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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 References This article cites 42 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/166/3/1730.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 expres- sion 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 con- ditions. 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.
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