an input of 0.5 Ag of DNA) indicates that IE generates approximately 100-fold more CAT activity than EP does. Conversely, in K562 cells (at input DNA levels up to 5 ,.g). EP generates 1.5-fold more CAT activity than IE does. Assuming that the factors responsible for IE expression are present at the same abundance in HeLa and K562 cells, it follows that the E-globin promoter is 150-fold more active in the embryonic erythroid K562 cell line than in HeLa cells. The experiment described in Fig. 3 has been repeated four times, using at least two independent DNA preparations. Similar results have also been obtained when the HSV-2 TK was used as control and when other nonerythroid cell lines such as Cos 7 and CV1 were used (not shown). To confirm that transcription from the EP-CAT construct did indeed originate from the correct E-globin initiation site, 20 ,g of EP-CAT was transfected into subconfluent K562 cells in a 75-cm2 flask; RNA was prepared 48 hr later and analyzed by S1 nuclease mapping using an end-labeled probe spanning the E-globin cap site. The 600-bp fragment extending from the HindIII site within the pUC9 polylinker to the EcoRI site within the CAT gene was gel purified and 5'-endlabeled with [y-32P]ATP and polynucleotide kinase, and the strands were separated (24). The single-stranded probe (shown in the line drawing of Fig. 4) was hybridized in probe excess at 57°C to 40 ,g of total RNA prepared from K562 cells transfected with EP-CAT. Hybrids were digested with 1000 units of S1 nuclease (Boehringer Mannheim) for 1.5 hr at 37°C and digestion products were separated on a denaturing 6% polyacrylamide gel. As shown in Fig. 4, a single digestion product 250 bp long is found, migrating exactly to the position expected for the fragment containing the E-globin major cap site. A similar S1 nuclease assay was performed on RNA derived from K562 cells after transfection with IE-CAT and, as shown in Fig. 4, a single band migrating to the predicted position of the immediate early promoter is found. Up-Regulation of the e-Globin Major Promoter by Interaction with Erythroid-Specific Activator(s). The experiments described above indicate that a 200-bp fragment containing the E-globin major promoter generated 150-fold higher levels of CAT activity after transfection into an embryonic erythroid cell line compared with any nonerythroid cell line tested. This could formally be due to (i) interaction of the E-globin promoter with erythroid-specific activator(s) or (ii) the specific absence in embryonic erythroid cells of repressor(s) present in all other cell types. Certain predictions can be made depending on which of these scenarios is correct. Thus, if the --globin promoter interacts with repressor in nonerythroid cells, competition with increasing amounts of itself should lead to an increase in transcription from the E-globin promoter in nonerythroid cells. If, on the other hand, the E-globin promoter interacts with erythroid-specific activator(s), self-competition in erythroid cells should lead to a
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decrease in transcription. To test these possibilities, we have cotransfected a fixed amount of EP-CAT with increasing amounts of eP-NEO in both K562 and HeLa cells. The fixed level of EP-CAT was that previously shown in Fig. 3 to produce maximum amounts of CAT activity (5 ug). As previously stated, at this level of input DNA, we assume that transcription factors interacting with the e-globin promoter are limiting. As shown in Fig. 5a, when 5 gg of EP-CAT is cotransfected with increasing amounts of eP-NEO, CAT activity decreases after addition of 5 ,g of EP-NEO and continues to decrease up to the maximum of 40 ,tg of input eP-NEO. To control for the possibility that increasing amounts of eP-NEO may compete for general transcription factors, EP-CAT was cotransfected with increasing amount of TK-NEO. As shown in Fig. 5a, at the highest levels of input TK-NEO (30 and 40 /ig) a slight decrease in CAT activity is found, probably indicating competition for general transcription factors. However, this curve is unlike the competition seen with OP-NEO, and we therefore suggest that the specific increase in transcription of the e-globin promoter after transfection into K562 cells is the direct result of interaction of cis-acting elements contained on this 200-bp fragment with erythroid-specific activator(s). This is further supported by the results shown in Fig. 5b, where competition of EP-CAT with EP-NEO was carried out in HeLa cells. In this case, only a slight reduction in CAT activity was observed at the highest levels of input competitor. Major E-Globin Promoter and -200 bp Promoter Interact with the Same Erythroid-Specific "Trans" Factor(s). We have used competition assays to determine whether the e-globin
Proc. Natl. Acad. Sci. USA 87 (1990)
Developmental Biology: Wu et al.
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major promoter (EP) and -200 bp promoter (0P2) can compete for the same erythroid-specific regulatory factor(s). K562 cells were transfected with increasing amounts of EP2-CAT (0.1-40 ,ug per 75-cm2 flask) to determine the level of input DNA at which maximum CAT activity was reached exactly as described in Fig. 3. Saturation was reached at 7.5 ,ug of input DNA (not shown). A fixed level of 7.5 ,ug of eP2-CAT was cotransfected with increasing amounts of EPNEO into K562 cells. As shown in Fig. 6a, as EP-NEO increases from 5 to 40 ,ug, CAT correspondingly increases, up to the maximum at 40 ,ug of input EP-NEO. It was not possible to increase EP-NEO levels further because cell death occurred at higher input. No change in CAT activity is seen when TK-NEO is used as competitor (Fig. 6a) or when this competition is carried out in HeLa cells (Fig. 6b). These results suggest that the EP fragment competes for an erythroid-specific factor which down-regulates transcription from EP2, the E-globin -200 bp promoter. S1 nuclease analysis confirmed that transcripts originated at the predicted position of the -200 bp promoter (Fig. 4). The reverse competition experiment, in which a fixed amount, 5 ,ug, of EP-CAT is cotransfected into K562 cells with increasing amounts of EP2-NEO, is shown in Fig. 7a. CAT activity derived from the major e-globin promoter decreases after competition with 5 ,ug of eP2-NEO and continues to decrease up to 30 ,ug of competitor DNA, indicating that EP2 competes for erythroid-specific factor(s) which up-regulate transcription from the major promoter. This competition is not seen when TK-NEO is used as competitor (Fig. Sa). The slight decrease in CAT activity seen at the highest level of eP2-NEO in HeLa cells (Fig. 7b) is not significantly different from that found with TK-NEO as
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Developmental Biology: Wu et al. DISCUSSION The human E-globin gene demonstrates a high degree of tissue specificity and developmental stage specificity of expression. This specificity can be somewhat mimicked by transfection experiments, since 100-fold greater transcription from the E-globin major cap site is found after transfection into K562 cells. In the current study, we have used a competition assay to determine the nature of the putative interaction(s) of the E-globin -200 bp and major promoters, in isolation, with erythroid-specific trans factor(s). Comparison of activity of the E-globin and intermediate early promoters within the linear range of the expression curve in K562 and HeLa cells (Fig. 3) indicates that the E-globin promoter is 150-fold more active in K562 cells than in HeLa cells. This conclusion makes the assumption that the factors responsible for IE transcription are present at the same abundance in both cell lines. We have proceeded on the assumption that either the K562 cell line possesses specific activator(s) which interact with element(s) within the 200-bp BamHI-Pvu II promoter fragment or the K562 cells specifically lack factor(s) which down-regulate this promoter in all other cell lines. The results shown in Fig. 5 strongly support the former hypothesis. Thus competition of 5 ,ug of EP-CAT (the level at which transacting factors are limiting in K562 cells) with increasing amounts of EP-NEO leads to a gradual reduction in CAT activity to approximately 10% of baseline. This reduction in CAT activity after self-competition suggests that the EP fragment interacts mainly with transcription activator(s) and when these factors are present in limiting amounts, selfcompetition leads to a reduction of transcription from the major E-globin promoter. Such a decrease is unlikely to result from competition for general transcription factors, since it is not seen either when TK-CAT is used as competitor or when the competition is carried out in HeLa cells (Fig. 5). S1 nuclease analysis (Fig. 4) indicates that initiation of transcription occurs from the correct E-globin cap site. The different kinetics of competition observed with EP-NEO and TK-NEO may suggest that different classes of factors are involved and that the slight reduction with CAT activity at the highest levels of input TK-NEO may reflect competition for general transcription factors required by all promoters. It is not possible to distinguish by this assay whether one or more trans-acting factors are involved in the erythroid-specific up-regulation of the E-globin promoter, and it is conceivable that the overall positive response is the summation of interactions with multiple positive and negative factors. Similarly, this assay does not indicate whether the same factor(s) is limiting in K562 and HeLa cells. Erythroid-specific factors have been directly demonstrated binding to the a-globin and /3-globin promoter regions (25-27), and work from these laboratories suggests that multiple factors may be involved. Previous experiments have indicated that cotransfection of the e-globin gene, in nonerythroid cells, with the adenovirus EIA gene leads to a 20- to 30-fold increase in transcription from the major cap site and a corresponding decrease from the -200 cap site (11). A similar switch in transcription from the -200 cap site to the major cap site is seen after transfection into the K562 cell line (19) and during induction of K562 cells with hemin (an event thought to be equivalent to normal red blood cell differentiation), suggesting that an erythroid equivalent of EIA may exist. The switch of transcription from the -200 cap site to the major cap site during erythroid differentiation is apparently a critical stage in activation of the gene. Recently Cao et al. (8) have demonstrated that the region between -177 to -392 relative to the e-globin cap site contains an element ('silencer") which down-regulates transcription from the e-globin cap site. Down-regulation is more pronounced in nonerythroid cells
Proc. Natl. Acad. Sci. USA 87 (1990)
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than in erythroid cells. The results shown in Fig. 6 indicate that cotransfection in K562 cells of a fixed (saturation) amount of EP2-CAT with increasing amounts of EP-NEO leads to an increase in CAT activity, suggesting that the EP fragment competes for an erythroid-specific factor(s) which represses transcription from the -200 bp promoter. The reverse competition, shown in Fig. 7 (fixed amount of EPCAT, increasing amounts of eP2-NEO), results in a reduction in CAT activity, suggesting that the EP2 fragment competes for a factor(s) which up-regulates transcription from the E-globin major cap site. These results suggest, but do not prove, that the same erythroid-specific factor may upregulate the major cap site and down-regulate the -200 cap site. Such a factor would bear a strong resemblance to the adenovirus EIA gene product. Thus, at least two of the multiple E-globin control elements may share regulatory trans-factors, and these factors may perform opposite functions when bound to different elements. We thank Nick Shelness for assistance in preparing the manuscript. Support for this research was provided by National Institutes
of Health Grant ROI HL37023. 1. Harrison, P. R. (1976) Nature (London) 262, 353-356. 2. Zhu, J., Allan, M. & Paul, J. (1984) Nucleic Acids Res. 12, 9191-9204. 3. Tuan, D. & London, I. M. (1984) Proc. Nati. Acad. Sci. USA 81, 2718-2722. 4. Bushel, P., Rego, K., Mendelsohn, L. & Allan, M. (1990) Mol. Cell. Biol. 10, 1199-1208. 5. Forrester, W. C., Thompson, C., Elder, J. T. & Groudine, M. (1986) Proc. NatI. Acad. Sci. USA 83, 1359-1363. 6. Allan, M., Lanyon, W. G. & Paul, J. (1983) Cell 35, 187-197. 7. Allan, M. & Paul, J. (1984) Nucleic Acids Res. 12, 1193-1200. 8. Cao, S. X., Gutman, P. D., Harish, P. G. & Schechter, A. N. (1989) Proc. Natl. Acad. Sci. USA 86, 1209-1216. 9. Wu, J., Grindlay, G. J., Bushel, P., Mendelsohn, L. & Allan, M. (1990) Mol. Cell. Biol. 10, 1199-1208. 10. Allan, M., Grindlay, G. J., Wu, J., Bushel, P. & Mendelsohn, L., Nucleic Acids Res., in press. 11. Allan, M., Montague, P., Zhu, J. D. & Paul, J. (1984) Cell 38, 399-407. 12. Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S. & Axel, R. (1979) Cell 76, 777-785. 13. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051. 14. Chirgwin, J. M., Pryzbyla, A. E., MacDonald, R. J. & Rutter, W. (1979) Biochemistry 18, 5294-5299. 15. Berk, A. J. & Sharp, P. A. (1977) Cell 12, 721-732. 16. Weaver, R. F. & Weissman, C. (1979) Nucleic Acids Res. 6, 1175-1193. 17. Gaffney, D. F., McLauchlan, J., Whitton, J. L. & Clements, J. B. (1985) Nucleic Acids Res. 13, 7847-7863. 18. Lang, J. C., Wilkie, N. M. & Davison, A. J. (1982) J. Gen. Virol. 64, 2679-26%. 19. Allan, M., Montague, P., Grindlay, G. J., Sibbet, G., DonovanPeluso, M., Bank, A. & Paul, J. (1985) Nucleic Acids Res. 13,
6125-6136.
20. Colbere-Gerapin, F., Horodniceau, F., Kourilsky, P. & Gerapin, A. C. (1981) J. Mol. Biol. 150, 1-14. 21. Scholar, H. R. & Gruss, P. (1984) Cell 36, 403-411. 22. Mercola, M., Goverman, J., Mirrell, C. & Calame, K. (1985) Science 277, 266-270. 23. Rutherford, T. R., Glegg, R. B. & Weatherall, D. J. (1979) Nature (London) 280, 164-165. 24. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 521-560. 25. Barnhart, K. M., Kim, C. G., Baneri, S. S. & Sheffrey, M. (1988) Mol. Cell. Biol. 8, 3215-3226. 26. Emerson, B. M., Lewis, C. D. & Felsenfeld, G. (1985) Cell 41, 21-30. 27. Kemper, B., Jackson, P. D. & Felsenfeld, G. (1987) Mol. Cell. Biol. 7, 2059-2069.
Vol. 87, pp. 8115-8119, October 1990 Developmental Biology
Interaction of c-globin cis-acting control elements with erythroid-specific regulatory macromolecules JING WU*, G. JOAN GRINDLAYt, CLARE JOHNSON*, AND MAGGI ALLAN*t *Departments of Genetics and Medicine, College of Physicians and Surgeons of Columbia University, 630 West 168th Street, New York, NY 10032; and
tBeatson Institute, Garscube Estate, Switchback Road, Bearsden, Scotland, United Kingdom Communicated by Gary Felsenfeld, July 3, 1990
ABSTRACT We have used a competition assay to investigate the influence of erythroid-specific cellular factors on transcription from the human E-globin major cap site promoter and the minor promoter located 200 base pairs (bp) upstream from the e-globin cap site. In the human erythroid cell line K562, competition of the E-globin major cap site promoter linked to the chloramphenicol acetyltransferase (CAT) gene (EP-CAT) with the same promoter fragment linked to a neomycin resistance gene (EP-NEO) leads to a reduction in CAT activity. This indicates the specific presence of K562 cells of factor(s) which interact with the 200-bp promoter fragment (isolated from the gene body or flanking sequences) to activate transcription from the E-globin major cap site. Competition of the e-globin major promoter (as EP-CAT) with the upstream minor E-globin promoter (as EP2-NEO) also leads to a reduction in CAT activity, indicating that both promoters share erythroid-specific trans-acting factors. The reverse competition (EP2-CAT with EP-NEO) leads to an increase in CAT activity, suggesting that the existence of erythroid-specific factor(s) which repress transcription from the 200-bpupstream E-globin promoter.
major mRNA cap site (the "-200 bp promoter") (6) and the major cap site promoter in response to the erythroid environment. The experimental approach is designed to determine the nature of erythroid-specific trans-acting factors interacting with these two promoters; specifically, whether they are activators or repressors and whether the same factor(s) can interact with different e-globin DHS/promoters.
MATERIALS AND METHODS Cell Culture and Transfections. Cell lines were grown on SLM medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO). Twenty-four hours prior to transfection cells were passaged to give 50%-confluent cultures and the medium was replaced 3-4 hr prior to addition of DNA. Calcium phosphate precipitates were formed as described by Wigler et al. (12). Cells were exposed to precipitates for 14-16 hr, after which medium was replaced. Cells were transfected with DNA at a range of concentrations (0.1-40 ,ug per 75-cm2 flask) for each construct to determine the point at which maximum expression of each promoter was obtained. In each case, input DNA was made up to 40 ,ug per 75-cm2 flask by the addition of pAT153, a pBR322 derivative. Chloramphenicol Acetyltransferase (CAT) Assays. CAT assays were used as an indirect means of determining the promoter activity of DNA fragments inserted immediately upstream of the bacterial CAT gene (13). Cell lines were transiently transfected with CAT constructs as described above and CAT assays were performed exactly as described by Wu et al. (9). Ascending chromatography on SIL-G silica gel thin layer plates (Kodak) was performed in 95% chloroform/5% methanol (vol/vol). Autoradiography was overnight at room temperature. The radioactive spots were cut out and their radioactivities were measured, and percentage conversion from unacetylated to acetylated [14C]chloramphenicol was calculated. The levels of CAT activity were then plotted against levels of input DNA. RNA Preparation and Analysis. Total RNA was prepared by a variation of the method of Chirgwin et al. (14). S1 Nuclease Mapping. S1 nuclease mapping was carried out by the method of Berk and Sharp (15) as modified by Weaver and Weissman (16). Construction of Plasmids. The promoterless CAT vector pCO contains the pUC9 multiple cloning site, the CAT gene, and a 100-bp herpes simplex virus (HSV)-2 immediate early terminator element at the 3' end of the CAT gene (Fig. 2 and ref. 9). Test promoters were inserted (using linkers if necessary) into the BamHI site of pCO. To perform competition experiments, test promoters were also inserted (by means of linkers where necessary) into the HindIlI site of the promoterless vector pNEO. pNEO con-
The human e-globin gene is expressed in embryonic erythroid cells during the first trimester of gestation. Activation of the gene is accompanied by a complex process of cell divisions and differentiation in which several intermediary cell types can be identified between the "committed" erythroid stem cell stage and the terminally differentiated reticulocyte (1). The biological complexity associated with the regulation of this highly tissue-specific gene may be reflected in the finding that the E-globin gene possesses at least 10 erythroid-specific regions of DNase 1 hypersensitivity (DHS) in the 5' flanking region (2, 3). These DHS vary in their pattern of sensitivity to DNase 1 at different stages during the activation and inactivation of this gene (4, 5). Six of the E-globin DHS possess minor promoters (2, 4, 6, 7), and recently at least three of these DHS/promoters have been shown to be implicated with controlling transcription of the E-globin gene at different stages of erythroid differentiation and development (8-10). (For a map of the E-globin gene region see Fig. 1.) In some cases, regulatory function occurs as a direct result of activity of the minor upstream E-globin promoters (9, 10), and the finding that several well-documented regulatory elements also possess minor promoters (discussed in ref. 11) suggests that this type of control mechanism may be of general significance. The finding that the E-globin gene possesses multiple regulatory elements raises the question of whether multiple trans-acting factors interact with these elements or whether the same factor can interact with more than one element. In this study, we investigate the nature of the relationships between the E-globin promoter 200 bp upstream from the
Abbreviations: DHS, region of DNase 1 hypersensitivity; HSV, herpes simplex virus; CAT, chloramphenicol acetyltransferase; NEO, neomycin resistance. 4To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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tains the neomycin resistance (NEO) gene coding for aminoglycoside 3'-phosphotransferase (3')-II from transposon TnS and therefore carries resistance to the antibiotic G418 (20). At its 3' end is the HSV-1 TK polyadenylylation site contained within a Sma I-Pvu II fragment (T).
RESULTS Experimental Strategy. To identify cellular factors specifically involved in transcriptional regulation, we have used a competition assay described by Scholar and Gruss (21) and Mercola et al. (22). Briefly, a test promoter, covalently linked to a gene whose product is readily monitored, is cotransfected into appropriate cell lines with increasing amounts of competitor DNA. The competitor sequence is covalently linked to a gene which does not interfere with the test gene. In this study, we have used the CAT gene as test and the NEO gene as competitor. If cellular factors controlling transcription of the test promoter are limiting, then increasing amounts of competitor should lead to a decrease in CAT activity. Transcription from the Major e-Globin Cap Site Is Specifically Up-Regulated 100- to 200-Fold after Transfection into K562 Cells. To ensure a valid comparison of promoter activity in different cell lines, two parameters of the assay must be established. First, it is necessary to determine the quantity of input promoter required to attain maximum CAT activity in each cell line. Second, to control for variable HB
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transfection efficiency in different cell lines, CAT activity of the test promoter must be expressed as a function of activity of a promoter whose activity is constant within a range of cell lines. The test promoter used in this study is a 200-bp BamHI-Pvu II fragment containing the E-globin major cap site, inserted by means of BamHI linkers into the promoterless CAT vector pCO (Materials and Methods and legend to Fig. 2). The control promoter is a 200-bp Sma I-Sau3A fragment containing the HSV-2 immediate early promoter (Materials and Methods and legend to Fig. 2). Increasing amounts of the constructs EP-CAT and IE-CAT (from 0.1 to 40 jig of input DNA per 75-cm2 flask) were introduced into the embryonic human erythroid cell line K562 (23) and into the human nonerythroid cell line HeLa. Fortyeight hours later, CAT activity was measured and plotted as a function of input DNA. The maximum level of CAT activity achieved by IE-CAT was given an arbitrary value of 100 in each cell line and levels of EP-CAT are expressed relative to this. After transfection into K562 cells (Fig. 3a), IE-CAT reaches saturation at 20 ,ug of input DNA, while eP-CAT reaches saturation at 5 ,ug of input DNA. This implies that a IE-CAT
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FIG. 2. Construction of CAT and NEO plasmids containing different promoters. The line drawings indicate the constructs used in competition assays. The promoterless CAT construct pCO contains the pUC9 multiple cloning site (mcs), the bacterial CAT gene, and an HSV-2 immediate early gene terminator element (T) inserted at the 3' end of the CAT gene (9). Test promoters were inserted, using BamHI linkers, into the BamHI site of pCO. The promoters used are (i) eP, a 200-bp BamHI-Pvu II fragment containing the E-globin major promoter (see Fig. 1); (ii) EP2, a 350-bpXba I-BamHI fragment containing the E-globin -200 bp promoter (see Fig. 1); (iii) IE, a 200-bp HSV-2 immediate early promoter (9, 17); and (iv) TK, an HSV-2 thymidine kinase promoter (18). The promoterless NEO construct pNEO contains the kanamycin-neomycin resistance gene coding for aminoglycoside 3'-phosphotransferase (3')-II from transposon Tn5 and is therefore resistant to the antibiotic G418 (19, 20). The EP, EP2, IE, and TK promoters were inserted by means of HindIII linkers into the HindIII site of pNEO.
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Developmental Biology: Wu et al. limiting levels of cellular trans-acting factors are present in K562 cells, sufficient to saturate 20 ,ug of IE-CAT and 5 ,ug of EP-CAT. In HeLa cells, IE-CAT reaches a maximum level of expression after transfection with 0.5 ,tg of DNA, while EP-CAT reaches maximum expression with 20 jig of input DNA (Fig. 3b), implying the presence of cellular factors sufficient to saturate 0.5 Ag of the IE construct and 20 ,ug of the E-globin construct. These results imply that, although the E-globin promoter can be transcribed in HeLa cells, the factor(s) mediating transcription are different from those interacting with the E-globin promoter in K562 cells. The finding that 40-fold higher input level of EP-CAT (compared with IE-CAT) are required to generate maximum promoter activity in HeLa cells suggests that the HeLa factor(s) interact only weakly with the E-globin promoter and therefore a large excess of --globin DNA is required to drive the reaction to completion. In HeLa cells, comparison of promoter activity at the linear part of the curve (up to an input of 0.5 Ag of DNA) indicates that IE generates approximately 100-fold more CAT activity than EP does. Conversely, in K562 cells (at input DNA levels up to 5 ,.g). EP generates 1.5-fold more CAT activity than IE does. Assuming that the factors responsible for IE expression are present at the same abundance in HeLa and K562 cells, it follows that the E-globin promoter is 150-fold more active in the embryonic erythroid K562 cell line than in HeLa cells. The experiment described in Fig. 3 has been repeated four times, using at least two independent DNA preparations. Similar results have also been obtained when the HSV-2 TK was used as control and when other nonerythroid cell lines such as Cos 7 and CV1 were used (not shown). To confirm that transcription from the EP-CAT construct did indeed originate from the correct E-globin initiation site, 20 ,g of EP-CAT was transfected into subconfluent K562 cells in a 75-cm2 flask; RNA was prepared 48 hr later and analyzed by S1 nuclease mapping using an end-labeled probe spanning the E-globin cap site. The 600-bp fragment extending from the HindIII site within the pUC9 polylinker to the EcoRI site within the CAT gene was gel purified and 5'-endlabeled with [y-32P]ATP and polynucleotide kinase, and the strands were separated (24). The single-stranded probe (shown in the line drawing of Fig. 4) was hybridized in probe excess at 57°C to 40 ,g of total RNA prepared from K562 cells transfected with EP-CAT. Hybrids were digested with 1000 units of S1 nuclease (Boehringer Mannheim) for 1.5 hr at 37°C and digestion products were separated on a denaturing 6% polyacrylamide gel. As shown in Fig. 4, a single digestion product 250 bp long is found, migrating exactly to the position expected for the fragment containing the E-globin major cap site. A similar S1 nuclease assay was performed on RNA derived from K562 cells after transfection with IE-CAT and, as shown in Fig. 4, a single band migrating to the predicted position of the immediate early promoter is found. Up-Regulation of the e-Globin Major Promoter by Interaction with Erythroid-Specific Activator(s). The experiments described above indicate that a 200-bp fragment containing the E-globin major promoter generated 150-fold higher levels of CAT activity after transfection into an embryonic erythroid cell line compared with any nonerythroid cell line tested. This could formally be due to (i) interaction of the E-globin promoter with erythroid-specific activator(s) or (ii) the specific absence in embryonic erythroid cells of repressor(s) present in all other cell types. Certain predictions can be made depending on which of these scenarios is correct. Thus, if the --globin promoter interacts with repressor in nonerythroid cells, competition with increasing amounts of itself should lead to an increase in transcription from the E-globin promoter in nonerythroid cells. If, on the other hand, the E-globin promoter interacts with erythroid-specific activator(s), self-competition in erythroid cells should lead to a
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decrease in transcription. To test these possibilities, we have cotransfected a fixed amount of EP-CAT with increasing amounts of eP-NEO in both K562 and HeLa cells. The fixed level of EP-CAT was that previously shown in Fig. 3 to produce maximum amounts of CAT activity (5 ug). As previously stated, at this level of input DNA, we assume that transcription factors interacting with the e-globin promoter are limiting. As shown in Fig. 5a, when 5 gg of EP-CAT is cotransfected with increasing amounts of eP-NEO, CAT activity decreases after addition of 5 ,g of EP-NEO and continues to decrease up to the maximum of 40 ,tg of input eP-NEO. To control for the possibility that increasing amounts of eP-NEO may compete for general transcription factors, EP-CAT was cotransfected with increasing amount of TK-NEO. As shown in Fig. 5a, at the highest levels of input TK-NEO (30 and 40 /ig) a slight decrease in CAT activity is found, probably indicating competition for general transcription factors. However, this curve is unlike the competition seen with OP-NEO, and we therefore suggest that the specific increase in transcription of the e-globin promoter after transfection into K562 cells is the direct result of interaction of cis-acting elements contained on this 200-bp fragment with erythroid-specific activator(s). This is further supported by the results shown in Fig. 5b, where competition of EP-CAT with EP-NEO was carried out in HeLa cells. In this case, only a slight reduction in CAT activity was observed at the highest levels of input competitor. Major E-Globin Promoter and -200 bp Promoter Interact with the Same Erythroid-Specific "Trans" Factor(s). We have used competition assays to determine whether the e-globin
Proc. Natl. Acad. Sci. USA 87 (1990)
Developmental Biology: Wu et al.
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major promoter (EP) and -200 bp promoter (0P2) can compete for the same erythroid-specific regulatory factor(s). K562 cells were transfected with increasing amounts of EP2-CAT (0.1-40 ,ug per 75-cm2 flask) to determine the level of input DNA at which maximum CAT activity was reached exactly as described in Fig. 3. Saturation was reached at 7.5 ,ug of input DNA (not shown). A fixed level of 7.5 ,ug of eP2-CAT was cotransfected with increasing amounts of EPNEO into K562 cells. As shown in Fig. 6a, as EP-NEO increases from 5 to 40 ,ug, CAT correspondingly increases, up to the maximum at 40 ,ug of input EP-NEO. It was not possible to increase EP-NEO levels further because cell death occurred at higher input. No change in CAT activity is seen when TK-NEO is used as competitor (Fig. 6a) or when this competition is carried out in HeLa cells (Fig. 6b). These results suggest that the EP fragment competes for an erythroid-specific factor which down-regulates transcription from EP2, the E-globin -200 bp promoter. S1 nuclease analysis confirmed that transcripts originated at the predicted position of the -200 bp promoter (Fig. 4). The reverse competition experiment, in which a fixed amount, 5 ,ug, of EP-CAT is cotransfected into K562 cells with increasing amounts of EP2-NEO, is shown in Fig. 7a. CAT activity derived from the major e-globin promoter decreases after competition with 5 ,ug of eP2-NEO and continues to decrease up to 30 ,ug of competitor DNA, indicating that EP2 competes for erythroid-specific factor(s) which up-regulate transcription from the major promoter. This competition is not seen when TK-NEO is used as competitor (Fig. Sa). The slight decrease in CAT activity seen at the highest level of eP2-NEO in HeLa cells (Fig. 7b) is not significantly different from that found with TK-NEO as
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Developmental Biology: Wu et al. DISCUSSION The human E-globin gene demonstrates a high degree of tissue specificity and developmental stage specificity of expression. This specificity can be somewhat mimicked by transfection experiments, since 100-fold greater transcription from the E-globin major cap site is found after transfection into K562 cells. In the current study, we have used a competition assay to determine the nature of the putative interaction(s) of the E-globin -200 bp and major promoters, in isolation, with erythroid-specific trans factor(s). Comparison of activity of the E-globin and intermediate early promoters within the linear range of the expression curve in K562 and HeLa cells (Fig. 3) indicates that the E-globin promoter is 150-fold more active in K562 cells than in HeLa cells. This conclusion makes the assumption that the factors responsible for IE transcription are present at the same abundance in both cell lines. We have proceeded on the assumption that either the K562 cell line possesses specific activator(s) which interact with element(s) within the 200-bp BamHI-Pvu II promoter fragment or the K562 cells specifically lack factor(s) which down-regulate this promoter in all other cell lines. The results shown in Fig. 5 strongly support the former hypothesis. Thus competition of 5 ,ug of EP-CAT (the level at which transacting factors are limiting in K562 cells) with increasing amounts of EP-NEO leads to a gradual reduction in CAT activity to approximately 10% of baseline. This reduction in CAT activity after self-competition suggests that the EP fragment interacts mainly with transcription activator(s) and when these factors are present in limiting amounts, selfcompetition leads to a reduction of transcription from the major E-globin promoter. Such a decrease is unlikely to result from competition for general transcription factors, since it is not seen either when TK-CAT is used as competitor or when the competition is carried out in HeLa cells (Fig. 5). S1 nuclease analysis (Fig. 4) indicates that initiation of transcription occurs from the correct E-globin cap site. The different kinetics of competition observed with EP-NEO and TK-NEO may suggest that different classes of factors are involved and that the slight reduction with CAT activity at the highest levels of input TK-NEO may reflect competition for general transcription factors required by all promoters. It is not possible to distinguish by this assay whether one or more trans-acting factors are involved in the erythroid-specific up-regulation of the E-globin promoter, and it is conceivable that the overall positive response is the summation of interactions with multiple positive and negative factors. Similarly, this assay does not indicate whether the same factor(s) is limiting in K562 and HeLa cells. Erythroid-specific factors have been directly demonstrated binding to the a-globin and /3-globin promoter regions (25-27), and work from these laboratories suggests that multiple factors may be involved. Previous experiments have indicated that cotransfection of the e-globin gene, in nonerythroid cells, with the adenovirus EIA gene leads to a 20- to 30-fold increase in transcription from the major cap site and a corresponding decrease from the -200 cap site (11). A similar switch in transcription from the -200 cap site to the major cap site is seen after transfection into the K562 cell line (19) and during induction of K562 cells with hemin (an event thought to be equivalent to normal red blood cell differentiation), suggesting that an erythroid equivalent of EIA may exist. The switch of transcription from the -200 cap site to the major cap site during erythroid differentiation is apparently a critical stage in activation of the gene. Recently Cao et al. (8) have demonstrated that the region between -177 to -392 relative to the e-globin cap site contains an element ('silencer") which down-regulates transcription from the e-globin cap site. Down-regulation is more pronounced in nonerythroid cells
Proc. Natl. Acad. Sci. USA 87 (1990)
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than in erythroid cells. The results shown in Fig. 6 indicate that cotransfection in K562 cells of a fixed (saturation) amount of EP2-CAT with increasing amounts of EP-NEO leads to an increase in CAT activity, suggesting that the EP fragment competes for an erythroid-specific factor(s) which represses transcription from the -200 bp promoter. The reverse competition, shown in Fig. 7 (fixed amount of EPCAT, increasing amounts of eP2-NEO), results in a reduction in CAT activity, suggesting that the EP2 fragment competes for a factor(s) which up-regulates transcription from the E-globin major cap site. These results suggest, but do not prove, that the same erythroid-specific factor may upregulate the major cap site and down-regulate the -200 cap site. Such a factor would bear a strong resemblance to the adenovirus EIA gene product. Thus, at least two of the multiple E-globin control elements may share regulatory trans-factors, and these factors may perform opposite functions when bound to different elements. We thank Nick Shelness for assistance in preparing the manuscript. Support for this research was provided by National Institutes
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