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Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry
Published on: June 21, 2018
Molecular cloning and identification of the human interleukin 13 alpha 2 receptor (IL-13Ra2) promoter
1Department of Neurosurgery, University of Minnesota Medical School, 2001 Sixth Street SE, Minneapolis, MN 55455, USA.
Abstract:
The interleukin 13 alpha 2 receptor (IL-13Ra2) has been shown to be expressed in most malignant glioblastoma cells. Recent studies suggest that IL-13Ra2 serves as a dominant negative inhibitor or a decoy receptor for IL-13. To investigate the transcriptional regulation of this receptor, we cloned and characterized the promoter for the human IL-13Ra2 gene. Our results demonstrate that this promoter contains three TATA boxes and one CCAAT site. Several putative transcriptional factor binding sites for nuclear factor of activated T cells 1, AP1 (c-JUN and c-FOS), AP2, GABP, OCT1, GATA3, PRE, and C-ETS1 were predicted in the promoter region. Using the secreted alkaline phosphate reporter gene assay, we investigated the functional activity of the human IL-13Ra2 promoter by transient transfection in glioma cell lines U118, U87, and T98, which differ in their expression of the human IL-13Ra2 protein. The different secreted alkaline phosphate activities among these 3 cell lines suggest that the expression of human IL-13Ra2 is regulated at the transcriptional level. Methylation analysis showed that expression of IL-13Ra2 may not be the result of methylation of the CpG dinucleotides in the promoter region of the gene. Deletion analysis identified a 64 base pair (bp) region that is necessary for human IL-13Ra2 promoter activity. This 64-bp sequence contains cis-elements for AP1, nuclear factor of activated T cells, and AP2. The possible role of AP1 in the regulation of human IL-13Ra2 promoter activity was suggested by in vitro mutagenesis and c-JUN N-terminal kinase inhibition analysis.
Insights
The interleukin 13 alpha 2 receptor (IL-13Ra2) promoter was characterized, revealing a 64-bp region crucial for its activity in glioblastoma cells. This finding sheds light on IL-13Ra2 transcriptional regulation.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- The interleukin 13 alpha 2 receptor (IL-13Ra2) is highly expressed in malignant glioblastoma.
- IL-13Ra2 may function as a dominant negative inhibitor or decoy receptor for IL-13.
Purpose of the Study:
- To clone and characterize the promoter region of the human IL-13Ra2 gene.
- To investigate the transcriptional regulation of IL-13Ra2 in glioma cell lines.
Main Methods:
- Promoter cloning and characterization, including TATA and CCAAT site identification.
- Reporter gene assays (secreted alkaline phosphatase) with transient transfection in U118, U87, and T98 glioma cells.
- Methylation analysis and deletion analysis to identify key regulatory regions.
Main Results:
- The human IL-13Ra2 promoter contains three TATA boxes and one CCAAT site.
- Functional analysis revealed cell-specific transcriptional regulation of IL-13Ra2 expression.
- A 64-bp minimal promoter region was identified, containing cis-elements for AP1, nuclear factor of activated T cells, and AP2.
- Methylation of CpG dinucleotides in the promoter region does not appear to regulate IL-13Ra2 expression.
Conclusions:
- Human IL-13Ra2 expression is regulated at the transcriptional level in glioma cells.
- The identified 64-bp region is essential for IL-13Ra2 promoter activity.
- AP1, nuclear factor of activated T cells, and AP2 are likely key regulators of IL-13Ra2 transcription, with a potential role for AP1 suggested by mutagenesis and kinase inhibition studies.
