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OP0098 The jak3 promoter region is stimulated by t cell activation and contains essential ap-1 and ets-1 elements
1. M Aringer1,
2. DM Frucht2,
3. M Chen2,
4. M Centola2,
5. JJ O’Shea2
1. 1Rheumatology/Internal Medicine III, University of Vienna, Austria, Wien, Austria
2. 2Arthritis and Rheumatism Branch, NIAMS/National Institutes of Health, Bethesda, MD, USA

## Abstract

Background Janus Kinase 3 (Jak3) is a non-receptor tyrosine kinase essential for interleukin- (IL-) 2, 4, 7, 9, and 15 signalling. In T lymphocytes, Jak3 expressed is clearly increased upon T cell activation. The regulatory regions of the Jak3 promoter, however, are unknown.

Objectives We therefore decided to characterise the promoter region of Jak3.

Methods A 5´race procedure was used to detect the beginning of Jak3 cDNA derived from both the Jurkat T cell line and an NK cell line (NK3.3). All sequencing was performed on an ABI 377 automated sequencer using DNA amplified by PCR and the ABI Big Dye terminator kit. Putative promoter pieces were amplified by PCR and subcloned into a luciferase vector. In addition, site-directed mutagenesis was used to change two AP-1 sites and a putative Stat binding site within the core promoter region. Jurkat T cells were transfected both with these constructs and a beta-galactosidase vector for normalisation; both activities were sequentially read on a luminometer using the Tropix Dual Light kit.

Results All Jak3 mRNA started 5´ of a 3515 basepair intron; the first basepair of the longest mRNA species found was denominated as +1. The 1039 basepair DNA region 5 of the mRNA start (-1013/+27) worked as a (TATA-less) promoter and could be activated by PMA, PHA, or immobilised anti-CD3, but not anti-CD28. As expected, the promoter did not work in the  reverse direction. Truncation constructs showed the active region situated within a 267 basepair (-240/+27) region, the core promoter was positioned between ?74 and ?16. Within this region, that contained also an SP-1 site, a double-AP1 site, but not the potential Stat site in between, was of major importance, as demonstrated by targeted point mutation. Surrounding the core promoter, two Ets-1 consensus elements had significant (around 6-fold) enhancer activity.

Conclusion The Jak3 promoter is situated around 3500 basepairs 5of its ATG-codon. The promoter can be activated by T cell receptor crosslinking and contains an active double AP-1 binding site and Ets-1 consensus elements that work as significant enhancers. This may lead to improved IL-2 (or ?4, -7- or ?15) signalling in disease states where T lymphocytes are being activated.

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