The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aringer, M.
Right arrow Articles by O’Shea, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aringer, M.
Right arrow Articles by O’Shea, J. J.
The Journal of Immunology, 2003, 170: 6057-6064.
Copyright © 2003 by The American Association of Immunologists

Characterization and Analysis of the Proximal Janus Kinase 3 Promoter1

Martin Aringer2,*,{dagger}, Sigrun R. Hofmann2,*, David M. Frucht*, Min Chen*, Michael Centola*, Akio Morinobu*, Roberta Visconti*, Daniel L. Kastner*, Josef S. Smolen{dagger} and John J. O’Shea3,*

* Molecular Immunology and Inflammation Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892; and {dagger} Department of Rheumatology, Internal Medicine III, University of Vienna, Vienna, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase essential for signaling via cytokine receptors that comprise the common {gamma}-chain ({gamma}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 ~1-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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Janus kinase 3 (Jak3)4 is a tyrosine kinase of major importance in the signaling pathways of the IL-2, -4, -7, -9, -15, and -21, whose receptors all comprise the common {gamma}-chain ({gamma}c) (1, 2, 3, 4, 5). Via its N terminus, Jak3 binds the intracellular portion of {gamma}c (6, 7), whereas another kinase, Jak1, binds the ligand-specific subunit. The importance of Jak3 is most vividly illustrated by the fact that mutations of human Jak3 are associated with SCID (8, 9, 10). Deficiency of Jak3 and {gamma}c in humans is typically associated with the lack of T cell and NK cell development; B cells develop, but are poorly functional. In some patients with leaky mutations, small numbers of T cells develop and, interestingly, exhibit activation markers (11). Jak3-/- mice also have profound immunodeficiency, but the phenotype is somewhat different from that of human Jak3 deficiency. These mice lack B cells and have small numbers of T cells, which also have an activated phenotype (12).

The major defects in lymphoid development that occur in Jak3 SCID can be explained in large measure by the function of three essential {gamma}c-using cytokines, for which Jak3 evidently plays an important role in signaling. IL-7 has been demonstrated to be essential for proper lymphoid survival and function in mice and humans (13, 14, 15, 16, 17, 18, 19). By contrast, IL-15 is essential for proper NK cell development, as is apparent in mice deficient in this cytokine receptor (20). The absence of signaling by both these cytokines in Jak3 SCID explains the block of lymphoid and NK cell development seen in this disorder. The absence of IL-2 or one of its receptor subunits is associated with lymphoproliferation and autoimmune disease (21, 22, 23); the absence of IL-2 signaling probably explains why the remnant T lymphocytes of some Jak3 SCID patients and of the knockout mice have an activated phenotype. The distinct effects of Jak3 deficiency on B cell development in humans and mice presumably reflect differences in cytokine requirements or cytokine expression in the two species, although the basis for the differences has not been elucidated. It is interesting, however, that IL-21 appears to be an important regulator of murine B cell function (24, 25). The expression of Jak3 is of interest in that it is predominantly expressed in cells of hemopoietic origin and in organs where these cells are found (26, 27, 28, 29). The abnormalities of patients with Jak3 defects are consistent with such a tissue distribution, as is the fact that successful bone marrow transplantation is essentially curative (9). Whereas the original reports on human and murine distribution indicated absent or very low level Jak3 expression in nonhemopoietic tissues (26, 28), other reports reached slightly different conclusions. Several studies have noted Jak3 expression in endothelium, glomeruli, and mesangial cells (30, 31, 32). Another study described wide expression of putative Jak3 mRNA species in a variety of tissues (29); however, there was no direct comparison with cells that express high levels of Jak3 (such as NK or activated T cells). Thus, whereas lymphoid and hemopoietic cells clearly express high levels of Jak3, it remains to be determined whether Jak3 is expressed in meaningful levels in other tissues. It is notable that neither Jak3-deficient patients nor knockout mice suffer from any other abnormalities than those caused by a lack of functional immune cells. Nonetheless, it is also pertinent that despite the high level expression of Jak3 in monocytes, no significant defects of this cell lineage have been found in Jak3 SCID patients (33). While it is clear that Jak3 is highly expressed in hemopoietic cells, its relative expression and physiologic or pathologic functional relevance in nonhemopoietic tissues remain to be determined.

Another interesting aspect of the expression of Jak3 is that it is highly regulated by cell activation and cell differentiation. That is, in T cells, lectins or phorbol esters induce Jak3 up-regulation (26, 34). Similarly, B cell receptor cross-linking or CD40 cross-linking in B lymphocytes up-regulates Jak3 expression (35), and LPS, IFN-{gamma}, and IL-2 induce its expression in monocytes (36). In addition, Jak3 was induced during the terminal differentiation of myeloid cell lines (37), whereas, in contrast to B cells, mature plasma cells express only very low levels of Jak3 (35). In thymus, Jak3 is highly expressed at all stages, as it is in adult, double-negative thymocytes (28). Given the activation-dependent regulation, it is tempting to speculate that Jak3 might be induced in a variety of cells by inflammatory stimuli. This could explain the expression of Jak3 in nonlymphoid cells, particularly in pathological settings (30, 31, 32).

Despite this wealth of interesting findings, the promoter elements of JAK3 have not yet been characterized. Because of the pivotal role of Jak3 in lymphoid development and because of the interesting aspects of its regulated expression, we therefore thought it important to identify the Jak3 promoter and to begin defining the mechanisms involved in its regulation. In particular, we wanted to find elements that could explain the hemopoietic and activation-dependent expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells, cell lines, and stimulation

PBMC were prepared from venous blood anticoagulated with preservative-free heparin sedimented over Ficoll-Paque Plus (Amersham Pharmacia Biotech, Piscataway, NJ) gradients. PBMC, Jurkat cells, JCAM1.1 (Lck-negative TCRbright Jurkat), and NK3.3 cells were grown in RPMI 1640 containing 10% FCS, glutamine, and antibiotics (Biofluids, Rockville, MD). Jurkat cells that express large T Ag (Jurkat T) were also used in these studies; these cells constitutively express more Jak3 than parental Jurkat cells (data not shown). Medium for NK3.3 was enriched with Lymphocult-T (Biotest, Denville, NJ) and 40 U of IL-2/ml. COS7 and HeLa cells were grown in DMEM containing 10% FCS, glutamine, and antibiotic/antimycotic. Where described, cells were stimulated with PMA (50 ng/ml), ionomycin (3.5 µg/ml), cyclosporin A (500 ng/ml), or PHA (1 µg/ml; all from Sigma-Aldrich, St. Louis, MO). For stimulation with specific Abs, six-well tissue culture plates (BD Biosciences, Franklin Lakes, NJ) were incubated with PBS containing anti-CD3 and/or anti-CD28 (10 µg/ml; both from R&D Systems, Minneapolis, MN) for 16 h.

5' RACE and DNA sequencing

Total RNA was purified from Jurkat T cells activated with PHA for 24 h and from NK3.3 cells using the RNeasy MiniKit (Qiagen, Valencia, CA) or RNA STAT-60 (Tel-Test, Friendswood, TX). 5' RACE was performed according to a commercial protocol (Life Technologies, Gaithersburg, MD) using the following primers: 5'-tgcttgatgctaatgtctacgatt-3', 5'-tccaggtccatgatgtacttggcc-3', 5'-gtggtacacaggcaggatgccgctg-3', and 5'-tcctccgtggccagagcaaa-3'. The F23402 cosmid was provided by L. K. Ashworth (Human Genome Center at the Lawrence Livermore National Laboratory). Genomic DNA was purified from an EBV-transformed B cell line of a healthy donor using the QiaAmp kit (Qiagen, Santa Clarita, CA). PCRs were performed using Elongase (Life Technologies) on a Gene Amp 9700 PCR system (PerkinElmer, Norwalk, CT); the PCR products were purified over microconcentrators (Amicon, Beverly, MA). Sequencing reactions were performed with the same thermocycler using the Big Dye Terminator Kit (PE Applied Biosystems, Foster City, CA). The products were then purified over G-50 columns (5 Prime 3 Prime, Inc., Boulder, CO), vacuum-dried, and sequenced on an ABI 377 automated sequencer as previously described (38). Candidate promoter regions were scanned for possible transcription factor binding sites using the programs of the TRANSFAC Internet site (39).

Subcloning and luciferase assays

Putative promoter constructs were generated by PCR from genomic DNA and cloned into the Topo TA vector (Invitrogen, Carlsbad, CA). The inserts were excised and subcloned into the pGL3-Basic vector (Promega, Madison, WI). The luciferase constructs were verified by sequencing. Two micrograms of purified plasmid DNA was used per data point in each transfection together with 0.3 µg of pEF1/His/lacZ plasmid ({beta}-galactosidase vector; Invitrogen) using Superfect transfection reagent (Qiagen, Valencia, CA). As positive control vector we used a CMV-promoter driven pGL3 vector. Alternatively, human PBMC and NK3.3 were transfected by electroporation using the human T cell Nucleofector Kit (Amaxa Biosystems, Cologne, Germany) according to the commercial protocol, using 5 µg of purified plasmid DNA together with 0.3 µg of pEF1/His/lacZ plasmid. The transfection efficiency of electroporation, determined by flow cytometry after transfecting a green fluorescence protein vector, was 30–35%. The following day the cells were activated as described and harvested after another 30–36 h of incubation. The cells were lysed in ice-cold Reporter Lysis Buffer (Promega, Madison, WI), and the lysates were consecutively analyzed for luciferase and {beta}-galactosidase activities with the Dual Light Kit (Tropix, Bedford, MA) on a Monolight 3010 luminometer (Analytical Luminescence Laboratory, Sparks, MD); luciferase expression is represented as normalized by {beta}-galactosidase activity (expressed as a percentage).

Site-directed mutagenesis

The -74/+27 and/or the -122/+27 truncated luciferase promoter constructs were used as a template for mutations disrupting the binding consensus of several sites, using the Transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA) as previously described (40). The following primers (mutated base pairs are underlined) were used for the construct lacking the Stat binding site and both AP-1 binding sites, respectively: 5'-CCTGACTTTCGGTTTATGACAGTGGCTCAGGAA-3' and 5'-CAGCTCCCGCAGGGCCCTTCCTTTCGGTAAATTCCAGTGGCTCAGACCAAGG-3'. To create the three Ets-1 mutations, we used the following primers: 5'-CGGCTGATGCAGAGGCTTCCGCTACTTTCTGCCCCTCCTTGCCCG-3', 5'-GAAACCAAGGGGCCCACACAGCTAGGAGCCGAGTGGGACTTTCCTC-3', and 5'-GGGCGGCTGATGCAGAGGCTGACGCTACTTTCTGCCCCTCCTTGC-3'. All mutations were verified by sequencing. Transfections and luciferase assays were performed as described above.

Chromatin immunoprecipitation (ChIP) assay

ChIP assays were performed according to protocol using a commercially available kit (Upstate Biotechnology, Lake Placid, NY) and polyclonal Abs against Ets-1/2 and Stat-6 (Santa Cruz Biotechnology, Santa Cruz, CA) as well as antiacetylated histone H3 and rabbit IgG (negative control; Upstate Biotechnology, Lake Placid, NY). Jurkat, HeLa, and COS7 cells were cross-linked with formaldehyde, lysed, and sonicated using an XL2020 sonicator (Heat Systems Ultrasonics, Farmingdale, NY); an aliquot of the sonicated cells was amplified as input and immunoprecipitated with Abs to acetylated histone H3, Ets-1/2, or control Ab (rabbit IgG). After reversing the cross-linking, PCR for the Jak3 gene was performed (35 cycles; 30 s at 95°C, 45 s at 59°C, 45 s at 72°C) using the following primers for the human Jak3 gene: 5'-ctcacacatgctacagatggg-3' and 5'-ggtttcctgagccactgtcat-3' (275-bp product; including Jak3 promoter region -6 to -281). The PCR products were visualized on an ethidium bromide gel.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of the Jak3 transcriptional start site and organization of the 5' region of the Jak3 gene

To identify the transcriptional start site in the Jak3 gene, we employed 5' RACE using mRNA from Jurkat T or NK3.3 cells. We identified multiple products corresponding to 5'-untranslated sequence, the largest segment containing 111 bp of untranslated mRNA (Fig. 1). The first base of this largest product was therefore assigned position +1 and is identical with base 27,800 of the human chromosome 19 cosmid R34383 as deposited in GenBank by Lamerdin et al. (AC007201). Lai et al. (41) identified the same 5'-untranslated segment of the Jak3 mRNA, but it was 20 bp shorter than the longest product found by us. These differences in length may be due to alternative transcriptional start sites.



View larger version (85K):
[in this window]
[in a new window]
  		FIGURE 1. The Jak3 mRNA start sites and the upstream region containing the putative promoter. The Jak3 mRNA is printed in bold letters interrupted by a 3515-bp intron; the initiation codon is heavily underlined. The first base pairs of all mRNAs found in this study are underlined; the first base pair of our longest mRNA piece is denoted +1. The first base pair of the study by Lai (41 ) is marked by a double underline; the common piece of all published mRNA sequences (26 41 ) upstream of the large intron is marked by a dotted underline.

 
Comparison of the obtained mRNA with genomic DNA showed a 3,515-bp intron with a splice acceptor site 13 bp upstream of the ATG codon (Fig. 1), again consistent with the reported R34383 sequence (intron from base 27,711 to base 24,197). The intron ends with a typical pyrimidine-rich stretch and AG. After identifying the region of the bona fide transcriptional start site, we sequenced roughly 1,000 bp 5' to identify potential promoter/enhancer elements. Fig. 1 shows 1,050 bp of genomic DNA upstream of the transcriptional start site containing the putative promoter region. With the exception of 1 bp (g instead of t at -947), this sequence is identical with the R34383 sequence (28,850 through 27,801 bp) as well as with bp 314,420 through 313,371 of the Homo sapiens 19p13.1 sequence (Hs19_11445) (42), in accordance with the known chromosomal localization of Jak3.

The upstream JAK3 region contains tissue-specific promoter activity

We next examined whether the DNA upstream of the transcriptional start site contained promoter activity. To this end we subcloned a 1,039-bp fragment of upstream DNA (-1013 to +27) into the pGL3 vector. Transfection of this construct into Jurkat T cells resulted in considerable basal promoter activity, >20-fold higher than that in the empty vector (Fig. 2A). Since promoters typically only function in the correct orientation, we also transfected a promoter construct that contained the same 1,039 bp but in reverse orientation. This reverse construct had only minimal promoter activity (Fig. 2A). These findings led us to conclude that the roughly 1,000 bp upstream of the Jak3 transcriptional start site do indeed contain promoter activity.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 2. The putative Jak3 promoter is active in T, but not in nonlymphoid, cells and is induced by lectin and phorbol esters. A, Jurkat T cells were transfected with empty vector, vector containing the putative Jak3 promoter region, or vector containing the putative promoter in the reverse direction. Bars show the mean ± SD of arbitrary light units normalized for {beta}-galactosidase ({beta}-Gal) light units (samples were measured as triplets). B, COS7 cells were cotransfected with a {beta}-galactosidase vector plus empty vector, vector containing the putative promoter, or a positive control vector (CMV-promoter driven pGL3 vector). Numbers given are luciferase light units normalized for {beta}-Gal light units. C, The acetylation status of histone H3 in the nucleosomes associated with the Jak3 core promoter region was assessed by ChIP assay in Jurkat and COS7 cells. Cells were lysed, and proteins were cross-linked with formaldehyde and immunoprecipitated with Ab to acetyl histone H3 (anti-acetyl H3) or control Ab (rabbit IgG). After reversing the cross-linking, PCR for the Jak3 gene was performed. Input represents PCR amplification of the total sample. Results are representative of two independent experiments. D, Jurkat T cells were cotransfected with a {beta}-galactosidase vector plus empty vector, vector containing the putative Jak3 promoter, or vector containing the putative promoter in the reverse orientation and were cultured without stimulation or with PHA or PMA. Results are expressed as luciferase light units normalized for {beta}-Gal light units. Results in A–D are representative of two independent experiments.

 
Previous data have indicated two features of Jak3 expression. First, Jak3 is abundantly expressed in lymphoid and hemopoietic cells, but is not highly expressed in other cell types. Second, Jak3 expression apparently is activation dependent. Therefore, we next wanted to address whether the putative Jak3 promoter region could be responsible for such regulation.

To test whether our promoter fragment also contained elements responsible for Jak3 tissue specificity, we transfected the identical construct into nonlymphoid cells. COS7, an embryonic kidney cell line, was useful, in that it does not express endogenous Jak3. The Jak3 promoter construct did not have activity in COS cells (Fig. 2B) or HeLa cells (not shown), in contrast to a control construct driven by the CMV promoter. Thus, the Jak3 promoter construct also contained tissue-specific elements.

Chromatin remodeling is an important regulator of accessibility of DNA to transcription factors and subsequent transcriptional activation. Post-translational modification of histones, especially histone acetylation, typically indicates remodeled genes, which are actively transcribed. We therefore examined whether histone acetylation in the Jak3 gene correlates with Jak3 expression. Using the ChIP assay, comparing lymphoid vs nonlymphoid cells, we noted histone H3 acetylation in the Jak3 promoter in Jurkat (Fig. 2C, lane 5) and NK3.3 cells (not shown), but not in COS7 (lane 2) or HeLa (not shown) cells. The input DNA was equivalent (lanes 1 vs 4).

The putative Jak3 promoter region is induced by T cell activation

Having demonstrated that the promoter construct was active in lymphocytes, we next investigated whether it was also regulated in an activation-dependent manner. We and others have shown that lectins and phorbol ester up-regulate Jak3 expression (26, 34). We therefore tested whether the candidate promoter segment we isolated could also be induced by these stimuli. As shown in Fig. 2D, in the absence of stimulation the promoter construct is expressed in Jurkat cells. Additionally, treatment of the cells with either PHA or PMA amplified expression (>4- and 6-fold, respectively). In contrast, the promoter construct in the reverse orientation was not activatable. Taken together, these experiments are consistent with the following conclusions. Jak3 gene promoter activity 1) resides in the region upstream of the Jak3 transcriptional start site and is orientation dependent; 2) is present in lymphoid, but not in COS7 or HeLa cells; and 3) is both basally active and inducible. Based on these properties we will therefore refer to the segment isolated as the proximal Jak3 promoter.

Signaling via the TCR activates the Jak3 promoter

PHA and PMA are nonspecific stimuli used experimentally to mimic signaling by the TCR. We next examined whether TCR cross-linking by an anti-CD3{epsilon} Ab bound to tissue culture plates also trans-activated the Jak3 reporter construct. As shown in Fig. 3A, TCR cross-linking was almost as efficient as PMA in activating the Jak3 promoter construct (>70% of the PMA-induced activation). Co-occupancy of the TCR and CD28 is a very potent stimulus for activating T cells. Some, but not all, T cell activation genes are synergistically induced by this combination. We therefore next investigated whether the cross-linking of CD28, either alone or in conjunction with TCR cross-linking, had an effect on the Jak3 promoter. As shown in Fig. 3A, CD28-dependent signals appeared to have little effect on the expression of the reporter construct.



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 3. The Jak3 promoter can be activated by TCR cross-linking. A, Jurkat T cells were cotransfected with a {beta}-galactosidase ({beta}-Gal) vector plus the luciferase vector containing the putative promoter region and were cultured in medium, in medium containing PMA, or on plates with immobilized anti-CD3, immobilized anti-CD28, or both. Results given are the mean ± SD of luciferase light units normalized for {beta}-Gal light units of the same sample. Results are representative of two independent experiments. B, Jurkat T cells and Lck-negative Jurkat cells (JCAM 1.1) expressing comparable levels of TCR (not shown) were cotransfected with a {beta}-Gal vector plus the luciferase vector containing the Jak3 promoter region and were cultured in medium without activating agents or on immobilized anti-CD3 or with PMA. Results are luciferase light units normalized for {beta}-Gal light units and are representative of two independent experiments.

 
Activation of the Src family kinase, Lck, is an early and essential step in TCR signal transduction. We therefore next wanted to investigate whether promoter activation followed the usual pathway downstream of TCR cross-linking, which is dependent on Lck. To this end we compared wild-type Jurkat T cells with Lck-negative JCAM1.1 cells (Fig. 3B). Both cell lines had equivalent high levels of CD3 surface expression as determined by flow cytometry (not shown). Whereas both PMA and anti-CD3 stimulation activated the promoter construct in wild-type Jurkat T cells, stimulation with PMA, but not with anti-CD3, led to promoter activity in JCAM1.1 cells (Fig. 3B). In conclusion, these findings indicated that TCR-dependent signals were capable of inducing this Jak3 promoter construct.

The major promoter activity resides within 240 bp upstream of the mRNA start site

We next tried to delimit the essential regions of the Jak3 promoter in terms of both basal and inducible activities. For this purpose a series of 5' and 3' truncation constructs were generated and transfected into Jurkat T cells. As shown in Fig. 4A, the promoter activity was similar after reducing the fragment to a 267-bp (-240/+27) segment, leaving the region upstream of the transcriptional start site intact. In contrast, a 938-bp construct lacking 75 bp upstream of the transcriptional start was completely inactive, indicating that this deletion disrupted the core promoter region. This indicated that the Jak3 promoter was located within 240 bp upstream of the longest obtained cDNA and contained essential elements within 75 bp 5' of position +1.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 4. Truncation defines the minimal active promoter region. A, Jurkat T cells were cotransfected with a {beta}-Gal vector and equimolar amounts of luciferase vector containing different pieces (base pairs above each column) of the Jak3 promoter region and cultured with or without activation on immobilized CD3 Abs. Results are representative of two independent experiments. B, Within the active promoter area, consensus transcription factor core binding sites are underlined, whereas the dashed underline denotes the binding matrix areas. C, The series of constructs used for delimiting the active promoter region are shown; important consensus binding sites still present on the respective construct are indicated.

 
The 267 bp of DNA containing the promoter activity are shown in Fig. 4B. This sequence was analyzed using the MatInspector program (43) and revealed a number of potential transcription factor binding sites. Using 5'-truncated mutants (Fig. 4C), the relative importance of the various elements was analyzed (Fig. 5A). Loss of the putative AP4 and AP1 sites between -240 and -172 had essentially no effect, whereas removal of the GATA and AP4 sites between -172 and -122 led to a mild increase in promoter activity. Removing the segment between -122 and -101, containing potential binding sites for Myb, Sp1, and AP4, did not appear to affect promoter activity. Further truncation (beyond -101), however, considerably reduced basal promoter activity. Specifically, removal of a putative double Ets-1 binding site overlapping a C/EBP site diminished the baseline activity by almost two-thirds, although the relative increase after TCR stimulation was unchanged. Continued truncation, which removed the GC/Sp1 sites overlapping with an Ikaros consensus site, completely abolished baseline activity and also led to a loss of inducibility, indicating that these GC-rich motifs are part of the Jak3 core promoter.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 5. Truncations and specific mutations confirm the important role of AP-1 and Ets-1 sites. A, Jurkat T cells were cotransfected with a {beta}-galactosidase ({beta}-Gal) vector and equimolar amounts of empty luciferase vector or luciferase vector containing 5' truncation constructs of the Jak3 promoter region and cultured in the presence or the absence of immobilized CD3 Abs. Results are the mean ± SD of luciferase light units normalized for {beta}-Gal light units and are representative of three independent experiments. B, The same approach as in A was used to test 3' truncation constructs. C, Using the same approach in the context of the -74/+27 construct, a 5',3' truncation mutant lacking Ets-1 binding sites, and constructs with both AP-1 sites or the Stat site mutated were transfected and compared with the -74/+27 construct and the empty vector. Results in B and C are representative of two independent experiments.

 
To further define the major elements that support Jak3 transcriptional control, a series of 3' truncation mutants (Fig. 4C) were made in the context of the active -240/+27 construct (Fig. 5B). Compared with the whole 267-bp construct, the baseline activity was reduced by 80% when the 3' Ets-1 binding site was lost (-240/+2 construct), also removing a distal Ikaros site, although the relative induction remained similar to that of the complete construct. Removal of another 18 bp containing NF-AT, C/EBP, Ets-1, and Ikaros binding sites (-240/-16 construct) reduced the remaining baseline activity by another 50%, but the relative induction stayed the same. Removing the AP1/Stat compound binding site and a putative Oct-1 site (-240/-40 construct; Fig. 5B) completely abolished both the baseline activity and the inducibility, arguing that this region contained part of the JAK3 core promoter.

Specific mutations confirm the roles of AP-1 sites and Ets-1 binding enhancers

To more specifically investigate the consensus AP-1/Stat/AP-1 compound site, point mutations were made in both AP-1 sites in the context of the -74/+27 construct. Mutation of the AP-1 sites reduced baseline promoter activity to about one-fourth, demonstrating the important role of AP-1 for the baseline activity of the Jak3 promoter (Fig. 5C). Mutation of these sites in the context of the larger -122/+27 construct significantly reduced luciferase activity (Fig. 6A), but did not affect inducibility. In contrast, mutation of the consensus Stat binding site did not diminish promoter activity; however, the cytokine inducibility of this construct was not assessed.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 6. AP-1 and Ets-1 sites are critical for expression of the Jak3 promoter. A, Jurkat T cells were cotransfected with a {beta}-galactosidase ({beta}-Gal) vector and equimolar amounts of empty luciferase vector or luciferase vectors containing the -122/+27 or the -74/+27 5' truncation constructs of the Jak3 promoter region with the AP-1 sites intact or mutated ({Delta}AP-1) and cultured in the presence or the absence of immobilized CD3 Abs. Results are the mean ± SD of luciferase light units normalized for {beta}-galactosidase light units. B, The same approach as in A was used in the context of the -122/+27 construct to test constructs where Ets-1 binding sites were mutated. C, Primary human T lymphocytes were transfected by electroporation with a {beta}-Gal vector and equimolar amounts of empty luciferase vector or luciferase vectors containing the whole promoter region or the -122/+27 construct with the 5' and 3' Ets-1 binding sites mutated and tested in the same way. D, ChIP assay with Jurkat cells and anti-Ets or control Ab. Cells were lysed, and proteins were cross-linked with formaldehyde and immunoprecipitated with Ab to Ets-1/2 (anti-Ets-1/2), Stat6 (anti-Stat6), or control Ab (rabbit IgG). After reversing the cross-linking, PCR for the Jak3 gene was performed. Input represents PCR amplification of the total sample. Results are representative of two independent experiments.

 
We also mutated the Ets-1 binding sites in the context of the -122/+27 fragment. Interestingly, the disruption of these sites led to a total loss in promoter activity (Fig. 6B). Moreover, it was sufficient to mutate either the 5' (-95/-91) or the 3' (+9) Ets-1 binding site to completely block promoter activity. These results confirm that the Ets-1 binding regions have an essential role in the regulation of the Jak3 promoter. Similar results were obtained using primary human lymphocytes. The basal Jak3 promoter activity in these cells, which shows about a 5-fold increase compared with the empty vector, is abrogated when the Ets sites are mutated (Fig. 6C). Comparable data were obtained using NK3.3 cells (not shown).

To confirm that Ets-1/2 binds the Jak3 promoter, we employed chromatin immunoprecipitation assays, which involve cross-linking of proteins to DNA in vivo, followed by immunoprecipitation of a specific protein and identification of its associated promoter DNA by PCR. By using Abs that are specific for individual Ets domain proteins, in vivo targets can be unambiguously identified. As shown in Fig. 6D, the Jak3 promoter was amplified when the immunoprecipitation was performed using a polyclonal Ab specific for Ets-1/2 (lane 4), but not with a control Ab.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we report the identification and characterization of the promoter region of the Jak3 gene, hoping to gain insight into two properties of Jak3: its propensity to be expressed in hemopoietic cells and its induction following cell activation.

All Jak3 mRNAs found in Jurkat T and NK cells begin 5' of a large 3,515 base-pair intron with a splice-acceptor site in the 5' untranslated region, 13 bp upstream of the ATG codon. This is similar to the putative murine Jak3 promoter, which also contains an intron in the 5' untranslated mRNA. All published human Jak3 mRNA sequences also contain sequence 5' of this large intron and start within the same region (26, 41). We therefore also expected to find promoter activity close to the transcriptional start sites.

Indeed, the 1-kbp region upstream of the transcriptional start site contained considerable promoter activity when cloned into a luciferase vector and transfected into Jurkat T cells. In contrast, no promoter activity was found in nonhemopoietic cells, such as COS7 or HeLa cells. Moreover, the promoter region was acetylated in lymphoid but not nonlymphoid cells, fitting the known tissue specificity of Jak3 (26, 27, 28, 29). Consistent with the expectation of finding the core promoter near the transcriptional start site, a 267-bp fragment (-240/+27) surrounding the mRNA start site contained most of the promoter function, suggesting that this region contains the major tissue-specific and TCR-inducible promoter elements. The described promoter region exhibited considerable baseline activity and was further induced by PMA, PHA, and direct TCR cross-linking. Moreover, the induction by TCR cross-linking was absent in the Lck-negative Jurkat cell line JCAM1.1. Therefore, the Jak3 promoter is responsive to TCR-mediated signals, and stimulation via the TCR clearly represents an important way of stimulating Jak3 expression.

Since COS cells do not express Jak3, the absence of promoter activity was expected. However, we considered the possibility that a negative control element might be operative in the vicinity of the promoter. Truncation would therefore be expected to eliminate such a repressor element, which would then lead to transcription, but none of a series of truncation constructs was expressed in COS7 cells (data not shown). These findings argue against the idea that Jak3 expression is down-regulated in nonlymphoid cells by engagement of a repressor. Rather, the data are consistent with the interpretation that the lack of Jak3 expression and the lack of promoter activity in nonlymphoid cells are probably due to the lack of positively acting transcription factors.

Several such transcription factors have binding sites in the 267-bp Jak3 promoter region. Particularly interesting among these are four consensus Ets-1 binding sites, mutation of which had dramatic effects on promoter activity. Importantly, Ets binding sites are also present in the putative promoters of the murine and pufferfish Jak3 gene (44, 45). Ets proteins are a large family of >50 evolutionary conserved transcription factors. These helix-turn-helix proteins do not typically dimerize, but, rather, form complexes with other transcription factors (46).

The founding member of this family, Ets-1, is expressed in human T, B, and NK cells and plays an important role in a variety of processes, including cell proliferation and differentiation, cell development, transformation, and apoptosis (47, 48, 49). Importantly, we identified Ets-1 binding to the Jak3 promoter using ChIP. This is notable because deficiency of Ets-1 (49, 50) has important effects on T cell survival and the development of NK cells (48); whether the absence of NK cells and the poor lymphoid survival in Ets-1-deficient conditions relate to Jak3 deficiency is an interesting possibility and needs to be explored. It may be relevant in this regard that hemopoietic cells express other Ets-1 family members, such as PU.1 (important for myeloid and lymphoid differentiation), SAP-1 (biological role in T cell differentiation), Fli-1 (megakaryocyte differentiation), and SpiB (B cell function), which might also regulate Jak3. While the ChIP assay showed that Ets-1/2 bind directly to the Jak3 promoter, it is conceivable that other Ets family members can also bind and might contribute to the regulation of Jak3; indeed, there might be cell type variation depending upon which family members are present in the greatest abundance.

The other motif present in the Jak3 promoter found to have significant effects were AP-1 sites. AP-1 complexes are heterodimers of Fos and Jun proteins, are ubiquitously expressed and downstream of mitogen-activated protein kinase cascades, and play an important role in the signaling response. The induction of Fos and the activation of Jun necessary to form the complex are well-known consequences of TCR signaling. Interestingly, Ets and AP-1 binding sites occur in a large number of promoter/enhancer elements, and functional cooperation between Ets and AP-1 is critical for controlled expression of many genes, including cytokines (47, 51, 52). It is notable, though, that AP-1 is not only activated in T cells following TCR cross-linking, but can be a consequence of a broad spectrum of stimuli in a variety of cells (53). Thus, it is still possible that under the appropriate circumstances activation of this pathway could lead to the expression of Jak3 in nonlymphoid cells.

In contrast to Ets-1 and AP-1 binding sites, the consensus NF-AT site also found 3' of the core promoter appeared to be of minor importance, given the modest loss in activity after removal of this region (Fig. 5B) and the minimal effect of cyclosporin A (data not shown). This is again consistent with data on the murine Jak3 promoter, where no NF-AT site was detected (44).

While the present studies were directed toward improving the understanding of Jak3 transcription, it should be noted that regulation of transcription alone is likely to be an incomplete explanation of the regulation of Jak3 expression. In addition, determinants such as the propensity to undergo ubiquitination and proteasomal degradation (54, 55) are likely to also affect the amount of Jak3 present in a given cell at a given time. It is also notable in this regard that the Jak3 gene has an AT-rich motif in its 3'-untranslated region, similar to cytokines; so message stability may also be an important, but unexplored, aspect of regulation. In view of the essential function of Jak3, it will definitely be of interest to define all the mechanisms involved in its regulation.

Taken together, the present data indicate that the Jak3 promoter is found 60 bp upstream of a large (roughly 3500 bp) intron in the nontranslated 5' portion of Jak3 mRNA. The promoter is tissue specific in that it is active in primary lymphocytes, Jurkat T cells, and NK3.3 cells, but not in COS or HeLa cells, consistent with the pattern of Jak3 expression. The core promoter region extends over roughly 60 bp and is surrounded by elements containing essential binding sites for Ets-1. These Ets-1 binding sites are of particular importance, as mutations of these sites lead to complete abrogation of promoter activity. Using this information, it will be important to define exactly which transcription factors regulate Jak3 expression in vivo in normal and pathologic states, in lymphoid and perhaps nonlymphoid cells.


   Footnotes
 
1 This work was supported by a Max Kade Foundation Postdoctoral Research Exchange Grant (to M.A.). Back

2 M.A. and S.R.H. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. John J. O’Shea, Molecular Immunology and Inflammation Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 10 Center Drive, MSC-1820, Building 10, Room 9N252, Bethesda, MD 20892-1820. E-mail address: osheajo{at}mail.nih.gov Back

4 Abbreviations used in this paper: Jak3, Janus kinase 3; {gamma}c, common {gamma}-chain; ChIP, chromatin immunoprecipitation. Back

Received for publication February 27, 2003. Accepted for publication April 7, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ihle, J. N., W. Thierfelder, S. Teglund, D. Stravapodis, D. Wang, J. Feng, E. Parganas. 1998. Signaling by the cytokine receptor superfamily. Ann. NY Acad. Sci. 865:1.[Medline]
  2. O’Shea, J. J.. 1997. Jaks, STATs, cytokine signal transduction, and immunoregulation: are we there yet?. Immunity 7:1.[Medline]
  3. Baird, A. M., D. C. Thomis, L. J. Berg. 1998. T cell development and activation in Jak3-deficient mice. J. Leukocyte Biol. 63:669.[Abstract]
  4. Aringer, M., A. Cheng, J. W. Nelson, M. Chen, C. Sudarshan, Y. J. Zhou, J. J. O’Shea. 1999. Janus kinases and their role in growth and disease. Life Sci. 64:2173.[Medline]
  5. Asao, H., C. Okuyama, S. Kumaki, N. Ishii, S. Tsuchiya, D. Foster, K. Sugamura. 2001. Cutting edge: the common gamma-chain is an indispensable subunit of the IL-21 receptor complex. J. Immunol. 167:1.[Abstract/Free Full Text]
  6. Chen, M., A. Cheng, Y. Q. Chen, A. Hymel, E. P. Hanson, L. Kimmel, Y. Minami, T. Taniguchi, P. S. Changelian, J. J. O’Shea. 1997. The amino terminus of JAK3 is necessary and sufficient for binding to the common {gamma} chain and confers the ability to transmit interleukin 2-mediated signals. Proc. Natl. Acad. Sci. USA 94:6910.[Abstract/Free Full Text]
  7. Cacalano, N. A., T. S. Migone, F. Bazan, E. P. Hanson, M. Chen, F. Candotti, J. J. O’Shea, J. A. Johnston. 1999. Autosomal SCID caused by a point mutation in the N-terminus of Jak3: mapping of the Jak3-receptor interaction domain. EMBO J. 18:1549.[Medline]
  8. Buckley, R. H., R. I. Schiff, S. E. Schiff, M. L. Markert, L. W. Williams, T. O. Harville, J. L. Roberts, J. M. Puck. 1997. Human severe combined immunodeficiency: genetic, phenotypic, and functional diversity in one hundred eight infants. J. Pediatr. 130:378.[Medline]
  9. Candotti, F., J. J. O’Shea, A. Villa. 1998. Severe combined immune deficiencies due to defects of the common {gamma} chain-JAK3 signaling pathway. Springer Semin. Immunopathol. 19:401.[Medline]
  10. Schumacher, R. F., P. Mella, R. Badolato, M. Fiorini, G. Savoldi, S. Giliani, A. Villa, F. Candotti, A. Tampalini, J. J. O’Shea, et al 2000. Complete genomic organization of the human JAK3 gene and mutation analysis in severe combined immunodeficiency by single-strand conformation polymorphism. Hum. Genet. 106:73.[Medline]
  11. Brugnoni, D., L. D. Notarangelo, A. Sottini, P. Airo, M. Pennacchio, E. Mazzolari, S. Signorini, F. Candotti, A. Villa, P. Mella, et al 1998. Development of autologous, oligoclonal, poorly functioning T lymphocytes in a patient with autosomal recessive severe combined immunodeficiency caused by defects of the Jak3 tyrosine kinase. Blood 91:949.[Abstract/Free Full Text]
  12. Thomis, D. C., C. B. Gurniak, E. Tivol, A. H. Sharpe, L. J. Berg. 1995. Defects in B lymphocyte maturation and T lymphocyte activation in mice lacking Jak3. Science 270:794.[Abstract/Free Full Text]
  13. Park, S. Y., K. Saijo, T. Takahashi, M. Osawa, H. Arase, N. Hirayama, K. Miyake, H. Nakauchi, T. Shirasawa, T. Saito. 1995. Developmental defects of lymphoid cells in Jak3 kinase-deficient mice. Immunity 3:771.[Medline]
  14. Freeden-Jeffry, U., P. Vieira, L. A. Lucian, T. McNeil, S. E. Burdach, R. Murray. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181:1519.[Abstract/Free Full Text]
  15. He, Y. W., T. R. Malek. 1996. Interleukin-7 receptor alpha is essential for the development of {gamma}{delta}+ T cells, but not natural killer cells. J. Exp. Med. 184:289.[Abstract/Free Full Text]
  16. Maraskovsky, E., M. Teepe, P. J. Morrissey, S. Braddy, R. E. Miller, D. H. Lynch, J. J. Peschon. 1996. Impaired survival and proliferation in IL-7 receptor-deficient peripheral T cells. J. Immunol. 157:5315.[Abstract]
  17. Candeias, S., J. J. Peschon, K. Muegge, S. K. Durum. 1997. Defective T-cell receptor {gamma} gene rearrangement in interleukin-7 receptor knockout mice. Immunol. Lett. 57:9.[Medline]
  18. Puel, A., S. F. Ziegler, R. H. Buckley, W. J. Leonard. 1998. Defective IL7R expression in T-B+NK+ severe combined immunodeficiency. Nat. Genet. 20:394.[Medline]
  19. Baird, A. M., J. A. Lucas, L. J. Berg. 2000. A profound deficiency in thymic progenitor cells in mice lacking Jak3. J. Immunol. 165:3680.[Abstract/Free Full Text]
  20. Lodolce, J. P., D. L. Boone, S. Chai, R. E. Swain, T. Dassopoulos, S. Trettin, A. Ma. 1998. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9:669.[Medline]
  21. Schorle, H., T. Holtschke, T. Hunig, A. Schimpl, I. Horak. 1991. Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting. Nature 352:621.[Medline]
  22. Kundig, T. M., H. Schorle, M. F. Bachmann, H. Hengartner, R. M. Zinkernagel, I. Horak. 1993. Immune responses in interleukin-2-deficient mice. Science 262:1059.[Abstract/Free Full Text]
  23. Suzuki, H., T. M. Kundig, C. Furlonger, A. Wakeham, E. Timms, T. Matsuyama, R. Schmits, J. J. Simard, P. S. Ohashi, H. Griesser. 1995. Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor {beta}. Science 268:1472.[Abstract/Free Full Text]
  24. Ozaki, K., R. Spolski, C. G. Feng, C. F. Qi, J. Cheng, A. Sher, H. C. Morse III, C. Liu, P. L. Schwartzberg, W. J. Leonard. 2002. A critical role for IL-21 in regulating immunoglobulin production. Science 298:1630.[Abstract/Free Full Text]
  25. Suto, A., H. Nakajima, K. Hirose, K. Suzuki, S. Kagami, Y. Seto, A. Hoshimoto, Y. Saito, D. C. Foster, I. Iwamoto. 2002. Interleukin 21 prevents antigen-induced IgE production by inhibiting germ line C{epsilon} transcription of IL-4-stimulated B cells. Blood 100:4565.[Abstract/Free Full Text]
  26. Kawamura, M., D. W. McVicar, J. A. Johnston, T. B. Blake, Y. Q. Chen, B. K. Lal, A. R. Lloyd, D. J. Kelvin, J. E. Staples, J. R. Ortaldo. 1994. Molecular cloning of L-JAK, a Janus family protein-tyrosine kinase expressed in natural killer cells and activated leukocytes. Proc. Natl. Acad. Sci. USA 91:6374.[Abstract/Free Full Text]
  27. Takahashi, T., T. Shirasawa. 1994. Molecular cloning of rat JAK3, a novel member of the JAK family of protein tyrosine kinases. FEBS Lett. 342:124.[Medline]
  28. Gurniak, C. B., L. J. Berg. 1996. Murine JAK3 is preferentially expressed in hematopoietic tissues and lymphocyte precursor cells. Blood 87:3151.[Abstract/Free Full Text]
  29. Safford, M. G., M. Levenstein, E. Tsifrina, S. Amin, A. L. Hawkins, C. A. Griffin, C. I. Civin, D. Small. 1997. JAK3: expression and mapping to chromosome 19p12–13.1. Exp. Hematol. 25:374.[Medline]
  30. Takahashi, T., T. Shirasawa, K. Miyake, Y. Yahagi, N. Maruyama, N. Kasahara, T. Kawamura, O. Matsumura, T. Mitarai, O. Sakai. 1995. Protein tyrosine kinases expressed in glomeruli and cultured glomerular cells: Flt-1 and VEGF expression in renal mesangial cells. Biochem. Biophys. Res. Commun. 209:218.[Medline]
  31. Takahashi, T., T. Shirasawa, K. Miyake, Y. Yahagi, O. Matsumura, A. Abe, Y. Natori, H. Yamabe, Y. Utsunomiya, N. Maruyama, et al 1996. Jak3 expression in glomerular epithelia of IgA nephropathy (IgA-N) patients. Clin. Exp. Immunol. 104:517.[Medline]
  32. Verbsky, J. W., E. A. Bach, Y. F. Fang, L. Yang, D. A. Randolph, L. E. Fields. 1996. Expression of Janus kinase 3 in human endothelial and other non-lymphoid and non-myeloid cells. J. Biol. Chem. 271:13976.[Abstract/Free Full Text]
  33. Villa, A., M. Sironi, P. Macchi, C. Matteucci, L. D. Notarangelo, P. Vezzoni, A. Mantovani. 1996. Monocyte function in a severe combined immunodeficient patient with a donor splice site mutation in the Jak3 gene. Blood 88:817.[Abstract/Free Full Text]
  34. Sofer, L., D. Kampa, J. Burnside. 1998. Molecular cloning of a chicken JAK homolog from activated T cells. Gene 215:29.[Medline]
  35. Tortolani, P. J., B. K. Lal, A. Riva, J. A. Johnston, Y. Q. Chen, G. H. Reaman, M. Beckwith, D. Longo, J. R. Ortaldo, K. Bhatia. 1995. Regulation of JAK3 expression and activation in human B cells and B cell malignancies. J. Immunol. 155:5220.[Abstract]
  36. Musso, T., J. A. Johnston, D. Linnekin, L. Varesio, T. K. Rowe, J. J. O’Shea, D. W. McVicar. 1995. Regulation of JAK3 expression in human monocytes: phosphorylation in response to interleukins 2, 4, and 7. J. Exp. Med. 181:1425.[Abstract/Free Full Text]
  37. Rane, S. G., E. P. Reddy. 1994. JAK3: a novel JAK kinase associated with terminal differentiation of hematopoietic cells. Oncogene 9:2415.[Medline]
  38. McDermott, M. F., I. Aksentijevich, J. Galon, E. M. McDermott, B. W. Ogunkolade, M. Centola, E. Mansfield, M. Gadina, L. Karenko, T. Pettersson, et al 1999. Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes. Cell 97:133.[Medline]
  39. Wingender, E., P. Dietze, H. Karas, R. Knuppel. 1996. TRANSFAC: a database on transcription factors and their DNA binding sites. Nucleic Acids Res. 24:238.[Abstract/Free Full Text]
  40. Chen, M., A. Cheng, F. Candotti, Y. J. Zhou, A. Hymel, A. Fasth, L. D. Notarangelo, J. J. O’Shea. 2000. Complex effects of naturally occurring mutations in the JAK3 pseudokinase domain: evidence for interactions between the kinase and pseudokinase domains. Mol. Cell. Biol. 20:947.[Abstract/Free Full Text]
  41. Lai, K. S., Y. Jin, D. K. Graham, B. A. Witthuhn, J. N. Ihle, E. T. Liu. 1995. A kinase-deficient splice variant of the human JAK3 is expressed in hematopoietic and epithelial cancer cells. J. Biol. Chem. 270:25028.[Abstract/Free Full Text]
  42. Jang, W., H. C. Chen, H. Sicotte, G. D. Schuler. 1999. Making effective use of human genomic sequence data. Trends Genet. 15:284.[Medline]
  43. Quandt, K., K. Frech, H. Karas, E. Wingender, T. Werner. 1995. MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res. 23:4878.[Abstract/Free Full Text]
  44. Gurniak, C. B., D. C. Thomis, L. J. Berg. 1997. Genomic structure and promoter region of the murine Janus-family tyrosine kinase, Jak3. DNA Cell Biol. 16:85.[Medline]
  45. Leu, J. H., S. J. Yan, T. F. Lee, C. M. Chou, S. T. Chen, P. P. Hwang, C. K. Chou, C. J. Huang. 2000. Complete genomic organization and promoter analysis of the round-spotted pufferfish JAK1, JAK2, JAK3, and TYK2 genes. DNA Cell Biol. 19:431.[Medline]
  46. Sharrocks, A. D.. 2001. The ETS-domain transcription factor family. Nat. Rev. Mol. Cell. Biol. 2:827.[Medline]
  47. Li, R., H. Pei, D. K. Watson. 2000. Regulation of Ets function by protein-protein interactions. Oncogene 19:6514.[Medline]
  48. Bories, J. C., D. M. Willerford, D. Grevin, L. Davidson, A. Camus, P. Martin, D. Stehelin, F. W. Alt. 1995. Increased T-cell apoptosis and terminal B-cell differentiation induced by inactivation of the Ets-1 proto-oncogene. Nature 377:635.[Medline]
  49. Muthusamy, N., K. Barton, J. M. Leiden. 1995. Defective activation and survival of T cells lacking the Ets-1 transcription factor. Nature 377:639.[Medline]
  50. Barton, K., N. Muthusamy, C. Fischer, C. N. Ting, T. L. Walunas, L. L. Lanier, J. M. Leiden. 1998. The Ets-1 transcription factor is required for the development of natural killer cells in mice. Immunity 9:5553.
  51. Bassuk, A. G., J. M. Leiden. 1995. A direct physical association between ETS and AP-1 transcription factors in normal human T cells. Immunity 3:223.[Medline]
  52. Wasylyk, B., C. Wasylyk, P. Flores, A. Begue, D. Leprince, D. Stehelin. 1990. The c-ets proto-oncogenes encode transcription factors that cooperate with c-Fos and c-Jun for transcriptional activation. Nature 346:191.[Medline]
  53. Foletta, V. C., D. H. Segal, D. R. Cohen. 1998. Transcriptional regulation in the immune system: all roads lead to AP-1. J. Leukocyte Biol. 63:139.[Abstract]
  54. Yu, C. L., S. J. Burakoff. 1997. Involvement of proteasomes in regulating Jak-STAT pathways upon interleukin-2 stimulation. J. Biol. Chem. 272:14017.[Abstract/Free Full Text]
  55. Ungureanu, D., P. Saharinen, I. Junttila, D. J. Hilton, O. Silvennoinen. 2002. Regulation of Jak2 through the ubiquitin-proteasome pathway involves phosphorylation of Jak2 on Y1007 and interaction with SOCS-1. Mol. Cell. Biol. 22:3316.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
J. A. Ross, Z. S. Nagy, and R. A. Kirken
The PHB1/2 Phosphocomplex Is Required for Mitochondrial Homeostasis and Survival of Human T Cells
J. Biol. Chem., February 22, 2008; 283(8): 4699 - 4713.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
C. M Smyth, S. L Ginn, C. T Deakin, G. J Logan, and I. E Alexander
Limiting {gamma}c expression differentially affects signaling via the interleukin (IL)-7 and IL-15 receptors
Blood, July 1, 2007; 110(1): 91 - 98.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
J. K. Mangan, S. G. Rane, A. D. Kang, A. Amanullah, B. C. Wong, and E. P. Reddy
Mechanisms associated with IL-6-induced up-regulation of Jak3 and its role in monocytic differentiation
Blood, June 1, 2004; 103(11): 4093 - 4101.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aringer, M.
Right arrow Articles by O’Shea, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aringer, M.
Right arrow Articles by O’Shea, J. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS