Promoter Janus Kinase 3 Proximal Characterization and Analysis Of

The Journal of Immunology Characterization and Analysis of the Proximal Janus Kinase 3 Promoter1 Martin Aringer,2*† Sigrun R. Hofmann,2* David M. Frucht,* Min Chen,* Michael Centola,* Akio Morinobu,* Roberta Visconti,* Daniel L. Kastner,* Josef S. Smolen,† and John J. O’Shea3* Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase essential for signaling via cytokine receptors that comprise the common ␥-chain (␥c), i.e., the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Jak3 is preferentially expressed in hemopoietic cells and is up-regulated upon cell differentiation and activation. Despite the importance of Jak3 in lymphoid development and immune function, the mechanisms that govern its expression have not been defined. To gain insight into this issue, we set out to characterize the Jak3 promoter. The 5؅-untranslated region of the Jak3 gene is interrupted by a 3515-bp intron. Upstream of this intron and the transcription initiation site, we identified an ϳ1-kb segment that exhibited lymphoid-specific promoter activity and was responsive to TCR signals. Truncation of this fragment revealed that core promoter activity resided in a 267-bp fragment that contains putative Sp-1, AP-1, Ets, Stat, and other binding sites. Mutation of the AP-1 sites significantly diminished, whereas mutation of the Ets sites abolished, the inducibility of the promoter construct. Chromatin immunoprecipitation assays showed that histone acetylation correlates with mRNA expression and that Ets-1/2 binds this region. Thus, transcription factors that bind these sites, especially Ets family members, are likely to be important regulators of Jak3 expression. The Journal of Immunology, 2003, 170: 6057–6064. anus kinase 3 (Jak3)4 is a tyrosine kinase of major impor- important role in signaling. IL-7 has been demonstrated to be es- tance in the signaling pathways of the IL-2, -4, -7, -9, -15, sential for proper lymphoid survival and function in mice and hu- J and -21, whose receptors all comprise the common ␥-chain mans (13–19). By contrast, IL-15 is essential for proper NK cell (␥c) (1–5). Via its N terminus, Jak3 binds the intracellular portion development, as is apparent in mice deficient in this cytokine re- of ␥c (6, 7), whereas another kinase, Jak1, binds the ligand-specific ceptor (20). The absence of signaling by both these cytokines in subunit. The importance of Jak3 is most vividly illustrated by the Jak3 SCID explains the block of lymphoid and NK cell develop- fact that mutations of human Jak3 are associated with SCID (8– ment seen in this disorder. The absence of IL-2 or one of its re- 10). Deficiency of Jak3 and ␥c in humans is typically associated ceptor subunits is associated with lymphoproliferation and auto- with the lack of T cell and NK cell development; B cells develop, immune disease (21–23); the absence of IL-2 signaling probably but are poorly functional. In some patients with leaky mutations, explains why the remnant T lymphocytes of some Jak3 SCID pa- small numbers of T cells develop and, interestingly, exhibit acti- tients and of the knockout mice have an activated phenotype. The Ϫ Ϫ vation markers (11). Jak3 / mice also have profound immuno- distinct effects of Jak3 deficiency on B cell development in humans deficiency, but the phenotype is somewhat different from that of and mice presumably reflect differences in cytokine requirements human Jak3 deficiency. These mice lack B cells and have small or cytokine expression in the two species, although the basis for numbers of T cells, which also have an activated phenotype (12). the differences has not been elucidated. It is interesting, however, The major defects in lymphoid development that occur in Jak3 that IL-21 appears to be an important regulator of murine B cell SCID can be explained in large measure by the function of three function (24, 25). The expression of Jak3 is of interest in that it is essential ␥c-using cytokines, for which Jak3 evidently plays an predominantly expressed in cells of hemopoietic origin and in or- gans where these cells are found (26–29). The abnormalities of patients with Jak3 defects are consistent with such a tissue distri- *Molecular Immunology and Inflammation Branch, National Institute of Arthritis and bution, as is the fact that successful bone marrow transplantation is Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892; and †Department of Rheumatology, Internal Medicine III, University of Vi- essentially curative (9). Whereas the original reports on human and enna, Vienna, Austria murine distribution indicated absent or very low level Jak3 expres- Received for publication February 27, 2003. Accepted for publication April 7, 2003. sion in nonhemopoietic tissues (26, 28), other reports reached The costs of publication of this article were defrayed in part by the payment of page slightly different conclusions. Several studies have noted Jak3 ex- charges. This article must therefore be hereby marked advertisement in accordance pression in endothelium, glomeruli, and mesangial cells (30–32). with 18 U.S.C. Section 1734 solely to indicate this fact. Another study described wide expression of putative Jak3 mRNA 1 This work was supported by a Max Kade Foundation Postdoctoral Research Ex- change Grant (to M.A.). species in a variety of tissues (29); however, there was no direct 2 M.A. and S.R.H. contributed equally to this work. comparison with cells that express high levels of Jak3 (such as NK 3 Address correspondence and reprint requests to Dr. John J. O’Shea, Molecular Im- or activated T cells). Thus, whereas lymphoid and hemopoietic munology and Inflammation Branch, National Institute of Arthritis and Musculoskel- cells clearly express high levels of Jak3, it remains to be deter- etal and Skin Diseases, National Institutes of Health, 10 Center Drive, MSC-1820, mined whether Jak3 is expressed in meaningful levels in other Building 10, Room 9N252, Bethesda, MD 20892-1820. E-mail address: [email protected] tissues. It is notable that neither Jak3-deficient patients nor knock- 4 Abbreviations used in this paper: Jak3, Janus kinase 3; ␥c, common ␥-chain; ChIP, out mice suffer from any other abnormalities than those caused by chromatin immunoprecipitation. a lack of functional immune cells. Nonetheless, it is also pertinent Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 6058 JAK3 PROMOTER CHARACTERIZATION AND ANALYSIS that despite the high level expression of Jak3 in monocytes, no Subcloning and luciferase assays significant defects of this cell lineage have been found in Jak3 Putative promoter constructs were generated by PCR from genomic DNA SCID patients (33). While it is clear that Jak3 is highly expressed and cloned into the Topo TA vector (Invitrogen, Carlsbad, CA). The inserts in hemopoietic cells, its relative expression and physiologic or were excised and subcloned into the pGL3-Basic vector (Promega, Mad- pathologic functional relevance in nonhemopoietic tissues remain ison, WI). The luciferase constructs were verified by sequencing. Two to be determined. micrograms of purified plasmid DNA was used per data point in each transfection together with 0.3 ␮g of pEF1/His/lacZ plasmid (␤-galactosi- Another interesting aspect of the expression of Jak3 is that it is dase vector; Invitrogen) using Superfect transfection reagent (Qiagen, Va- highly regulated by cell activation and cell differentiation. That is, lencia, CA). As positive control vector we used a CMV-promoter driven in T cells, lectins or phorbol esters induce Jak3 up-regulation (26, pGL3 vector. Alternatively, human PBMC and NK3.3 were transfected by 34). Similarly, B cell receptor cross-linking or CD40 cross-linking electroporation using the human T cell Nucleofector Kit (Amaxa Biosys- tems, Cologne, Germany) according to the commercial protocol, using 5 in B lymphocytes up-regulates Jak3 expression (35), and LPS, ␮g of purified plasmid DNA together with 0.3 ␮g of pEF1/His/lacZ plas- IFN-␥, and IL-2 induce its expression in monocytes (36). In ad- mid. The transfection efficiency of electroporation, determined by flow dition, Jak3 was induced during the terminal differentiation of my- cytometry after transfecting a green fluorescence protein vector, was 30– eloid cell lines (37), whereas, in contrast to B cells, mature plasma 35%. The following day the cells were activated as described and harvested cells express only very low levels of Jak3 (35). In thymus, Jak3 is after another 30–36 h of incubation. The cells were lysed in ice-cold Re- porter Lysis Buffer (Promega, Madison, WI), and the lysates were consec- highly expressed at all stages, as it is in adult, double-negative utively analyzed for luciferase and ␤-galactosidase activities with the Dual thymocytes (28). Given the activation-dependent regulation, it is Light Kit (Tropix, Bedford, MA) on a Monolight 3010 luminometer (An- tempting to speculate that Jak3 might be induced in a variety of alytical Luminescence Laboratory, Sparks, MD); luciferase expression is cells by inflammatory stimuli. This could explain the expression of represented as normalized by ␤-galactosidase activity (expressed as a percentage). Jak3 in nonlymphoid cells, particularly in pathological settings (30–32). Site-directed mutagenesis Despite this wealth of interesting findings, the promoter ele- The Ϫ74/ϩ27 and/or the Ϫ122/ϩ27 truncated luciferase promoter con- ments of JAK3 have not yet been characterized. Because of the structs were used as a template for mutations disrupting the binding con- pivotal role of Jak3 in lymphoid development and because of the sensus of several sites, using the Transformer site-directed mutagenesis kit interesting aspects of its regulated expression, we therefore (Clontech, Palo Alto, CA) as previously described (40). The following primers (mutated base pairs are underlined) were used for the construct thought it important to identify the Jak3 promoter and to begin lacking the Stat binding site and both AP-1 binding sites, respectively: defining the mechanisms involved in its regulation.

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