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Stat5 and Sp1 regulate transcription of the cyclin D2 gene in response to IL-2
A Martino1, J H Holmes, J D Lord
1Virginia Mason Research Center, Seattle, WA 98101, USA. tmartino@vmresearch.org
Abstract:
The IL-2R promotes rapid expansion of activated T cells through signals mediated by the adaptor protein Shc and the transcription factor Stat5. The mechanisms that engage the cell cycle are not well defined. We report on the transcriptional regulation of the cell cycle gene cyclin D2 by the IL-2R. IL-2-responsive induction of a luciferase reporter gene containing 1624 bp of the cyclin D2 promoter/enhancer was studied in the murine CD8(+) T cell line CTLL2. Reporter gene deletional analysis and EMSAs indicate an IL-2-regulated enhancer element flanks nucleotide -1204 and binds a complex of at least three proteins. The enhancer element is bound constitutively by Sp1 and an unknown factor(s) and inducibly by Stat5 in response to IL-2. The Stat5 binding site was essential for IL-2-mediated reporter gene activity, and maximum induction required the adjacent Sp1 binding site. Receptor mutagenesis studies in the pro-B cell line BA/FG (a derivative of the BA/F3 cell line) demonstrated a correlation between Stat5 activity and cyclin D2 mRNA levels when the Stat5 signal was isolated, disrupted, and then rescued. Further, a dominant-negative form of Stat5 lacking the trans-activation domain inhibited induction of cyclin D2 mRNA. We propose that the IL-2R regulates the cyclin D2 gene in part through formation of an enhancer complex containing Stat5 and Sp1.
Insights
Interleukin-2 receptor (IL-2R) signaling regulates T cell proliferation by controlling cyclin D2 gene expression. This involves the transcription factor Stat5 binding to an enhancer element, crucial for T cell cycle progression.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Interleukin-2 receptor (IL-2R) signaling is critical for T cell expansion.
- The precise mechanisms linking IL-2R to cell cycle engagement remain unclear.
- Cyclin D2 is a key cell cycle gene, but its transcriptional regulation by IL-2R is not well defined.
Purpose of the Study:
- To investigate the transcriptional regulation of the cyclin D2 gene by the IL-2R.
- To identify the specific regulatory elements and transcription factors involved in IL-2-mediated cyclin D2 induction.
Main Methods:
- Utilized a luciferase reporter gene assay with a cyclin D2 promoter/enhancer construct in CTLL2 T cells.
- Performed deletional analysis and electrophoretic mobility shift assays (EMSAs) to map regulatory elements.
- Conducted receptor mutagenesis studies in BA/FG cells and used dominant-negative Stat5 to assess functional roles.
Main Results:
- Identified an IL-2-regulated enhancer element between nucleotides -1204 and -1116 of the cyclin D2 promoter.
- This enhancer binds Sp1 constitutively and Stat5 inducibly upon IL-2 stimulation.
- Stat5 binding was essential for IL-2-mediated reporter gene activity, with maximal induction requiring an adjacent Sp1 binding site; Stat5 activity correlated with cyclin D2 mRNA levels.
Conclusions:
- The IL-2R regulates cyclin D2 gene expression, a key driver of T cell proliferation.
- This regulation occurs through the formation of an enhancer complex involving Stat5 and Sp1.
- Findings elucidate a critical molecular mechanism linking IL-2 signaling to T cell cycle progression.