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A putative silencer element in the IL-5 gene recognized by Bcl6
Masafumi Arima1, Hirochika Toyama, Hirohito Ichii
1Department of Developmental Genetics (H2), Graduate School of Medicine, Chiba University, Chiba, Japan.
Journal of Immunology (Baltimore, Md. : 1950)
|July 5, 2002
Summary
The Bcl6 gene represses Interleukin-5 (IL-5) production by binding to a specific DNA sequence. This mechanism helps control Th2 cytokine levels and prevent eosinophilic inflammation.
Area of Science:
- Immunology
- Molecular Biology
- Gene Regulation
Background:
- The Bcl6 gene product is a transcriptional repressor found in adult murine tissues.
- Bcl6 deficiency in mice leads to eosinophilic inflammation due to excessive Th2 cytokine production.
- The precise mechanism by which Bcl6 regulates these cytokine productions remains debated.
Purpose of the Study:
- To investigate the regulatory role of Bcl6 in Th2 cytokine production, specifically Interleukin-5 (IL-5).
- To identify and characterize the binding site of Bcl6 on the IL-5 gene.
- To elucidate how Bcl6 binding affects IL-5 gene expression.
Main Methods:
- Stimulation of CD4(+) T cells from Bcl6-deficient and transgenic mice with anti-CD3 antibodies.
- Identification of a putative Bcl6-binding sequence (IL5BS) in the 3' untranslated region of IL-5 genes.
- Gel retardation assays and chromatin immunoprecipitation assays to confirm Bcl6 binding.
- Reporter gene assays in K562 cells to assess repression activity.
- Analysis of Bcl6 binding activity in Th2 and Th1 clones post-stimulation.
Main Results:
- IL-5 production was preferentially affected by Bcl6 levels in stimulated CD4(+) T cells.
- A specific Bcl6-binding sequence (IL5BS) was identified in the 3' UTR of murine and human IL-5 genes.
- Bcl6 protein directly binds to the IL5BS, confirmed by gel retardation and ChIP assays.
- Endogenous Bcl6 binding activity decreased in Th2 clones but not Th1 clones after stimulation.
- Exogenous Bcl6 repressed reporter gene expression containing IL5BS, with repression lost upon IL5BS mutation.
- The IL5BS is essential for Bcl6-mediated repression of IL-5 cDNA expression.
Conclusions:
- Bcl6 directly represses IL-5 gene expression through binding to the IL5BS element in the 3' UTR.
- The IL5BS acts as a silencer element, mediating Bcl6's regulatory function on IL-5.
- This mechanism provides insight into the control of Th2 cytokine production and the pathogenesis of eosinophilic inflammation.