Related Experiment Videos
Sp1 and Sp3 function as key regulators of leukotriene C(4) synthase gene expression in the monocyte-like cell line,
K J Serio1, C R Hodulik, T D Bigby
1Department of Veteran Affairs Medical Center, San Diego, CA 92161, USA.
Insights
Leukotriene C4 (LTC4) synthase gene expression in immune cells is regulated by specific DNA elements. Signal protein Sp1 and Sp3 transcription factors bind to a key promoter region, activating LTC4 synthase gene transcription.
Area of Science:
- Molecular Biology
- Immunology
- Gene Regulation
Background:
- Leukotriene C4 (LTC4) synthase plays a crucial role in inflammatory responses.
- Understanding the transcriptional regulation of LTC4 synthase is vital for controlling inflammatory processes.
Purpose of the Study:
- To investigate the molecular mechanisms governing leukotriene C4 (LTC4) synthase gene expression in mononuclear phagocytes.
- To identify key regulatory elements and transcription factors involved in LTC4 synthase gene transcription.
Main Methods:
- Utilized promoter-reporter constructs and deletion analysis in THP-1 cells to map regulatory regions.
- Employed electrophoretic mobility shift assays (EMSAs) to identify protein binding to the promoter.
- Used supershift EMSAs and transfection assays in Sp-deficient SL-2 cells to confirm the role of Sp1 and Sp3.
Main Results:
- The first 1.3 kb of the LTC4 synthase promoter significantly increased reporter activity.
- A critical region between -92 and -23 bp, containing an Sp1 consensus site, was identified as essential for promoter activity.
- Electrophoretic mobility shift assays confirmed binding of Sp1 and Sp3 transcription factors to this region.
- Transfection studies demonstrated that Sp1 and Sp3 transactivate LTC4 synthase gene transcription.
Conclusions:
- The Sp1 consensus site is a necessary element for LTC4 synthase gene transcription.
- Sp1 and Sp3 transcription factors positively regulate LTC4 synthase gene expression through this site.
- LTC4 synthase gene expression is transcriptionally controlled in mononuclear phagocytes via Sp1 and Sp3.
Abstract:
The goal of this study was to examine the mechanisms of leukotriene C(4) (LTC(4)) synthase gene expression in mononuclear phagocytes. Transfection of the monocyte-like cell line THP-1 with LTC(4) synthase promoter-reporter constructs demonstrated that the first 1.3 kb of the promoter mediated a 21.1-fold increase in reporter activity. Deletion analysis revealed that the region between -92 and -23 bp, which contains a signal protein (Sp)1 consensus site at -42 to -37 bp, mediated an 11.5-fold increase in reporter activity. Using a probe from -56 to -17 bp, electrophoretic mobility shift assays (EMSAs) demonstrated that Sp1 and THP-1 and HeLa nuclear extracts bind to this region. Binding was eliminated by mutation of the Sp1 consensus site. Supershift EMSAs using anti-Sp1 and anti-Sp3 antibodies demonstrated that these Sp family members bind to the region. Transfection of the Sp-deficient Drosophila SL-2 cell line with a construct containing the -92 to -23 bp promoter region and Sp expression vectors revealed that Sp1 and Sp3 transactivate gene transcription. We conclude that the Sp1 site is a necessary element for LTC(4) synthase gene transcription. Sp1 and Sp3 function through this site to positively regulate transcription. Thus, we provide evidence that the LTC(4) synthase gene is transcriptionally regulated in mononuclear phagocytes.