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Multifactor cis-dominant negative regulation of IL-2 gene expression in anergized T cells
S Kitagawa-Sakakida1, R H Schwartz
1Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
Abstract:
The molecular mechanism underlying IL-2 transcriptional blockade in anergic T cell clones is not fully understood. To examine whether an active negative regulatory process occurs, we created a reporter construct containing as an enhancer four copies of the NF-AT site and one copy of the octamer site (4X NF-AT-Oct). This construct was only slightly reduced (1.3-fold) in its expression when stimulated under anergic conditions, while a whole mouse IL-2 enhancer construct showed a reduction of 4.3-fold. Addition of the -176 to -96 sequence to the 4X NF-AT-Oct construct did not impart the ability to be affected by anergy, but addition of the -236 to -96 sequence did, demonstrating that anergy is an active inhibitory process and that more than the presence of the -150 AP-1 binding site (-152 to -147) is required to mediate the effect. Mutational studies of the -236 to -96 sequence indicated that the presence of both the -130 AP-1-like site (-187 to -181) and the -150 proximal AP-1 site were necessary to observe anergy. Because the -180 site is not required for trans-activation, it was possible to confirm by mutation in the normal mouse IL-2 enhancer that this site is absolutely essential for anergy induction. The simplest model to explain these results is that anergy is mediated by a complex of multiple transcription factors that exert a cis-acting dominant negative regulatory effect on the trans-activation of the IL-2 gene.
Insights
T cell anergy involves an active inhibitory process that blocks interleukin-2 (IL-2) gene transcription. Specific AP-1 binding sites within the IL-2 enhancer are crucial for mediating this anergy effect.
Area of Science:
- Immunology
- Molecular Biology
- Gene Regulation
Background:
- The molecular mechanisms of interleukin-2 (IL-2) transcriptional blockade in anergic T cells remain unclear.
- Investigating whether an active negative regulatory process contributes to this blockade is essential for understanding T cell function.
Purpose of the Study:
- To elucidate the molecular mechanisms of IL-2 transcriptional blockade in anergic T cells.
- To determine if an active negative regulatory process is involved in suppressing IL-2 gene expression during T cell anergy.
Main Methods:
- Creation of a reporter construct (4X NF-AT-Oct) to assess enhancer activity under anergic conditions.
- Comparison of reporter construct expression with a whole mouse IL-2 enhancer construct.
- Deletion and mutational analyses of specific DNA sequences within the IL-2 enhancer (-236 to -96 region).
Main Results:
- The 4X NF-AT-Oct construct showed only a minor reduction in expression during anergy, unlike the whole IL-2 enhancer.
- A specific region (-236 to -96) was identified as critical for mediating the anergy-induced reduction in IL-2 expression.
- Both the -130 AP-1-like site and the -150 proximal AP-1 site were found to be necessary for anergy induction, with the -180 site being essential for this process.
Conclusions:
- T cell anergy involves an active inhibitory process that suppresses IL-2 gene transcription.
- The IL-2 enhancer contains critical regulatory elements, including specific AP-1 binding sites, that mediate this anergy-induced suppression.
- A model proposing a multi-transcription factor complex exerting dominant negative regulation on IL-2 gene trans-activation is suggested.