Transgene Expression That Confers Position-Independent a New

A New Regulatory Region of the IL-2 Locus That Confers Position-Independent Transgene Expression

This information is current as Mary A. Yui, Gabriela Hernández-Hoyos and Ellen V. of September 29, 2021. Rothenberg J Immunol 2001; 166:1730-1739; ; doi: 10.4049/jimmunol.166.3.1730 http://www.jimmunol.org/content/166/3/1730 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. A New Regulatory Region of the IL-2 Locus That Confers Position-Independent Transgene Expression1

Mary A. Yui,2* Gabriela Herna´ndez-Hoyos,2*† and Ellen V. Rothenberg3*

Although the promoter/enhancer of the IL-2 gene mediates inducible reporter gene expression in vitro, it cannot drive consistent expression in transgenic mice. The location and existence of any regulatory elements that could open the IL-2 locus in vivo have remained unknown, preventing analysis of IL-2 regulation in developmental contexts. In this study, we report the identification of such a regulatory region, marked by novel DNase-hypersensitive sites upstream of the murine IL-2 promoter in unstimulated and stimulated T cells. Inclusion of most of these sites in an 8.4-kb IL-2 promoter green fluorescent protein transgene gives locus control region-like activity. Expression is efficient, tissue specific, and position independent. This transgene is expressed not only in peripheral T cells, but also in immature thymocytes and thymocytes undergoing positive selection, in agreement with endog-

enous IL-2 expression. In contrast, a 2-kb promoter green ﬂuorescent protein transgene, lacking the new hypersensitive sites, is Downloaded from expressed in only a few founder lines, and expression is dysregulated in CD8؉ cells. Thus, the 6.4 kb of additional upstream IL-2 sequence contains regulatory elements that provide integration site independence and differential regulation of transgene expression in CD8 vs CD4 cells. The Journal of Immunology, 2001, 166: 1730–1739.

nterleukin-2 is the primary cytokine produced by newly ac- impossible until now to attempt mapping sequence elements that tivated naive T cells, and it plays a key role in the regulation restrict IL-2 expression to particular cell types. http://www.jimmunol.org/ I of immune responses. IL-2 expression is stringently con- Such a pattern of poor expression is characteristic of transgene trolled, requiring multiple signals for induction, and is activated constructs that are known to contain incomplete positive regula- only in a subset of mature cells that express other activation mark- tory regions (12–14). The regulatory regions of these transgenes ers (1). IL-2 is also induced in certain stages of thymic develop- are missing sequence elements that can cooperate with proximal ment in which its significance is still unknown (2–5). promoters/enhancers by opening the local chromatin and maintain- Although the regulation of IL-2 expression in response to stim- ing a transcriptionally competent domain. Some such elements are ulation has been studied in great detail in transient transfection designated locus control regions (LCRs),4 genetic regulatory ele- assays (reviewed in Refs. 6 and 7), we know very little about the ments that confer tissue-specific and physiological levels of tran- mechanisms regulating IL-2 expression in a developmental con- scription on linked genes irrespective of integration site (15). It is by guest on September 29, 2021 text. Available cell lines are wholly inadequate to address this only recently that the potential roles of elements affecting chro- question by in vitro transfection studies because they do not rep- matin structure have been addressed for regulation of any cytokine resent normal developmental states. However, the well-character- genes (reviewed in Ref. 16). Distal regulatory elements have been ized promoter/enhancer of the IL-2 gene is notoriously poor at identified by DNase hypersensitivity for the IL-3/GM-CSF locus allowing expression of reporter genes in transgenic mice. Use of (17, 18), and differential chromatin remodeling has been described the known promoter/enhancer (600 bp) to drive transgene expres- for IFN-␥, IL-4, and IL-13 in Th1 vs Th2 cells (19). In addition, sion resulted in only 1 of 17 and 2 of 26 founder mice expressing histone acetylation has been shown to be important for IL-4 in- the transgene properly (8, 9). Use of a more extended 5Ј regulatory ducibility in mature T cells (20). The addition of a heterologous region (up to 2700 bp), including all sequences tested for function (CD2) LCR to the 2.7-kb IL-2 promoter/enhancer region was re- by transfection (10), resulted in expression in only two of five cently shown to yield a high incidence of transgene expression, in founders (11). In several of the transgenic founders that express the 6 of 7 founders (21), which suggests that an LCR-like element is transgenes, there is evidence for insertional appropriation of host indeed missing from the IL-2 promoter regions tested to date. Con- regulatory sequences: i.e., the transgene gives ectopic expression, structs with exogenous LCRs, however, cannot be assumed to re- causes lethality when homozygous, or both (8, 9, 11) (our unpub- capitulate all aspects of normal developmental regulation that may lished results). In this context of variable expression, it has been be mediated by different protein-DNA interactions. Therefore, in this study, we sought molecular and functional evidence to locate any natural LCR-like elements that control the normal develop- *Division of Biology, California Institute of Technology, Pasadena, CA 91125; and mental regulation of the IL-2 gene. †Stowers Institute for Medical Research, Kansas City, MO 64110 Regions of DNA-protein interactions are often hypersensitive to Received for publication August 22, 2000. Accepted for publication November 13, 2000. DNase I digestion, and this characteristic has proven to be useful The costs of publication of this article were defrayed in part by the payment of page in detecting new and distant transcriptional regulatory sites. An charges. This article must therefore be hereby marked advertisement in accordance early study using this approach found evidence for hypersensitive with 18 U.S.C. Section 1734 solely to indicate this fact. (HS) sites beyond the minimal enhancer of the human IL-2 gene, 1 This work was supported by National Institutes of Health Grant AG13108 and the although they were not precisely mapped (22). We therefore used Stowers Institute for Medical Research. 2 M.A.Y. and G.H.-H. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Ellen V. Rothenberg, Division 4 Abbreviations used in this paper: LCR, locus control region; CBSS, Coffman’s of Biology, California Institute of Technology, 1200 East California Boulevard, Pas- balanced salt solution; GFP, green flluorescent protein; HS, hypersensitive site; HSA, adena, CA 91125. E-mail address: [email protected] heat-stable Ag (CD24).

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 1731 this method to find a series of new HS sites several kilobases followed by a final 5 min at 72°C, using an MJ PTC-200 DNA Engine upstream of the known IL-2 promoter/enhancer. The inclusion of Thermal Cycler (MJ Research, Watertown, MA). Founder mice were prop- these newly identified distal HS sites in green fluorescent protein agated by serial backcrosses to C57BL/6 mice. All conventional transgenic mice used for this study were generated and maintained in the Caltech (GFP) reporter gene constructs, along with the known IL-2 pro- Transgenic and Knockout Core Facility under specific pathogen-free con- moter/enhancer region, resulted in dramatically improved and conditions. Transgene copy number was determined by Southern blot hybrid- sistent expression of GFP in independent transgenic founders over ization of an IL-2 promoter region probe to genomic DNA cleaved with a wide range of transgene copy numbers. Expression was inducible XbaI and EcoRI. The 600-bp probe was generated by amplifying a PCR product using the tail-typing primers, which was then digested with PstIto and cell type specific in all expressing lines. This is the first study to remove the GFP sequence by gel purification. The intensities of the 3.7-kb identify a distal region of the IL-2 locus containing cis-acting ele- transgenic and the 2.4-kb endogenous (two-copy) IL-2 bands were deter- ments that are sufficient for developmentally regulated expression of mined by use of a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) a transgene independent of the site of chromosomal insertion. and ImageQuant software. To obtain transgenic SCID mice, transgene-positive male mice from two founder lines (lines 12 and 4) were crossed with C57BL/6-scid/scid Materials and Methods (B6-SCID) mice from the Rothenberg laboratory breeding colony at Cell culture and stimulations Caltech (24). To prevent infection of the immunodeficient animals, breed- ers and their pups were kept on prophylactic antibiotic (Baytril) treatment Splenocytes and purified T cells were cultured in DMEM supplemented Ϫ5 and transferred to sterile microisolator cages. Heterozygous offspring were with 10% FBS, 2 mM L-glutamine, 5 ϫ 10 M 2-ME, 100 U/ml peni- ␮ typed for the transgene by PCR analysis and then backcrossed to B6-SCID cillin, and 100 g/ml streptomycin (Life Technologies, Gaithersburg, MD) mice. Homozygous scid/scid pups were selected by flow cytometry of pe- at 37°C in 6% CO2. For stimulations via the TCR, 96-well flat-bottom ripheral blood samples to determine the absence of peripheral lymphocytes, plates (Corning, Corning, NY) were coated with 10 ␮g/ml purified ␣CD3 and transgene-positive animals were identified by PCR analysis of tail Downloaded from and ␣CD28 (PharMingen, San Diego, CA) in PBS for2hat37°C and 6 DNA. The transgene-positive SCID lines were then maintained on sterile washed with medium before the addition of 10 cells. For chemical stim- food, water, and bedding, in sterile microisolator cages in ventilated racks. ulations, 106 cells were incubated with 175 nM A23187 and 10 ng/ml PMA, both from Sigma (St. Louis, MO), and prepared as stock solutions Flow cytometric staining and cell sorting in DMSO. Single-cell suspensions of 106 RBC-depleted blood, splenocytes, or thy- DNase I-hypersensitive assays mocytes were stained on ice in 96-well U-bottom plates. Abs were diluted

in Coffman’s balanced salt solution (CBSS) buffer (1.25ϫ HBSS without http://www.jimmunol.org/ For purified T cells, C57BL/6 (B6) splenocytes were stained with biotin- phenol red) supplemented with 0.25% BSA (CBSS/BSA) (Pentax Fraction conjugated anti-B220 Ab (PharMingen), incubated with streptavidin-con- ϩ V; Miles, Kankakee, IL) with or without 0.1% sodium azide, as previously jugated MACs Microbeads, and passed over a VS MACS magnetic sep- described (24). Cells were preincubated in Fc block (PharMingen), fol- aration column (Miltenyi Biotec, Auburn, CA) to remove B cells. Purified lowed by staining with specific Abs and washing with CBSS/BSA. The T cells were then subjected to DNase I treatment either immediately or following staining reagents were used: CD3-APC, TCR␤-CyChrome, after6hofstimulation with PMA and A23187. Syngeneic melanoma CD44-PE, CD62L-PE, CD69-PE, NK1.1-PE, Thy-1-PE, CD8-APC, HSA- MCA102 cells were used for a non-T cell line (kindly provided by Michael biotin, streptavidin-APC (PharMingen), CD4-PE (Becton Dickinson, Nishimura, University of Chicago, Chicago, IL). Mountain View, CA), and Sca-1-TC (Caltag Laboratories, South San Fran- DNase I HS assays were conducted, as described previously (23), using cisco, CA). FACS analysis was performed using a FACSCalibur (Becton aliquots of 100 ϫ 106 cells permeabilized with lysolecithin for DNase Dickinson). Cells were sorted using a Coulter Elite (Coulter, Hialeah, FL). digestion and DNA extraction. Twenty micrograms of each DNA sample by guest on September 29, 2021 were digested with XbaI, electrophoresed in agarose gels, blotted, and hy- RNA purification and real-time quantitative RT-PCR bridized, as described previously. The three probes used were as follows (shown in Fig. 2): the distal upstream region, the 842-bp EcoRI to XbaI Total RNA was extracted using RNAzol (Leedo Medical, Houston, TX) in fragment, the promoter region, the 784-bp AccItoXbaI fragment including accordance with the manufacturer’s instructions. Stimulated cells were in- part of the second intron and third exon, and for the downstream region, the cubated for 6 h with plate-bound ␣CD3/␣CD28. RNA from 2 ϫ 106 cells 727-bp XbaItoEcoRI fragment in the third intron. was treated with RNase-free DNase to remove residual genomic DNA. First-strand cDNA synthesis reactions were then conducted using Super- Transgene construction script reverse transcriptase II (Life Technologies) and 250 ng random hex- amers (Pharmacia, Uppsala, Sweden) following standard protocols. DNA from the IL-2 upstream sequence was cloned from a C57BL/6 ␭ Real-time fluorescent PCR analysis (25) was conducted using the Ap- genomic library (Stratagene, La Jolla, CA) using a probe specific for the plied Biosystems Prism 7700 Sequence Detection System (PE Applied proximal promoter region of the IL-2 gene. Upstream sequences, from the Biosystems). Taqman probes and primers for murine GAPDH (VIC la- XhoI site at Ϫ8.4 kb to the PstI site at ϩ45 bp, and from the HindIII site beled) and IL-2 (FAM labeled) were purchased as sets from PE Applied at Ϫ2kb to the PstI site, were subcloned into an enhanced GFP vector Biosystems, and a GFP-specific oligo probe, 6FAM-GCTTTACTTGTA plasmid (EGFP-1) (Clontech Laboratories, Palo Alto, CA). In addition, the CAGCTCGTCCATGCCGA-TAMRA, was custom synthesized by the SV40 poly(A) site in the vector was deleted from NotItoAflII, and replaced same company. Primers used to specifically amplify the transgenic mes- with the 3Ј splice and poly(A) site from human ␤-globin, as the BamHI to sage crossed the intron between GFP (5Ј-CACATGGTCCTGCTG EcoRI fragment from plasmid pES4 (kindly provided by Stephen Hedrick, GAGTTC-3Ј) and the 3Ј ␤-globin sequences (5Ј-CAGCACACAGAC University of California, San Diego, CA). CAGCACGTT-3Ј). Thirty-microliter reactions were conducted in Taqman The 8.4-kb upstream IL-2 region used in this study was sequenced by Universal PCR buffer (1ϫ Taqman buffer A; 5 mM MgCl ; 200 ␮M each subcloning progressive unidirectional deletions using the Erase-A-Base 2 of dATP, dTTP, and dGTP; 400 ␮M dUTP; 8% glycerol; 200 ␮M primers; system (Promega, Madison, WI). Dideoxy sequencing on the subclones 100 ␮M fluorescent-labeled probe; 0.01 U/␮l AmpErase UNG; 0.05 U/␮l was then conducted using Applied Biosystems Prism Dye Terminator Cy- AmpliTaq Gold) using the following thermocycling conditions: 50°C, 2 cle Sequencing Ready Reaction and an Applied Biosystems Prism 9600 min; 95°C, 10 min; then 40 cycles of 95°C, 15 s and 60°C, 1 min. The cycle Sequence Detector System kit (PE Applied Biosystems, Foster City, CA). thresholds (C ) were determined by measurement of the amount of fluo- These sequence data have been submitted to the GenBank database under T rescent dye released after PCR amplification at each cycle using cDNA accession number AF290391. samples and specific primers and probes for GFP, IL-2, and GAPDH in Generation and characterization of transgenic mice separate duplicate tubes and selecting a threshold in the linear range of the amplification. Control GAPDH CTs were subtracted from GFP and IL-2 ϭ⌬ Transgenic mice were generated in the Caltech Transgenic Animal Facility CTs for each sample ( CT). Each value was then adjusted by subtracting ϫ ⌬ (Pasadena, CA) by pronuclear injection of (B6 DBA/2)F2 zygotes using the CT for a reference sample within each experiment (the transgene- ϭ⌬⌬ standard methods, and transgene positive mice were identified by PCR negative control for IL-2 and transgenic line 4 for GFP) ( CT). Rela- analysis of tail DNA. PCR primers in the IL-2 proximal promoter (IL2-1F: tive amounts of mRNA were calculated as 2Ϫ⌬⌬CT. 5Ј-CATCCTTAGATGCAACCCTTCC-3Ј) and the GFP coding sequence To convert GFP and IL-2 expression to an absolute scale, standard (GFP-1R: 5Ј-GCTGAACTTGTGGCCGTTTAC-3Ј) were used, amplify- curves of control cDNA plasmids were analyzed in parallel in some ex- ing a 830-bp product in transgene-positive mice. PCR conditions were as periments. The IL-2 cDNA standard was pGmIL2 (26). For the transgene, follows: 94°C, 5 min, then 35 cycles of 93°C, 30 s; 62°C, 15 s; 72°C, 45 s, a PCR product was amplified from cDNA of stimulated 8kbIL2p-GFP-175 1732 DISTAL IL-2 REGULATORY REGION splenocytes, using a GFP-specific primer incorporating an XbaI site for cloning (5Ј-AGACTCTAGACCACATGAAGCAGCACGACT-3Ј), and a reverse primer specific for exon 3 of the ␤-globin 3Ј end (5Ј-TGATAG- GCAGCCTGCACTGGT-3Ј). The product was then digested with XbaI and EcoRI to generate a 562-bp product that was cloned into pBlueScript (Stratagene). Results DNase I-HS sites are found upstream, but not downstream of the IL-2 gene in unstimulated and stimulated purified T cells Because protein-DNA contacts in the known 2700 bp of IL-2 5Ј- flanking DNA are insufficient to mediate full LCR-like activity, we sought evidence for other regions of the gene that might confer position-independent expression on transgenes. To identify distal sites of potential protein-DNA interactions in the IL-2 locus, we performed DNase I-hypersensitivity assays on unstimulated and stimulated purified mouse splenic T cells. Unstimulated T cells displayed a prominent HS site at ϳ4.5 kb upstream of the IL-2 start site, with a series of five weaker HS sites seen between Ϫ3 and Ϫ4.5 kb (Fig. 1A, left panel). The same HS sites were also Downloaded from found in unstimulated EL4 thymoma cells that express IL-2 only after stimulation (data not shown). These sites appeared to be cell type specific; the region from Ϫ3toϪ4.5 kb was not hypersensitive in a syngeneic melanoma cell line (Fig. 1A, right panel). These six T cell-specific HS sites are of particular interest as they indicate that the region of the IL-2 gene upstream of Ϫ2 kb has an http://www.jimmunol.org/ accessible chromatin structure in resting T cells and is bound by putative transcription factors that may play a role in maintaining the locus in an open but transcriptionally silent state. Six hours after stimulation with PMA and the calcium ionophore A23187, extensive chromatin remodeling was seen in the upstream region (Fig. 1A, middle panel). Four new HS sites appeared between Ϫ2.5 and Ϫ3 kb. At the same time, the six HS sites found in the unstimulated cells were still present, although the Ϫ4.5-kb site became less prominent. Three new, more distal sites by guest on September 29, 2021 also appeared, one strong band at approximately Ϫ8 kb, and two others at Ϫ7 and Ϫ10 kb. Again, a similar pattern of HS sites was also found in stimulated EL4 cells (data not shown). FIGURE 1. DNase I-HS sites in the IL-2 locus in unstimulated and In sharp contrast, no HS sites were observed in the region from Ϫ Ј stimulated purified splenic T cells. A, HS sites located from 2.5 kb to the third intron to 5 kb 3 of the IL-2 gene in unstimulated or Ͼ14 kb upstream of the IL-2 gene in unstimulated and stimulated purified stimulated purified T cells (Fig. 1B) or in EL4 cells (data not T cells. No HS sites were detected in a melanoma line, MCA205 (non-T shown). HS sites in the promoter-proximal region that have been cells). HS sites are indicated by arrows. Locations, given in kb, are relative reported in human and mouse T cell lines (4, 22, 27) were also to the IL-2 gene start site. B, No HS sites were detected in the 6-kb XbaI observed in purified T cells but not in non-T cells, and no new sites fragment extending from within the third intron to 5 kb 3Ј of IL-2 coding were revealed (Fig. 1C). A summary of IL-2 locus HS sites found sequence in stimulated or unstimulated T cells. C, HS sites in the region in resting and activated T cells is shown in Fig. 2. Overall, these from Ϫ2 kb in the promoter to ϩ2 kb in the second intron. Two sites in the results show that the distal upstream region of the IL-2 gene is proximal promoter region have been reported previously in EL4 cells (23). maintained in an open chromatin structure in resting peripheral T An unlabeled arrow indicates a weak site of hypersensitivity in the second intron of the stimulated cells. cells, but not in non-T cells, and that this region undergoes rapid and extensive remodeling upon activation. The presence of at least 13 new distal HS sites upstream of the IL-2 gene suggests that this region has many previously uncharacterized DNA-protein interactions that may play in vivo roles in mediating IL-2 locus accessi- optimal inducible expression in transiently transfected cells (10). bility or providing tissue-specific enhancer and/or silencer The 8kbIL2p-GFP construct also includes an additional 6.4 kb of functions. flanking DNA containing all of the novel HS sites found in resting T cells, as well as most, but not all, of the activation-induced HS Construction and in vitro testing of the IL2p-GFP transgenes sites (Fig. 2). The SV40 poly(A) in the original plasmid was re- utilizing 8.4 kb vs 2 kb of upstream IL-2 sequence placed with the human ␤-globin 3Ј-splice site and poly(A) tail, To characterize and test this upstream region for function in vitro which reproducibly improved the inducible expression of GFP by and in vivo, 5Ј-flanking sequence from the IL-2 gene was cloned 2–2.5ϫ after transient transfections in EL4 cells (data not shown). from a C57BL/6 genomic DNA library and sequenced. Two frag- The 2- and 8.4-kb GFP constructs gave similar GFP expression ments, 2 and 8.4 kb, were subcloned into a GFP-expressing vector, levels (within 20%) in these assays (data not shown), indicating to give constructs designated 2kbIL2p-GFP and 8kbIL2p-GFP, re- that the distal sites included in 8kbIL2p-GFP do not mediate con- spectively. Both constructs include the proximal 2 kb of 5Ј-flank- ventional enhancer activity in a nondevelopmental, nonchromatin ing sequence (to the PstI site at ϩ45) that was found to drive context. The Journal of Immunology 1733

FIGURE 2. Summary of HS sites found in stimulated and unstimulated puriﬁed splenic T cells, mapped relative to the IL-2 locus, and diagrams of the 2kbIL2p-GFP and 8kbIL2p-GFP transgene constructs used in this Downloaded from study. The XbaI sites and the probes (gray boxes) used for HS assays (Fig. 1, A–C) are as shown. Features of an analysis of the upstream DNA sequence from Ϫ2kbtoϪ8.4 kb (Genbank AF290391) are also indicated: a putative matrix attachment region identiﬁed by use of MARFinder (43) (M), a region of high homology with an intronic sequence in the Bruton’s thymidine kinase gene (btk), and a region of high homology with an upstream sequence in the human IL-2 gene (Œ). To construct the transgenes http://www.jimmunol.org/ used in this study, the 2-kb HindIII-PstI and the 8.4-kb XhoI-PstI frag- ments were subcloned into a GFP reporter vector with a 1.7-kb BamHI-PstI human ␤-globin 3Ј-splice site and poly(A) (transgene constructs 2kbIL2p- GFP and 8kbIL2p-GFP, respectively). Upstream sequences included in these constructs are shown relative to the IL-2 gene and HS sites (X, XbaI; H, HindIII; P, PstI).

The 8kbIL2p-GFP-transgenic mouse lines express the transgene by guest on September 29, 2021 more consistently than 2kbIL2p-GFP lines FIGURE 3. High frequency and magnitude of transgene expression in T The two constructs, 2kbIL2p-GFP and 8kbIL2p-GFP, were in- cells from 8kbIL2p-GFP transgenic lines: comparison with 2kbIL2p-GFP transgenic lines. A, Flow cytometric analysis of GFP expression in CD3 jected into mouse embryo pronuclei, and transgenic founders were cells, from two 8kbIL2p-GFP and two 2kbIL2p-GFP lines, without stim- produced. Eleven independent 8kbIL2p-GFP and twelve indepen- ulation (Unst) and 16 h after stimulation with PMA and A23187 (Stim). dent 2kbIL2p-GFP founders were obtained. Founders were bred to Trangenic line number and transgene copy numbers (in parentheses) are C57BL/6 mice and more detailed analyses performed on cells from given above each plot. Percentages of cells in each quadrant are indicated. transgene-positive founders and/or their progeny. Transgene copy B, GFP expression levels in stimulated and unstimulated peripheral T cells numbers ranged from 1 to Ͼ40 for both constructs, as determined from 11 independent 8kbIL2p-GFP and 12 independent 2kbIL2p-GFP were stained (ء) by Southern blot analysis. transgenic lines. Splenocytes and/or peripheral blood cells Transgene-positive mice were screened for activation-induced with anti-CD3 Abs and analyzed by flow cytometry either without stimu- ϩ lation (open bars) or after 16 h of stimulation with PMA and A23187 (filled GFP expression among CD3 cells. GFP expression was readily ϩ detected in CD3-positive cells from both kinds of founders, as bars). Both the percentages of GFP-positive cells among CD3 -gated cells and the mean fluorescence values among those GFPϩCD3ϩ cells are given shown in Fig. 3A. Although spontaneous GFP expression was ob- for each transgenic line (ϩ, missing data). served in some CD3ϩ cells (see below), the percentage and mean fluorescence of the GFPϩ cells increased dramatically with PMA/ A23187 stimulation. Some GFPϩ CD3Ϫ cells were observed in stimulated cells from all lines of both 2- and 8-kb transgenics transgenics, with 10 of 11 (91%) lines expressing GFP in stimu- which are capable of expressing GFP, and there is no consistent lated CD3ϩ cells (Ͼ3% positive under the conditions used in Fig. difference between the two constructs in the percentages of these 3A). The only 8kbIL2p-GFP line that failed to express GFP carries populations. These cells do not appear to be B cells or macro- only one copy of the transgene. By contrast, only 4 of 12 (33%) of phages (data not shown) and may represent T cells that have down- the 2kbIL2p-GFP lines expressed GFP at that level; the remainder regulated the TCR-CD3 complex. Ectopic transgene expression expressed the transgene very poorly or failed to express it at all was not observed with either construct, as major nonlymphoid or- over a wide range of copy numbers (Fig. 3B, left panels). The gans of the transgenic mice do not express GFP. characteristics of the expression patterns seen in different 8kbIL2p- Although lines from both the 2kbIL2p-GFP and 8kbIL2p-GFP GFP lines were qualitatively very consistent between lines and constructs were capable of expressing the transgene in a tissue- among the progeny of most individual lines (see below, and M. A. specific and inducible manner, the frequency of animals that ex- Yui and E. V. Rothenberg, in preparation). The expression in these pressed the transgene and the levels of expression differed between lines seemed unlikely to be a result of insertional mutagenesis, the two constructs (Fig. 3B). The 8kbIL2p-GFP lines expressed since there was no evidence of morbidity in transgene-positive GFP more consistently than any previously reported IL-2 promoter animals. These results show that the additional 6.4 kb of IL-2 gene 1734 DISTAL IL-2 REGULATORY REGION upstream sequence in the 8kbIL2p-GFP transgene sharply in- quantitated using real time fluorescent RT-PCR (25) in samples creases the probability that an individual cell will open and express from TCR/CD28-stimulated cells. RNA was prepared from the locus, independent of integration site. splenocytes from nontransgenic and transgenic mice, with or without 6-h stimulation with anti-CD3/anti-CD28, and cDNA from Ϫ ␣␤ GFP induction in purified GFP T cells these samples was analyzed in parallel with cDNA plasmid stan- Transgenic TCR␣␤ϩ splenocytes that are initially GFP negative dards. Fig. 5A shows that with this assay, the threshold cycle num- synthesize GFP de novo in response both to TCR ligation and to ber (CT) (the PCR cycle at which the product of each sample PMA/A23187 stimulation, as shown for two independent crossed a detection threshold) was linear with the log of sample 8kbIL2p-GFP transgenic lines in Fig. 4. A subset of TCR␣␤ϩ dilution over five orders of magnitude, for three independent sets GFPϪ cells was seen to express GFP by 16 h after stimulation, and of plasmid standard dilutions. These standards were used to cal- the percentage of GFPϩ cells increased further by 44 h. GFP was culate absolute levels of IL-2 and GFP mRNA in the cDNA from readily induced using either plate-bound anti-CD3/anti-CD28 or transgenic and control mice. As shown in Fig. 5B, the lower copy PMA/A23187 stimulation, although a higher percentage of GFPϩ number transgenic lines (three to four copies) could express GFP cells was always observed in PMA/A2387-stimulated cells, with mRNA at similar levels to the two-copy endogenous IL-2 genes, the mean fluorescence reaching peak levels at earlier time points. with ratios of about 1. Higher GFP:IL-2 ratios were seen at higher These results show that, as with endogenous IL-2, a subset of T transgene copy numbers. This finding suggests that the 8.4 kb of cells that are initially GFP negative respond rapidly to specific upstream sequence in the 8kbIL2p-GFP transgene encompasses activating signals by producing high levels of GFP. most, if not all, of the positive regulatory elements required for

inducible expression, in addition to conferring positional Downloaded from Quantitative analysis of transgene expression: similar independence. efficiencies of expression of transgenes and endogenous IL-2 In the 2kbIL2p-GFP transgenics, only a few lines (with 10–17 genes copies) show significant expression. Among these few 2-kb lines To assess whether the 8kbIL2p-GFP transgene includes regulatory that do express well, the levels of GFP mRNA induced at 6 h elements allowing transgene expression at a level comparable with spanned a 10-fold range that overlapped the expression levels seen that of the endogenous IL-2 genes, GFP and IL-2 mRNA were in 8kbIL2p-GFP transgenic lines (Fig. 5C). Thus, the elements http://www.jimmunol.org/ mapping between Ϫ2 and Ϫ8.4 kb affect the likelihood that transgenes will be expressed more than they affect the level of expression once induced. Effect of copy number on expression of 8kbIL2p-GFP transgenes in response to stimulation With the exception of line 175 (23 copies), the ratio of GFP:IL-2 expression showed only a modest increase over a 10-fold increase in copy number (Fig. 5B). Such a nonlinear response to copy num- by guest on September 29, 2021 ber could be a result of competition for a limiting pool of trans- acting positive regulatory factors. However, GFP expression did not occur at the expense of endogenous IL-2 expression, even at high transgene copy numbers (Fig. 5C, left panel). In agreement with this result, we have found that at both low and high copy numbers, IL-2 and GFP protein can both be detected in individual stimulated T cells (data not shown). GFP mRNA expression in the 8kbIL2p-GFP lines (Fig. 5C, right panel) generally paralleled protein expression (Fig. 3B), re- flecting a composite of the percentage of T cells that becomes GFPϩ and the mean fluorescence intensities of the GFPϩ cells. The two lines with the lowest transgene copy numbers also displayed the lowest mean fluorescence intensities among GFPϩ T cells (Fig. 3B, right panel). However, the percentage of cells expressing GFP did not show a consistent increase as a function of copy number (Fig. 3B, left panel). This suggests that the number of transgene copies (per integrated array) has only limited effect on the likelihood that these genes will be activated transcriptionally in response to a particular episode of stimulation.

Spontaneous GFPϩ cells have an effector/memory cell FIGURE 4. GFP expression is inducible in purified GFP-negative ␣␤ T phenotype cells. Splenocytes from 8kbIL2p-GFP lines 4 and 175 were stained with Although expression of the GFP transgenes is highly responsive to ␤ ␤ϩ TCR -CyChrome Abs, and GFP-negative TCR cells were sorted to activation in the population as a whole, there is a reproducible Ͼ99.9% purity using a Coulter Elite flow cytometer (shown on left). Sorted background of transgene expression, at both the RNA and protein cells were stimulated with either immobilized anti-CD3/anti-CD28 or PMA and A23187. Cells were harvested at 16 and 44 h and analyzed by levels, before stimulation (Figs. 3B and 5C). This background ex- flow cytometry. Percentages of GFPϩ cells and the mean fluorescence of pression is seen in all the expressing transgenic lines, regardless of the GFPϩ cells are given above each cytometric plot. Unstimulated cul- whether they are derived with the 2-kb or the 8.4-kb promoter tured T cells had low viability and did not become GFP positive (data not construct. Fig. 5C shows that background GFP RNA expression shown). can be seen even in populations of unstimulated cells that express The Journal of Immunology 1735

no detectable IL-2 RNA (see data for lines 4, 8, and 214, one experiment each, and for lines 186, 177, and 17). The spontaneous transgene expression, however, is associated with the same cell types that express IL-2 in response to induction. As shown in Fig. 3A (and data not shown), spontaneously GFPϩ cells are over- whelmingly concentrated in the CD3ϩ population, in both CD4ϩ and CD8ϩ subsets (see below). To determine whether this spontaneous expression of GFP is dependent on prior immunological activation, freshly isolated transgenic splenocytes were analyzed for surface phenotype markers that distinguish naive from previously activated cells. All of the GFPϩ cells in the CD4ϩ population were found in the minority subset that is CD44high (Fig. 6), and predominantly CD62Llow (data not shown), in every 8kb- and 2kbIL2p-GFP line tested. This phenotype is a hallmark of previously activated effector/memory cells. The majority of the GFPϩ cells were found to be small, resting cells, and most were also CD69low, indicating that only a minority of GFPϩ cells were recently activated. The cells express-

ing GFP spontaneously in vivo can still respond to TCR stimula- Downloaded from tion in vitro by turning on even higher levels of GFP expression (data not shown), which is consistent with a memory cell phenotype. These results indicate that the background GFP expression, in peripheral CD4 cells at least, is primarily associated with effector/

memory type T cells, presumably elicited by environmental stim- http://www.jimmunol.org/ ulation. The 8kbIL2p-GFP transgene is also expressed very consistently in TCR␥␦ and NKT cells, both of which are known to be activatible by endogenous Ags (data not shown and M.A.Yui and E.V.Rothenberg, in preparation).

Expression of the 8kbIL2p-GFP transgene in a developmental context: expression in primitive thymocyte subsets in SCID- transgenic mice

IL-2 is spontaneously induced both in TCRhigh thymocytes under- by guest on September 29, 2021 going positive selection to CD4 and CD8 lineages (2–5) and in much more primitive cells before TCR gene rearrangement (4). All of our IL2-GFP-transgenic lines that express in activated peripheral T cells also show spontaneous expression in TCRhigh thymo- FIGURE 5. Quantitative analysis of GFP and IL-2 mRNA induction in cytes (data not shown; and see below). It was of particular interest 8kbIL2p-GFP and 2kbIL2p-GFP transgenic lines. A, Threshold cycle data (the PCR cycle number at which the specific amplification product crossed to determine whether the 8kbIL2p-GFP transgene is also expressed in the more primitive thymocytes, in which expression cannot be a threshold (CT)) from real-time fluorescent RT-PCR analysis for IL-2 (left panel) and GFP (right panel) for mRNA quantitation in unstimulated and driven by conventional TCR-dependent signals. However, the im- anti-CD3/anti-CD28-stimulated splenocyte samples using plasmid stan- mature populations in which IL-2 mRNA is found are rare, alto- Ͻ dard curves. Shown are CT values for 10-fold serial dilutions of three gether representing 0.1% of normal thymocytes (4). Therefore, independent plasmid preparations. Standard curves were adjusted for the to enrich these subsets ϳ100-fold, we crossed the 8kbIL2-GFP difference in plasmid size, with the 3.65-kb pGmIL2 starting at 0.1 ng and transgenes from two separate founder lines onto the B6-SCID ho- ϳ ϫ 6 the 3.46-kb pGFP-Bg at 0.95 ng/reaction ( 2.6 10 molecules). Also mozygous background. GFP-expressing thymocytes from these shown are the no-template controls (NTC), and cDNA from unstimulated mice could then be identified by four-color flow cytometry, using (Unst) and stimulated (Stim) splenocytes (ϳ104 cell equivalents/reaction). Sca-1 (Ly6-A/E) and HSA (CD24) expression for primary subdi- Samples are from 8kbIL2p-GFP lines 4, 8, 17, 186, 175, 177, 2kbIL2p- Ϫ vision of cell types (reviewed in Ref. 28), as shown in Fig. 7. GFP line 214, and a transgene-negative (Tg ) mouse. The maximum CT value is 40 cycles. Standard curves for both IL-2 and GFP were linear over Both lines of SCID 8kbIL2p-GFP transgenics showed clear GFP ϩ low Ϫ the entire dilution range used. Duplicate values are shown for each sample expression that was restricted to the Sca-1 HSA and Sca-1 (dark circles and hollow triangles). B, Graph showing the ratios of GFP to HSAlow populations. These two populations express IL-2 mRNA IL-2 mRNA levels relative to transgene copy numbers for stimulated in vivo and are thought to represent pluripotent precursors and splenocytes from 8kbIL2p-GFP lines 4, 8, 186, 175, 17, and 177. The low NK-like cells, respectively (28). GFPϩ cells constituted a fraction copy lines have ratios close to 1, indicating that the trangene is ex- of each of these subsets: 25–30% of the Sca-1ϩ HSAlow subset and pressed at levels close to those of endogenous IL-2. C, Relative amounts of IL-2 (left panel) and GFP (right panel) mRNA determined by real-time fluorescent RT-PCR analysis, for RNA extracted from unstimulated (gray bars) and ␣CD3/CD28 stimulated (filled bars) spleno- GFP expression are presented on a scale with the levels for stimulated cells cytes. Transgene line and copy number (in parentheses) are given for of line 4 arbitrarily set as 1. Data from two independent experiments are each sample. CT values were adjusted for control GAPDH levels and presented: lines 4, 8, 175, and 214 were analyzed in both. Samples with no percentages of CD3ϩ cells in the input cell population. The relative detectable levels of IL-2 or GFP RNA are starred; these were frequent in values for IL-2 expression are presented on a scale with the average the first experiment, but not seen in the second experiment. Note that the level in activated nontransgenic cells set as 1. The relative values for scale of expression in each panel is logarithmic. 1736 DISTAL IL-2 REGULATORY REGION Downloaded from

FIGURE 6. Spontaneously GFPϩ CD4ϩ T cells are CD44high, previously activated cells. Freshly isolated splenocytes from two 8kbILp-GFP, two 2kbIL2p-GFP, and transgene-negative lines were analyzed by ﬂow- cytometric analysis for expression of GFP, CD4, and CD44, an effector/ memory marker for cells within the CD4ϩ population. The correlation of FIGURE 7. Spontaneous expression of the 8kbIL2p-GFP transgene in

GFP and CD44 fluorescence is shown in cells gated for CD4 expression. immature thymocytes of SCID mice is under correct developmental regu- http://www.jimmunol.org/ Percentages of cells in each quadrant are given; GFPϩCD4ϩ cells were lation. Four-color flow cytometric analyses of cells from line 4 crossed disproportionately CD44high in all transgenic lines tested (Ͼ85% CD44high; onto a SCID homozygous background are shown. Top panel, Sca-1 vs cf Ͻ25% CD44high for the rest of the CD4ϩ population). Trangenic line HSA staining for all cells. A total of 3.9% of these cells was GFPϩ. Lower number and transgene copy numbers (in parentheses) are given above each panels, Relationship between GFP fluorescence and each of three other plot. markers, NK1.1, Thy-1, or CD69, within the three Sca-1/HSA subsets indicated in the upper panel: A, Sca-1ϩ HSAlow population (precursor-like cells); B, the Sca-1Ϫ HSAlow population (NK-like cells); and C, the major HSAϩ population (specified/committed pro-T cells). Percentages shown in Ϫ low 35–40% of the Sca-1 HSA subset, in the example shown in the lower panels refer to each gated subset as 100%. ϩ Fig. 7. In each of these subsets, GFP cells were typical of the by guest on September 29, 2021 predominant cell type: the precursor-like Sca-1ϩ subset was Thy-1ϩ and NK1.1Ϫ (Fig. 7A), while the NK-like Sca-1Ϫ subset ϩ was Thy-1 heterogeneous and NK1.1 (Fig. 7B). Most, but not all, biased pattern of endogenous expression of GFP was observed in ϩ ϩ of the GFP cells were also CD69 , again in agreement with IL-2 the 2-kb transgenic lines, favoring the CD8ϩ cells (Fig. 8). This mRNA expression data (4). In sharp contrast, the population in- contrasts with the pattern of expression of endogenous IL-2, which ϩ cluding the majority of transgenic SCID thymocytes, the HSA is expressed at least as efficiently in CD4ϩ cells (29–31). Of the ϩ subset, did not express the transgene (Fig. 7C). The HSA subset four 2kbIL2p-GFP-transgenic lines that express GFP, three ex- consists of cells undergoing specification and commitment to the T press the transgene in percentages of splenic CD8ϩ cells 5–28 ϩ ϩ Ϫ cell lineage, corresponding to the CD25 CD44 / pro-T cell times higher than in CD4ϩ cells (Fig. 8C). This effect is most populations in the conventional fractionation scheme (24, 28). De- pronounced in 2kbIL2p-GFP line 227. By contrast, 8kbIL2p-GFP- spite the more advanced developmental state of these cells, they do transgenic lines express GFP in CD4ϩ and CD8ϩ cells at similar not express IL-2 mRNA in vivo (4). Results in SCID progeny of frequencies. Line 227 expresses GFP in 10 times more CD8ϩ cells the other 8kbIL2p-GFP transgenic line, line 12, were similar ex- than an 8kbIL2p-GFP line matched for spontaneous expression in ϩ cept that the percentage of cells expressing GFP in the Sca-1 CD4ϩ cells (Fig. 8A). population was about 3-fold lower (data not shown). The expres- This preferential expression in CD8 cells is not an artifact of sion of the transgene thus recapitulates faithfully the developmen- response to peripheral Ag exposure because it appears before the tal expression pattern of the endogenous gene, even in a stage of cells leave the thymus, in single-positive thymocytes emerging lymphocyte development in which the inducing signal must be from positive selection (Fig. 8, B and D). In agreement with the TCR independent. transient expression of endogenous IL-2 at this stage (2–5), small percentages of CD4ϩ and CD8ϩ single-positive thymocytes ex- Qualitative distortions in expression pattern of the 2kbIL2p-GFP ϩ press GFP, in all of the expression-positive transgenic lines with transgene: preferential expression in CD8 T cells either the 8- or 2-kb construct (Fig. 8, B and D; and data not In those lines in which it is expressed, the 2kbIL2p-GFP transgene shown). However, the frequency of GFP expression in CD8ϩ cells correctly directs expression to T cells and thymocytes, similarly to relative to CD4ϩ cells is elevated in three of four 2-kb lines (Fig. the 8kbIL2p-GFP transgene, but the detailed pattern of transgene 8D), and the percentage of GFPϩ cells among CD8ϩ thymocytes expression in T cell subsets differs. The most conspicuous differ- is greatly expanded in line 227 (Fig. 8B). ence is that the shorter transgene frequently drives high level ex- These results suggest that IL-2 may be regulated in part through pression in CD8ϩ, but not CD4ϩ T cells. Whereas transgenic lines distinct mechanisms in CD4 vs CD8 cells. The additional 6.4 kb of utilizing the 8.4-kb promoter construct showed similar low levels upstream sequence appears to be needed to exert a negative reg- of background expression in CD4ϩ and CD8ϩ T cells, a distinctly ulatory effect to limit expression in CD8ϩ, but not CD4ϩ, cells. The Journal of Immunology 1737 Downloaded from http://www.jimmunol.org/

FIGURE 8. The 2kbIL2p-GFP lines express GFP preferentially in CD8 cells in thymus and spleen. A and B, Representative comparison between splenocytes and thymocytes from a 2- and an 8.4-kb transgenic line. Freshly isolated splenocytes (and thymocytes from 2kbIL2p-GFP line 227 and 8kbIL2p-GFP line 175) were stained with CD4-PE and CD8-CyChrome and analyzed by ﬂow cytometry. Percentages of cells in the CD4ϩ and CD8ϩ populations that express GFP are indicated in the upper right quadrant of the plots. The two transgenic lines spontaneously express GFP in a similar percentage of CD4 splenocytes and thymocytes, but line 227 expresses GFP in higher percentages of CD8ϩ splenocytes (A) and CD8ϩ CD4Ϫ thymocytes (B). C and D, Summarize the ratios of the percentage of GFPϩCD4ϩ cells to the percentage of GFPϩCD8ϩ cells for freshly isolated populations from the spleens (C) and thymuses (D) of several 8kbIL2p-GFP and 2kbIL2p-GFP transgene-expressing lines. The data show that three of four of the expression- positive 2kbIL2p-GFP lines express GFP in a higher percentage of CD8 than CD4 cells (ratios in spleen Ն5), while the 8kbIL2p-GFP lines generally express the transgene equally well in both populations (ratios ϳ1). by guest on September 29, 2021

Discussion firm this prediction. A construct utilizing 2 kb of IL-2 upstream The well-characterized 600-bp IL-2 promoter/enhancer includes sequence results in transgene expression in only 4 of 12 indepen- cis-acting elements required for inducible and T cell-specific ex- dently derived lines, consistent with previous reports (11). In sharp pression in transient transfection studies. Nevertheless, constructs contrast, when the new 8.4-kb transgene construct is used, GFP is utilizing this promoter region consistently express transgenes very expressed in 10 of 11 independent lines. Thus, the addition of 6.4 poorly, demonstrating that this region is missing key regulatory kb of upstream sequence, including all of the 5Ј-flanking HS sites elements needed for proper gene expression in a developmental found in resting T cells and most of the sites observed in stimu- context. This is the first study directed to localize and functionally lated T cells, greatly increases the likelihood of transgene expres- characterize these missing regulatory elements. We have now sion, suggesting a relative independence from local integration site identified a 6.4-kb region upstream from the IL-2 promoter/en- positional effects. By contrast, expression from the 2-kb promoter hancer, encompassing multiple HS sites in resting and activated T construct appears to depend acutely upon factors related to the site cells, which confers integration site-independent and developmen- of integration. Furthermore, the 8.4-kb promoter transgene was tally consistent transgene expression. The ability to obtain repro- found to be expressed over a broad range of copy numbers (3–45) ducible, position-independent expression of a transgene utilizing in our studies, while the shorter 2-kb promoter transgene was not. IL-2 regulatory sequences makes it possible to map regulatory el- Position-independent expression and resistance of transgenes to ements and to test the significance of IL-2 expression in distinct heterochromatization of high copy number arrays are both prop- cell types and developmental states. erties correlated with the presence of an LCR (13, 14, 32), or of an Inclusion of the novel upstream HS sites in a transgene confers enhancer mediating LCR-like functions (33, 34). strong, position-independent activity Normally, sites of regulatory importance are phylogenetically The 8.4-kb flanking sequence transgene was designed to include a conserved, e.g., the murine and human versions of the proximal ϳ set of candidate regulatory sites that we have located by DNase- 600-bp IL-2 promoter/enhancer, which are 85% identical. This hypersensitivity analysis. The presence of at least 13 tissue-spe- comparison cannot be made for most of the new upstream region, cific HS sites upstream of any previously identified regulatory se- however, because the human sequence is interrupted at about quences provided the first evidence that multiple, uncharacterized Ϫ3300 by a Ͼ6-kb LINE element (35), and 5Ј sequence beyond regulatory elements in this region may contribute to normal IL-2 this is currently unavailable. Only one significantly conserved re- gene regulation in vivo, although they do not appear to be impor- gion is seen between Ϫ2000 and this breakpoint, corresponding to tant in transient transfection in vitro. Our transgenic studies con- Ϫ2206 to Ϫ2262 in the murine gene (27, 35). Transfection studies 1738 DISTAL IL-2 REGULATORY REGION in a transformed cell line have not revealed any net enhancer ac- sibly including cells transiently activated in the course of tivity in the new 6.4-kb upstream region (data not shown), but homeostatic proliferation. chromatin-opening functions need not be detectable in transient In contrast to our results, when a single copy of the GFP gene expression assays. In view of the activities reported for certain was knocked in to the IL-2 locus, background GFP was not ob- nuclear matrix attachment regions (12), it is of interest that the served (37). This could indicate that there are additional regulatory 8.4-kb upstream region of the murine IL-2 gene includes a poten- sequences mapping outside the 8.4 kb included in our transgene tial matrix attachment region (see Fig. 2). Alternatively, the new that are required for repression in memory cells or partially acti- sequences could exert their main effects through enhancer activity vated cells. The consistency of phenotype in our 8kbIL2p-GFP specific for particular, early developmental stages in the thymus transgenics should be a substantial aid toward functional identifi- when IL-2 is first expressed (cf Ref. 13). cation of such distal silencing sequences, if they exist. Other factors are also likely to contribute to the difference in background. GFP transgene expression in response to stimulation in vivo The fluorescence intensity of GFP in our trangenics is much higher Expression of the 8kbIL2p-GFP transgenes (and 2kbIL2p-GFP than that of the single knock-in allele, and this increased sensitivity transgenes, in lines that allow expression at all) is cell type specific could make the cells spontaneously expressing GFP easier to de- and inducible by TCR/CD28 ligation. All 8kbIL2p-GFP and tect. GFP mRNA is also likely to be more stable than IL-2 mRNA, 2kbILp-GFP transgenic lines that are capable of inducibly express- as it is designed to lack the 3Ј untranslated region destabilization ing the transgene also express GFP spontaneously in specific sub- motif found in the IL-2 gene (40, 41). This would exempt trans- sets of thymic and peripheral cells. In the thymus, these are the gene expression from any negative regulatory mechanism in mem- same cell populations that are known to express IL-2 in response ory cells that acts at the level of IL-2 mRNA stability. In any case, Downloaded from to developmental signals (2–5), including two subsets of TCR- the 8kbIL2p-GFP construct reveals a possible regulatory difference negative thymocytes, as well as mature TCR␣␤ and TCR␥␦ T cells between naive and memory T cells, and offers a tool to dissect its (M. A. Yui and E. V. Rothenberg, manuscript in preparation). mechanism. Thus, both transgenic constructs appear to be expressed in the compartments in which IL-2 is normally expressed. Cell specificity in the 8kbIL2p-GFP and 2kbIL2p-GFP ϩ ϩ IL-2 regulation is complex, and it will require further work to transgenic lines: differential regulation in CD4 and CD8 http://www.jimmunol.org/ determine all the respects in which the transgenes may or may not cells reproduce the regulation of the endogenous genes. However, based In the few 2kbIL2p-GFP transgenic lines that do express the trans- on quantitative PCR analysis of mRNA from 8-kb transgenic gene, the inducibility of GFP and fidelity of transgene expression splenocytes after stimulation, GFP mRNA appears to be expressed are largely similar to those of the 8kbIL2p-GFP transgenics. How- from three to four transgene copies at a comparable level to IL-2 ever, this work presents evidence of one regulatory defect shown mRNA from the two endogenous gene copies. This suggests that by the 2kbIL2p-GFP lines even when they permit expression. Most the gross efficiency of 8-kb transgene expression can approach that 2kbIL2p-GFP lines have relatively high background levels of GFP of endogenous IL-2. Still under investigation are the responses of ϩ

in CD8 cells in thymus as well as spleen, while expression in by guest on September 29, 2021 the transgenes to different stimulation conditions, and the role of ϩ CD4 cells in both tissues remains indistinguishable from that in transgene copy number in the magnitude of the response. Under ϩ ϩ the 8.4-kb lines. Although both CD4 and CD8 cells can express certain conditions, the endogenous IL-2 gene can be expressed IL-2 normally (29–31), there is no evidence for any natural situ- monoallelically (36), through a mechanism that is not understood. ϩ ϩ ation in which CD8 cells express more IL-2 than CD4 cells. More recently, IL-2 expression has been found to be mono- or This suggests that there is a regulatory element or elements with biallelic, depending on the conditions of stimulation and the timing ϩ ϩ distinct activities in CD8 and CD4 cells, which map(s) between of the assay (37, 38), suggesting independent and stochastic reg- 2 and 8.4 kb upstream of the IL-2 promoter. Alternatively, the ulation of the two alleles. We do not know whether the allelic unit ϩ CD8 cell bias could be an artifact of the integration sites per- is each individual gene (or transgene) or a larger chromatin do- missive for 2kbIL2p-GFP expression. In either case, these results main. Transgenes generally integrate at a single site in a tandem offer the first evidence that a component of IL-2 regulation in array that may act as a single locus, e.g., due to local chromatin ϩ CD8 cells can be mediated by cis elements that are physically effects, or each transgene copy may act independently. In addition, ϩ separable from those that regulate expression in CD4 cells. the orientation of the transgenes relative to one another may affect the efficiency of transcription. Such mechanisms could complicate the relationship between GFP and IL-2 expression and may help to A tool for analysis of IL-2 developmental regulation explain why GFP does not show a strictly linear relationship with IL-2 is expressed in various developmental contexts that may ul- transgene copy number. timately make it most interesting as a marker for early hemopoietic In one respect, expression of the transgenes appears to deviate lineage decisions (4, 5, 42). The signals driving the earliest intra- from expression of endogenous IL-2. There is background GFP thymic IL-2 expression are unknown, but the transcription factors mRNA expression well above the detection threshold in freshly mediating this programmed induction could be identified, once the isolated splenocytes of every transgenic mouse tested, although in critical cis-regulatory sequences are characterized. The 8kbIL2p- many of these samples there is no detectable IL-2 mRNA expres- GFP transgene clearly includes these sequences and can be used as sion. A variable percentage of fresh splenic T cells also expresses a starting point for this search. In addition, the ability to isolate low levels of GFP protein, albeit at much lower levels than seen in primitive IL-2-expressing cells, without perturbation, based on experimentally stimulated cells, in all transgene-expressing 2- and strong, unambiguous GFP fluorescence, will make it possible to 8-kb lines. This analysis shows no evidence for spontaneous acti- determine precisely their range of developmental potentials. vation of the transgene in naive T cells in any of the transgenic In conclusion, the results shown in this study locate element(s) lines. Instead, in the spleen, spontaneously GFP-expressing CD4ϩ conferring efficient, reproducible, position-independent expression cells were found to be confined to the minority subset that ex- in vivo that is separate from most of the elements in the IL-2 gene presses effector/memory markers (39), apparently elicited as a re- that mediate inducibility and T cell specificity. The identification sult of specific immune responses to environmental Ags, or pos- of novel regulatory sequences between Ϫ8.4 and Ϫ2 kb makes it The Journal of Immunology 1739 possible to begin to dissect the molecular basis of in vivo devel- 18. Duncliffe, K. N., A. G. Bert, M. A. Vadas, and P. N. Cockerill. 1997. A T opmental regulation of IL-2 for the first time. In addition, these cell-specific enhancer in the interleukin-3 locus is activated cooperatively by Oct and NFAT elements within a DNase I-hypersensitive site. Immunity 6:175. new transgenic lines, which provide highly sensitive detection of 19. Agarwal, S., and A. Rao. 1998. Modulation of chromatin structure regulates individual live GFP/IL-2-expressing cells, should be a valuable cytokine gene expression during T cell differentiation. Immunity 9:765. 20. Bird, J. J., D. R. Brown, A. C. Mullen, N. H. Moskowitz, M. A. Mahowald, tool to define the developmental significance of IL-2 induction and J. R. Sider, T. F. Gajewski, C. R. Wang, and S. L. Reiner. 1998. Helper T cell inducibility in lymphocyte subsets and lymphoid precursors. differentiation is controlled by the cell cycle. Immunity 9:229. 21. Saparov, A., F. H. Wagner, R. Zheng, J. R. Oliver, H. Maeda, R. D. Hockett, and C. T. Weaver. 1999. Interleukin-2 expression by a subpopulation of primary T Acknowledgments cells is linked to enhanced memory/effector function. Immunity 11:271. We thank Stephen Hedrick for kindly providing the plasmid containing the 22. Siebenlist, U., D. B. Durand, P. Bressler, N. J. Holbrook, C. A. Norris, human ␤-globin 3Ј splicing and poly(A) addition sites and Michael Nish- M. Kamoun, J. A. Kant, and G. R. Crabtree. 1986. Promoter region of interleu- imura for the melanoma cell line MCA102. We gratefully acknowledge kin-2 gene undergoes chromatin structure changes and confers inducibility on chloramphenicol acetyltransferase gene during activation of T cells. Mol. Cell. help from Robert Chen for screening the genomic library for the IL-2 Biol. 6:3042. promoter clone and to Xiao Sun for sequencing assistance. Shelley Dia- 23. Ward, S. B., G. Hernańdez-Hoyos, F. Chen, M. Waterman, R. Reeves, and mond and Pat Koen of the Flow Cytometry Core Facility provided invalu- E. V. Rothenberg. 1998. Chromatin remodeling of the interleukin-2 gene: distinct able help with flow cytometry and cell sorting. We also thank Shirley alterations in the proximal versus distal enhancer regions. Nucleic Acids Res. 26:2923. Pease, Xin Yu, Bruce Kennedy, and Alba Granados of the Caltech Trans- 24. Diamond, R. A., S. B. Ward, K. Owada-Makabe, H. Wang, and genic and Knockout Core Facility for the generation and maintenance of E. V. Rothenberg. 1997. Different developmental arrest points in RAG-2Ϫ/Ϫ and the transgenic mice. All primers were made at the Caltech Biopolymer SCID thymocytes on two genetic backgrounds: developmental choices and cell Synthesis Facility, and sequencing was performed at the Caltech DNA death mechanisms before TCR gene rearrangement. J. Immunol. 158:4052. 25. Heid, C. A., J. Stevens, K. J. Livak, and P. M. Williams. 1996. Real time quan- Sequencing Core Facility. titative PCR. Genome Res. 6:986. Downloaded from 26. McGuire, K. L., and E. V. Rothenberg. 1987. Inducibility of interleukin-2 RNA References expression in individual mature and immature T lymphocytes. EMBO J. 6:939. 1. Weaver, C. T., A. Saparov, L. A. Kraus, W. O. Rogers, R. D. Hockett, and 27. Rothenberg, E. V., S. B. Ward, M. Yui, G. Hernańdez-Hoyos, F. Chen, R. P. Bucy. 1998. 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