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Adjacent sequence controls the response polarity of nitric oxide-sensitive Sp factor binding sites
Jianhua Zhang1, Shuibang Wang, Robert A Wesley
1Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA.
The Journal of Biological Chemistry
|May 22, 2003
Summary
Nitric oxide (NO) and protein kinase A (PKA) signaling influence gene expression by altering Sp1 binding. Adjacent elements, like AP1 sites, determine whether NO/PKA acts as an activator or repressor, controlling TNFalpha and eNOS gene output.
Area of Science:
- Molecular Biology
- Cell Signaling
- Gene Regulation
Background:
- Nitric oxide (NO) and cAMP-dependent protein kinase (PKA) signaling pathways are crucial in cellular responses.
- Sp1 transcription factor binding to GC-rich elements is a key regulatory mechanism for gene expression.
- Differential regulation of tumor necrosis factor alpha (TNFalpha) and endothelial NO synthase (eNOS) by NO and PKA suggests complex upstream control.
Purpose of the Study:
- To investigate the role of elements flanking Sp1 binding sites in determining the response of TNFalpha and eNOS promoters to NO and PKA signaling.
- To elucidate the mechanism by which NO and PKA signaling modulate Sp1 binding and gene transcription.
- To define the functional interplay between Sp1 and adjacent regulatory elements in conferring stimulus-dependent gene expression polarity.
Main Methods:
- Promoter-reporter assays in human cell lines to assess transcriptional activity.
- Site-directed mutagenesis to investigate the role of specific DNA elements (Sp sites, AP1 sites).
- Chromatin immunoprecipitation (ChIP) assays to determine in vivo protein-DNA interactions.
Main Results:
- NO and PKA signaling differentially regulated TNFalpha (up-regulated) and eNOS (down-regulated) expression.
- Sp1 binding to both promoters was modulated by NO and PKA signaling, with mutations of Sp sites abolishing these responses.
- An upstream AP1 site in the TNFalpha promoter, absent in eNOS, was critical for determining the direction of NO/PKA effects, converting repression to activation.
- Reciprocal binding changes at contiguous AP1 and Sp sites were observed, with Sp1 and Sp3 binding to GC-rich regions in both promoters.
Conclusions:
- A composite regulatory module involving Sp1 and adjacent elements (like AP1) dictates the polarity of NO and cAMP-mediated gene expression.
- This module acts as a sensor that can convert 'off' signals to 'on' signals based on secondary inputs, adding complexity to gene regulation.
- The findings reveal a sophisticated mechanism for differential gene control by NO and PKA signaling through context-dependent modulation of transcription factor binding.