Vol. 10, No. 2
MOLECULAR AND CELLULAR BIOLOGY, Feb. 1990, p. 850-853 0270-7306/90/020850-04$02.00/0
Delineation of an Enhancerlike Positive Regulatory Element in the Interleukin-2 Receptor oc-Chain Gene BERTHA B. LIN, SHARON L. CROSS, NANCY F. HALDEN, DRAGOS G. ROMAN, MICHEL B. TOLEDANO, AND WARREN J. LEONARD* Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892 Received 31 July 1989/Accepted 27 October 1989
We have delineated a positive regulatory element in the interleukin-2 receptor a-chain gene (IL-2Ra) between positions -299 and -243 that can potently activate a heterologous (herpesvirus thymidine kinase [tk]) promoter in phorbol myristate acetate (PMA)-induced Jurkat T cells and is functional when cloned in either orientation. This enhancerlike element contains a site (-268/-257) that can bind NF-KB; however, unlike the immunoglobulin K gene KB enhancer element, the IL-2Ra KB-like site alone can only weakly activate a heterologous promoter. Adjacent 5' and 3' sequences also weakly activate the tk-CAT vector, but constructs combining the IL-2Ra KB-like site plus adjacent 5' and 3' sequences potently activate gene expression. This combination of regions is essential for potent PMA-induced transcription from the tk promoter. Experiments using constructs in which IL-2Ra upstream sequences are sequentially deleted suggested that there is a region 5' of position -299 which can suppress IL-2Ra promoter and/or enhancer activity. Thus, it is possible that both positive and negative elements may be important in the regulation of IL-2Ra gene transcription.
nested 5' endpoints beginning at position -472 were tested for the ability to activate the thymidine kinase (tk) promoter in PMA-induced Jurkat cells. Recombinant constructs were made by digesting 5' and 3' deletion constructs with XbaI and XmnI. Fragments were isolated by gel electrophoresis, joined at their common XmnI site at position -228, and cloned into the XbaI site in pBLCAT2 (a tk-chloramphenicol acetyltransferase [CAT] vector [11]). Plasmid DNAs were purified on two successive cesium chloride gradients and transfected with DEAE-dextran as previously described (3). Cell extracts were assayed for CAT activity at 37°C for 1 to 4 h by using acetyl coenzyme A (Pharmacia) and 14Cchloramphenicol (New England Nuclear Corp.), analyzed on silica gel 1B thin-layer chromatography plates (J. T. Baker) (5), and subjected to autoradiography. The spots corresponding to nonacetylated and acetylated chloramphenicol were excised and counted by liquid scintillation. Activities relative to pBLCAT2 were determined (Fig. 1). Although the -472/-136 and -430/-136 constructs induced tk promoter activity less than twofold, deletion of sequences between positions -430 and -299 significantly increased tk-CAT activity. In the positive orientation, this effect was most dramatic with deletions of sequences between positions -311 and -299, suggesting that these sequences can negatively influence tk-CAT expression. Since these transfections are in PMA-induced Jurkat cells, a setting in which IL-2Ra expression is induced, the physiologic significance of such negative effects in IL-2Ra gene regulation is unclear. The -228/-136 construct only minimally activated tk-CAT. Thus, deletion of sequences between positions -299 and -228 virtually abolished tk-CAT activity, indicating that these sequences constitute a positive regulatory region. Since sequences 5' of position -299 were not essential for positive regulatory activity, we examined constructs with a fixed 5' endpoint at position -299 and nested 3' endpoints in order to define a more limited sequence capable of mediating this activity (Fig. 1, 3' Deletions). The -299/-228 and -299/-243 constructs were able to induce tk-CAT activity approximately 15- to 20-fold in PMA-induced Jurkat cells,
The interaction of interleukin-2 (IL-2) with high-affinity IL-2 receptors on stimulated T cells represents a critical control mechanism in T-cell proliferation. High-affinity IL-2 receptors contain both a and ,B chains (14, 16; W. J. Leonard and M. Sharon, in J. Cossman, ed., The Molecular Genetics of Cancer Diagnosis, in press). a chains (IL-2Ra, p55, or Tac antigen) are not expressed on resting T cells but are induced after activation with mitogen or antigen (17) or after infection with human T-cell lymphotropic virus type I (HTLV-I) (6, 18). a chains can bind IL-2 with low affinity or can associate with ,B chains to form high-affinity IL-2 receptors (reviewed in Leonard and Sharon, in press). IL-2Ra gene expression is significantly regulated at the level of transcription initiation (7, 8). Experiments using 5' deletion mutations have indicated the importance of sequences between positions -327 and -244 for IL-2Ra promoter activity (3). This region contains a KB-like site (between positions -268 and -257) which can bind phorbol myristate acetate (PMA)-inducible factor(s) in PMA-induced Jurkat extracts and also can bind purified nuclear factor NF-KB (2, 4, 9, 13). Although the KB-like site is important in HTLV-I/Taxinduced transcription regulation of the IL-2Ra gene (1, 4, 9), its role in PMA-induced activation is controversial (2, 4, 9, 10). In PMA-stimulated Jurkat cells, transiently transfected constructs containing the immunoglobulin KB site linked to a heterologous promoter manifest high levels of activity (12). In contrast, the IL-2Ra KB site flanked by immunoglobulin K gene sequences does not significantly activate expression from a heterologous promoter (4). In this study, we used a heterologous promoter to define an enhancerlike element in the IL-2Ra gene. Maximal activity and PMA inducibility were found in constructs which include sequences both 5' and 3' of the KB site. Defining the boundaries of a positive regulatory element. In order to determine the boundaries of a positive regulatory element in the 5' upstream sequence of the IL-2Ra gene, constructs with a fixed 3' endpoint at position -136 and *
Corresponding author. 850
NOTES
VOL. 10, 1990
-1136
a472 IL-2R1 alpha Lm.
851
Jurkat + PMA Mean Rel Activity (#expt)
+1,09
Orientation
2';I
r p TK CAT : t12 pBLCAT 2. jlIJ
(+)
(-)
1.0
1.0
1.8 (7)
1.5 (5)
1.6 (2)
1.1 (6)
1.7 (2)
3.3 (5)
1.4 (2)
8.3 (6)
11.5 (3)
26.5 (7)
0.9 (2)
1.9 (4)
11.5 (3)
26.5 (7)
ND
19.0 (2)
5' Deletions
-472/-136 [ -430/-136 -369/-136 -311/-136
-299/-136 -228/-136 3' Deletions -299/-136 -299/-186
El ]I lI KIl ]I~l EIli]I I EII ]I:xwsws lI:*, . .>is.B ~~~~II~~~~~~~~~~z E~l0Z ilILI S>-
16.7 (4) llW El ~ R~~~~~~r>[; IIi R-U L~~~~~~~~~~~~~{SE>.IIII1..a.~f.. 14.8 (3) fsy
1
-299/-228 -299/-243 -299/-254 -299/-269
19.8 (10) 19.9 (4)
a
ND
10.6 (9)
5.1 (4)
3.4 (5)
FIG. 1. Summary of IL-2Ra 5' and 3' deletion constructs with tk-CAT in PMA-induced Jurkat T cells. Average relative activities of IL-2Ra tk-CAT constructs are compared with that of pBLCAT2 (no IL-2Ra insert), which is assigned a value of 1.0. (+) and (-), The orientation of the IL-2Ra insert in pBLCAT2. The number of times each construct was tested is indicated in parentheses.
while the -299/-254 and -299/-269 constructs had activities of approximately 11- and 4-fold, respectively. In the positive orientation, the activities of the -299/-228 and -299/-243 constructs were comparable to each other and were more potent than that of the -299/-136 construct. These results indicate that the 57-base-pair IL-2Rcx fragment, -299/-243, is sufficient for potent activation of a heterologous promoter in PMA-induced Jurkat T cells. To further study the -299 to -228 region, oligonucleotides corresponding to sequences within the region were prepared on an Applied Biosystems, Inc., 381A DNA synthesizer with XbaI-compatible ends, cloned into the XbaI site of pBLCAT2, and tested for the ability to activate tk-CAT expression (Fig. 2). A construct containing only sequences 5' of the KB site (-299/-269) increased tk-CAT activity approximately fourfold in PMA-induced Jurkat cells, whereas constructs containing the KB site (-268/-254) or sequences 3' of the KB site (-256/-228) increased activity approximately two- and threefold, respectively. Constructs spanning more than one of these smaller regions, -299/-254 (5' + KB) and -268/-243 (KB + 3'), increased CAT activity approximately 11- and 4-fold, respectively. The -299/-243 construct (5' + KB + 3') induced transcription approximately 15- to 20-fold and is similar in activity to the -299/ -228 construct. These data suggest that the sequences required for maximal activity of the -299/-243 fragment are not localized to a discrete element within this region but require the presence of sequences both 5' and 3' of the KB-like site.
Only constructs containing sequences both 5' and 3' of the NF-KB site were significantly PMA-inducible. The activities of these constructs were next evaluated in the presence and absence of PMA stimulation (Fig. 3). All of the constructs tested, including the vector control, pBLCAT2, displayed
increased activity in PMA-induced Jurkat cells. The IL-2Ra KB-like site alone conferred only a negligible increase in activity, even though NF-KB DNA binding activity is PMA inducible. Constructs spanning the KB site plus 5' sequences (-299/-254) or only 5' sequences (-299/-269) were not significantly PMA inducible. The two longest constructs, -299/-228 and -299/-243, which contain the KB-like site and sequences both 5' and 3' of the site, showed PMA inducibility significantly greater than that seen with pBLCAT2 alone, even though constructs with the KB site, 5' sequences (Fig. 3), and 3' sequences (4) by themselves were not significantly PMA inducible. This result suggests that these three regions may have an additive or synergistic effect for PMA induction of IL-2Ra expression. The positive regulatory activity of these constructs was not strictly dependent on PMA in that some constructs (e.g., -299/-243) significantly increased basal tk-CAT activity even in the absence of PMA. Negative orientation data are shown since we did not obtain the -299/-254 construct in the positive orientation; however, when the -299/-228 and -299/-269 positive-orientation constructs were tested with and without PMA, virtually identical results to those shown in Fig. 3 were obtained. Previous studies have indicated that the presence of
852
NOTES
MOL. CELL. BIOL.
Jurkat PMA Rel Activity (#expt) Mean +109
IL-2R alpha Promoter
+
-299 -228
-472
Orientation -268 -257 -2
-2
l
l
l
8
l
(+)
TK
CAT
N
s
pBLCAT 2
L
-299/-228
-299/-2691
rB|E
|
LII a
-299/-2541
1
-2681-243
L
___
1
l
1III
1.0
16.7 (4)
19.8 (10)
(4) 2.7 (3) 4.0 (1)
3.4 (5)
ND
10.6 (9)
3.3(1) 14.8 (3)
4.5(3) 19.9 (4)
5.1
L
-268/-254 -256/-228
-299/-2431
1.0
( )
2.0 (4)
2.3 (3)
FIG. 2. Relative activities of IL-2Rot tk-CAT constructs in PMA-induced Jurkat T cells. Average relative activities of IL-2Ra tk-CAT constructs are compared with that of pBLCAT2, which is assigned a value of 1.0. (+) and (-), The orientation of the IL-2Ra insert in pBLCAT2. The number of times each construct was tested is indicated in parentheses.
sequences upstream of position -347 decreased IL-2RaCAT activity in HTLV-I-infected cells but did not appear to influence IL-2Rco expression in PMA-induced Jurkat cells (3). We now show that IL-2Ra sequences between positions -430 and -299 (Fig. 1, in the positive orientation, particularly the sequences between positions -311 and -299) can diminish tk-CAT activity in PMA-induced Jurkat cells. It is conceivable that negative regulation may play an important 100
80-
-PMA U +PMA
C
o
60
2
0
0
E
L
20
co
0D
CO)
cm
cm
V cm
0 N
N~
m0
0)
0m
CD
a)
FIG. 3. Effect of PMA on IL-2Ra -299/-228 subclones in Jurkat T cells. Percent '4C-chloramphenicol conversion for the IL-2RcX constructs in the absence or presence of PMA stimulation is shown. Percent conversion (average of two independent experiments with similar results) values without and with PMA are as follows: pBLCAT2, 2.9 and 6.7%; -299/-228(-), 13.7 and 69.4%; -299/ -243(-), 20.8 and 85.5%; -299/-254(-), 15.5 and 30.5%; -299/ -269(-), 16.1 and 18.7%; -268/-254(-), 4.7 and 8.5%.
role in controlling the activation and cell-type specific transcription of the IL-2Ra gene, although the elements involved in such negative regulation may not be identical in different cell types and require further investigation. Using nested 5' and 3' deletion constructs, we have identified a region in the IL-2Rot gene, -299/-243, that potently activates transcription from a heterologous promoter in PMA-induced Jurkat cells. This finding is consistent with a previous study defining a positive element in a large IL-2Ra fragment (15). Here, we have more narrowly delineated a positive regulatory region and analyzed the component parts of this region and their PMA inducibility. In addition to an NF-KB-binding site, electrophoretic mobility shift analyses have identified other DNA-binding protein recognition sites in this region (M. B. Toledano et al., submitted for publication). These include (i) a serum-responsive element (SRE-like sequence) (-253/-244) 3' of the KB site and (ii) a site 5' to the KB site between positions -290 and -269. Since the IL-2Ra KB site and adjacent 5' and 3' sequences together are required for potent PMA-induced activation of a heterologous promoter, these additional nuclear factor binding regions may be required for maximal positive regulatory activity. Thus, we have defined an enhancerlike element in the IL-2Rot gene. Maximal PMA inducibility requires the combination of adjacent elements to obtain an additive or synergistic effect. Further elucidation of both the positive element and the potential negative regulatory regions noted above should help in understanding the precise and exquisitely controlled cell-type-specific expression of the IL-2Ra gene. B.B.L. was the recipient of a Howard Hughes Medical Institute Research Scholar award. M.B.T. was the recipient of a grant from the Ligue Nationale Franraise Contre le Cancer. S.L.C. was supported by a fellowship from the Cancer Research Institute of New York. This work was supported in part by a Public Health Service Intramural AIDS grant from the National Institutes of Health. We thank Richard D. Klausner and Kuan-Teh Jeang for critical comments.
NOTES
VOL. 10, 1990
LITERATURE CITED 1. Ballard, D. W., E. Bdhnlein, J. W. Lowenthal, Y. Wano, B. R. Franza, and W. C. Greene. 1988. HTLV-I Tax induces cellular proteins that activate the KB element in the IL-2 receptor a gene. Science 241:1652-1655. 2. Bohnlein, E., J. W. Lowenthal, M. Siekevitz, D. W. Ballard, B. R. Franza, and W. C. Greene. 1988. The same inducible nuclear proteins regulates mitogen activation of both the interleukin-2 receptor-alpha gene and type 1 HIV. Cell 53:827-836. 3. Cross, S. L., M. B. Feinberg, J. B. Wolf, N. J. Holbrook, F. Wong-Staal, and W. J. Leonard. 1987. Regulation of the human interleukin-2 receptor a chain promoter: activation of a nonfunctional promoter by the transactivator gene of HTLV-I. Cell 49:47-56. 4. Cross, S. L., N. F. Halden, M. J. Lenardo, and W. J. Leonard. 1989. Functionally distinct NF-KB binding sites in the immunoglobulin K and IL-2 receptor a chain genes. Science 244: 466-469. 5. Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell Biol. 2:1044-1051. 6. Kronke, M., W. J. Leonard, J. M. Depper, and W. C. Greene. 1985. Deregulation of interleukin-2 receptor gene expression in HTLV-I induced adult T-cell leukemia. Science 228:1215-1217. 7. Leonard, W. J., J. M. Depper, G. R. Crabtree, M. Kronke, P. B. Svetlik, N. J. Peffer, T. A. Waldmann, and W. C. Greene. 1984. Molecular cloning and expression of cDNAs for the human interleukin-2 receptor. Nature (London) 311:626-631. 8. Leonard, W. J., M. Kronke, N. J. Peffer, J. M. Depper, and W. C. Greene. 1985. Interleukin-2 receptor gene expression in normal human T-lymphocytes. Proc. Natl. Acad. Sci. USA 82:6281-6485. 9. Leung, K., and G. Nabel. 1988. HTLV-I transactivator induces interleukin-2 receptor expression through an NF-KB-like factor. Nature (London) 333:776-778. 10. Lowenthal, J. W., D. W. Ballard, E. Bohnlein, and W. C. Greene. 1989. Tumor necrosis factor a induces proteins that
11.
12.
13.
14.
15.
16.
17.
18.
853
bind specifically to KB-like enhancer elements and regulate interleukin-2 receptor alpha-chain gene expression in primary human T-lymphocytes. Proc. Natl. Acad. Sci. USA 86:23312335. Luckow, B., and G. Schutz. 1987. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 15:5490. Pierce, J. W., M. Lenardo, and D. Baltimore. 1988. Oligonucleotide that binds nuclear factor NF-KB acts as a lymphoidspecific and inducible enhancer element. Proc. Natl. Acad. Sci. USA 85:1482-1486. Ruben, S., H. Poteat, T. H. Tan, K. Kawakami, R. Roeder, W. Haseltine, and C. Rosen. 1988. Cellular transcription factors and regulation of IL-2 receptor gene expression by HTLV-I tax gene product. Science 241:89-92. Sharon, M., R. D. Klausner, B. R. Cullen, R. Chizzonite, and W. J. Leonard. 1986. Novel interleukin-2 receptor subunit detected by cross-linking under high-affinity conditions. Science 234:859-863. Suzuki, N., N. Matsunami, H. Kanamori, N. Ishida, A. Shimizu, Y. Yaoita, T. Nikaido, and T. Honjo. 1987. The human IL-2 receptor gene contains a positive regulatory element that functions in cultured cells and cell-free extracts. J. Biol. Chem. 262:5079-5086. Tsudo, M., R. W. Kozak, C. K. Goldman, and T. A. Waldmann. 1986. Demonstration of a non-Tac peptide that binds interleukin-2: a potential participant in a multichain interleukin-2 receptor complex. Proc. Natl. Acad. Sci. USA 83:9694-9698. Uchiyama, T., S. Broder, and T. A. Waldmann. 1981. A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T-cells. I. Production of anti-Tac monoclonal antibody and distribution of Tac (+) cells. J. Immunol. 126: 1393-1397. Wong-Staal, F., and R. C. Gallo. 1985. Human T-lymphotropic retroviruses. Nature (London) 317:395-403.
MOLECULAR AND CELLULAR BIOLOGY, Feb. 1990, p. 850-853 0270-7306/90/020850-04$02.00/0
Delineation of an Enhancerlike Positive Regulatory Element in the Interleukin-2 Receptor oc-Chain Gene BERTHA B. LIN, SHARON L. CROSS, NANCY F. HALDEN, DRAGOS G. ROMAN, MICHEL B. TOLEDANO, AND WARREN J. LEONARD* Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892 Received 31 July 1989/Accepted 27 October 1989
We have delineated a positive regulatory element in the interleukin-2 receptor a-chain gene (IL-2Ra) between positions -299 and -243 that can potently activate a heterologous (herpesvirus thymidine kinase [tk]) promoter in phorbol myristate acetate (PMA)-induced Jurkat T cells and is functional when cloned in either orientation. This enhancerlike element contains a site (-268/-257) that can bind NF-KB; however, unlike the immunoglobulin K gene KB enhancer element, the IL-2Ra KB-like site alone can only weakly activate a heterologous promoter. Adjacent 5' and 3' sequences also weakly activate the tk-CAT vector, but constructs combining the IL-2Ra KB-like site plus adjacent 5' and 3' sequences potently activate gene expression. This combination of regions is essential for potent PMA-induced transcription from the tk promoter. Experiments using constructs in which IL-2Ra upstream sequences are sequentially deleted suggested that there is a region 5' of position -299 which can suppress IL-2Ra promoter and/or enhancer activity. Thus, it is possible that both positive and negative elements may be important in the regulation of IL-2Ra gene transcription.
nested 5' endpoints beginning at position -472 were tested for the ability to activate the thymidine kinase (tk) promoter in PMA-induced Jurkat cells. Recombinant constructs were made by digesting 5' and 3' deletion constructs with XbaI and XmnI. Fragments were isolated by gel electrophoresis, joined at their common XmnI site at position -228, and cloned into the XbaI site in pBLCAT2 (a tk-chloramphenicol acetyltransferase [CAT] vector [11]). Plasmid DNAs were purified on two successive cesium chloride gradients and transfected with DEAE-dextran as previously described (3). Cell extracts were assayed for CAT activity at 37°C for 1 to 4 h by using acetyl coenzyme A (Pharmacia) and 14Cchloramphenicol (New England Nuclear Corp.), analyzed on silica gel 1B thin-layer chromatography plates (J. T. Baker) (5), and subjected to autoradiography. The spots corresponding to nonacetylated and acetylated chloramphenicol were excised and counted by liquid scintillation. Activities relative to pBLCAT2 were determined (Fig. 1). Although the -472/-136 and -430/-136 constructs induced tk promoter activity less than twofold, deletion of sequences between positions -430 and -299 significantly increased tk-CAT activity. In the positive orientation, this effect was most dramatic with deletions of sequences between positions -311 and -299, suggesting that these sequences can negatively influence tk-CAT expression. Since these transfections are in PMA-induced Jurkat cells, a setting in which IL-2Ra expression is induced, the physiologic significance of such negative effects in IL-2Ra gene regulation is unclear. The -228/-136 construct only minimally activated tk-CAT. Thus, deletion of sequences between positions -299 and -228 virtually abolished tk-CAT activity, indicating that these sequences constitute a positive regulatory region. Since sequences 5' of position -299 were not essential for positive regulatory activity, we examined constructs with a fixed 5' endpoint at position -299 and nested 3' endpoints in order to define a more limited sequence capable of mediating this activity (Fig. 1, 3' Deletions). The -299/-228 and -299/-243 constructs were able to induce tk-CAT activity approximately 15- to 20-fold in PMA-induced Jurkat cells,
The interaction of interleukin-2 (IL-2) with high-affinity IL-2 receptors on stimulated T cells represents a critical control mechanism in T-cell proliferation. High-affinity IL-2 receptors contain both a and ,B chains (14, 16; W. J. Leonard and M. Sharon, in J. Cossman, ed., The Molecular Genetics of Cancer Diagnosis, in press). a chains (IL-2Ra, p55, or Tac antigen) are not expressed on resting T cells but are induced after activation with mitogen or antigen (17) or after infection with human T-cell lymphotropic virus type I (HTLV-I) (6, 18). a chains can bind IL-2 with low affinity or can associate with ,B chains to form high-affinity IL-2 receptors (reviewed in Leonard and Sharon, in press). IL-2Ra gene expression is significantly regulated at the level of transcription initiation (7, 8). Experiments using 5' deletion mutations have indicated the importance of sequences between positions -327 and -244 for IL-2Ra promoter activity (3). This region contains a KB-like site (between positions -268 and -257) which can bind phorbol myristate acetate (PMA)-inducible factor(s) in PMA-induced Jurkat extracts and also can bind purified nuclear factor NF-KB (2, 4, 9, 13). Although the KB-like site is important in HTLV-I/Taxinduced transcription regulation of the IL-2Ra gene (1, 4, 9), its role in PMA-induced activation is controversial (2, 4, 9, 10). In PMA-stimulated Jurkat cells, transiently transfected constructs containing the immunoglobulin KB site linked to a heterologous promoter manifest high levels of activity (12). In contrast, the IL-2Ra KB site flanked by immunoglobulin K gene sequences does not significantly activate expression from a heterologous promoter (4). In this study, we used a heterologous promoter to define an enhancerlike element in the IL-2Ra gene. Maximal activity and PMA inducibility were found in constructs which include sequences both 5' and 3' of the KB site. Defining the boundaries of a positive regulatory element. In order to determine the boundaries of a positive regulatory element in the 5' upstream sequence of the IL-2Ra gene, constructs with a fixed 3' endpoint at position -136 and *
Corresponding author. 850
NOTES
VOL. 10, 1990
-1136
a472 IL-2R1 alpha Lm.
851
Jurkat + PMA Mean Rel Activity (#expt)
+1,09
Orientation
2';I
r p TK CAT : t12 pBLCAT 2. jlIJ
(+)
(-)
1.0
1.0
1.8 (7)
1.5 (5)
1.6 (2)
1.1 (6)
1.7 (2)
3.3 (5)
1.4 (2)
8.3 (6)
11.5 (3)
26.5 (7)
0.9 (2)
1.9 (4)
11.5 (3)
26.5 (7)
ND
19.0 (2)
5' Deletions
-472/-136 [ -430/-136 -369/-136 -311/-136
-299/-136 -228/-136 3' Deletions -299/-136 -299/-186
El ]I lI KIl ]I~l EIli]I I EII ]I:xwsws lI:*, . .>is.B ~~~~II~~~~~~~~~~z E~l0Z ilILI S>-
16.7 (4) llW El ~ R~~~~~~r>[; IIi R-U L~~~~~~~~~~~~~{SE>.IIII1..a.~f.. 14.8 (3) fsy
1
-299/-228 -299/-243 -299/-254 -299/-269
19.8 (10) 19.9 (4)
a
ND
10.6 (9)
5.1 (4)
3.4 (5)
FIG. 1. Summary of IL-2Ra 5' and 3' deletion constructs with tk-CAT in PMA-induced Jurkat T cells. Average relative activities of IL-2Ra tk-CAT constructs are compared with that of pBLCAT2 (no IL-2Ra insert), which is assigned a value of 1.0. (+) and (-), The orientation of the IL-2Ra insert in pBLCAT2. The number of times each construct was tested is indicated in parentheses.
while the -299/-254 and -299/-269 constructs had activities of approximately 11- and 4-fold, respectively. In the positive orientation, the activities of the -299/-228 and -299/-243 constructs were comparable to each other and were more potent than that of the -299/-136 construct. These results indicate that the 57-base-pair IL-2Rcx fragment, -299/-243, is sufficient for potent activation of a heterologous promoter in PMA-induced Jurkat T cells. To further study the -299 to -228 region, oligonucleotides corresponding to sequences within the region were prepared on an Applied Biosystems, Inc., 381A DNA synthesizer with XbaI-compatible ends, cloned into the XbaI site of pBLCAT2, and tested for the ability to activate tk-CAT expression (Fig. 2). A construct containing only sequences 5' of the KB site (-299/-269) increased tk-CAT activity approximately fourfold in PMA-induced Jurkat cells, whereas constructs containing the KB site (-268/-254) or sequences 3' of the KB site (-256/-228) increased activity approximately two- and threefold, respectively. Constructs spanning more than one of these smaller regions, -299/-254 (5' + KB) and -268/-243 (KB + 3'), increased CAT activity approximately 11- and 4-fold, respectively. The -299/-243 construct (5' + KB + 3') induced transcription approximately 15- to 20-fold and is similar in activity to the -299/ -228 construct. These data suggest that the sequences required for maximal activity of the -299/-243 fragment are not localized to a discrete element within this region but require the presence of sequences both 5' and 3' of the KB-like site.
Only constructs containing sequences both 5' and 3' of the NF-KB site were significantly PMA-inducible. The activities of these constructs were next evaluated in the presence and absence of PMA stimulation (Fig. 3). All of the constructs tested, including the vector control, pBLCAT2, displayed
increased activity in PMA-induced Jurkat cells. The IL-2Ra KB-like site alone conferred only a negligible increase in activity, even though NF-KB DNA binding activity is PMA inducible. Constructs spanning the KB site plus 5' sequences (-299/-254) or only 5' sequences (-299/-269) were not significantly PMA inducible. The two longest constructs, -299/-228 and -299/-243, which contain the KB-like site and sequences both 5' and 3' of the site, showed PMA inducibility significantly greater than that seen with pBLCAT2 alone, even though constructs with the KB site, 5' sequences (Fig. 3), and 3' sequences (4) by themselves were not significantly PMA inducible. This result suggests that these three regions may have an additive or synergistic effect for PMA induction of IL-2Ra expression. The positive regulatory activity of these constructs was not strictly dependent on PMA in that some constructs (e.g., -299/-243) significantly increased basal tk-CAT activity even in the absence of PMA. Negative orientation data are shown since we did not obtain the -299/-254 construct in the positive orientation; however, when the -299/-228 and -299/-269 positive-orientation constructs were tested with and without PMA, virtually identical results to those shown in Fig. 3 were obtained. Previous studies have indicated that the presence of
852
NOTES
MOL. CELL. BIOL.
Jurkat PMA Rel Activity (#expt) Mean +109
IL-2R alpha Promoter
+
-299 -228
-472
Orientation -268 -257 -2
-2
l
l
l
8
l
(+)
TK
CAT
N
s
pBLCAT 2
L
-299/-228
-299/-2691
rB|E
|
LII a
-299/-2541
1
-2681-243
L
___
1
l
1III
1.0
16.7 (4)
19.8 (10)
(4) 2.7 (3) 4.0 (1)
3.4 (5)
ND
10.6 (9)
3.3(1) 14.8 (3)
4.5(3) 19.9 (4)
5.1
L
-268/-254 -256/-228
-299/-2431
1.0
( )
2.0 (4)
2.3 (3)
FIG. 2. Relative activities of IL-2Rot tk-CAT constructs in PMA-induced Jurkat T cells. Average relative activities of IL-2Ra tk-CAT constructs are compared with that of pBLCAT2, which is assigned a value of 1.0. (+) and (-), The orientation of the IL-2Ra insert in pBLCAT2. The number of times each construct was tested is indicated in parentheses.
sequences upstream of position -347 decreased IL-2RaCAT activity in HTLV-I-infected cells but did not appear to influence IL-2Rco expression in PMA-induced Jurkat cells (3). We now show that IL-2Ra sequences between positions -430 and -299 (Fig. 1, in the positive orientation, particularly the sequences between positions -311 and -299) can diminish tk-CAT activity in PMA-induced Jurkat cells. It is conceivable that negative regulation may play an important 100
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FIG. 3. Effect of PMA on IL-2Ra -299/-228 subclones in Jurkat T cells. Percent '4C-chloramphenicol conversion for the IL-2RcX constructs in the absence or presence of PMA stimulation is shown. Percent conversion (average of two independent experiments with similar results) values without and with PMA are as follows: pBLCAT2, 2.9 and 6.7%; -299/-228(-), 13.7 and 69.4%; -299/ -243(-), 20.8 and 85.5%; -299/-254(-), 15.5 and 30.5%; -299/ -269(-), 16.1 and 18.7%; -268/-254(-), 4.7 and 8.5%.
role in controlling the activation and cell-type specific transcription of the IL-2Ra gene, although the elements involved in such negative regulation may not be identical in different cell types and require further investigation. Using nested 5' and 3' deletion constructs, we have identified a region in the IL-2Rot gene, -299/-243, that potently activates transcription from a heterologous promoter in PMA-induced Jurkat cells. This finding is consistent with a previous study defining a positive element in a large IL-2Ra fragment (15). Here, we have more narrowly delineated a positive regulatory region and analyzed the component parts of this region and their PMA inducibility. In addition to an NF-KB-binding site, electrophoretic mobility shift analyses have identified other DNA-binding protein recognition sites in this region (M. B. Toledano et al., submitted for publication). These include (i) a serum-responsive element (SRE-like sequence) (-253/-244) 3' of the KB site and (ii) a site 5' to the KB site between positions -290 and -269. Since the IL-2Ra KB site and adjacent 5' and 3' sequences together are required for potent PMA-induced activation of a heterologous promoter, these additional nuclear factor binding regions may be required for maximal positive regulatory activity. Thus, we have defined an enhancerlike element in the IL-2Rot gene. Maximal PMA inducibility requires the combination of adjacent elements to obtain an additive or synergistic effect. Further elucidation of both the positive element and the potential negative regulatory regions noted above should help in understanding the precise and exquisitely controlled cell-type-specific expression of the IL-2Ra gene. B.B.L. was the recipient of a Howard Hughes Medical Institute Research Scholar award. M.B.T. was the recipient of a grant from the Ligue Nationale Franraise Contre le Cancer. S.L.C. was supported by a fellowship from the Cancer Research Institute of New York. This work was supported in part by a Public Health Service Intramural AIDS grant from the National Institutes of Health. We thank Richard D. Klausner and Kuan-Teh Jeang for critical comments.
NOTES
VOL. 10, 1990
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