https://doi.org/10.1007/s00392-025-02625-4
1Universität Heidelberg Herz- und Kreislaufphysiologie Heidelberg, Deutschland
Background
Nitric oxide (NO), a key vasodilator and anti-inflammatory agent, is produced by endothelial nitric oxide synthase (NOS3), the expression of which is maintained by unidirectional shear stress (USS). A single nucleotide polymorphism (SNP) within the promoter of the human NOS3 gene (T-786C) limits its responsiveness to USS. Homozygosity for this SNP is an independent and strong predictor for coronary heart disease (CHD), which is predominantly associated with the development of atherosclerosis in coronary arteries. Another SNP at position -922 (A-922G) is in strong linkage disequilibrium with the -786 SNP. A member of the signal transducers and activator of transcription (STAT) family binds to the NOS3 promoter region between these two polymorphic sites, acting as a transcription factor that maintains NOS3 expression. A generic inhibitor of histone deacetylases, trichostatin A (TSA), attenuates USS-dependent NOS3 expression in human cultured endothelial cells (HUVEC) harboring the -922AA/-786TT genotype (homozygous wildtype) but disinhibited in cells with the -922GG/-786CC genotype (homozygous mutant). Bioinformatic analysis suggests a tight binding of the 11-zinc finger protein CTCF and the transcription factor Yin Yang 1 (YY1) to the DNA sequence at position -922 in HUVEC isolated from -922GG genotype individuals, potentially affecting chromatin remodeling and NOS3 expression. Our study investigates the role of these two SNPs in USS-dependent expression of the human NOS3 gene aiming at characterizing the epigenetic control mechanisms involved therein.
Nitric oxide (NO), a key vasodilator and anti-inflammatory agent, is produced by endothelial nitric oxide synthase (NOS3), the expression of which is maintained by unidirectional shear stress (USS). A single nucleotide polymorphism (SNP) within the promoter of the human NOS3 gene (T-786C) limits its responsiveness to USS. Homozygosity for this SNP is an independent and strong predictor for coronary heart disease (CHD), which is predominantly associated with the development of atherosclerosis in coronary arteries. Another SNP at position -922 (A-922G) is in strong linkage disequilibrium with the -786 SNP. A member of the signal transducers and activator of transcription (STAT) family binds to the NOS3 promoter region between these two polymorphic sites, acting as a transcription factor that maintains NOS3 expression. A generic inhibitor of histone deacetylases, trichostatin A (TSA), attenuates USS-dependent NOS3 expression in human cultured endothelial cells (HUVEC) harboring the -922AA/-786TT genotype (homozygous wildtype) but disinhibited in cells with the -922GG/-786CC genotype (homozygous mutant). Bioinformatic analysis suggests a tight binding of the 11-zinc finger protein CTCF and the transcription factor Yin Yang 1 (YY1) to the DNA sequence at position -922 in HUVEC isolated from -922GG genotype individuals, potentially affecting chromatin remodeling and NOS3 expression. Our study investigates the role of these two SNPs in USS-dependent expression of the human NOS3 gene aiming at characterizing the epigenetic control mechanisms involved therein.
Methods
Reporter gene analyzes were performed by transfecting an immortalized HUVEC cell line with various NOS3 promoter luciferase reporter gene constructs followed by exposure to USS (30 dyn/cm2) or static conditions for 24 hours. Additionally, HUVECs with the -922AA/-786TT or -922GG/-786CC genotype were transfected with CTCF siRNA to knockdown CTCF expression, followed by analyzing its effect on NOS3 expression. To examine genotype-dependent chromatin remodeling, HUVECs harboring either genotype were exposed to USS or static conditions followed by performing a chromatin accessibility assay.
Results
Construct harboring the -922GG/-786CC genotype revealed diminished reporter gene expression at baseline and in response to USS as compared to their -922AA/-786TT counterparts. Mutation of the STAT binding site at position -854 to -835 led to decreased reporter gene expression in cells transfected with the -922AA/-786TT genotype reporter construct under both conditions. Following mutation of the CTCF binding site around position -922 (-922GG/-786CC genotype construct), reporter gene expression was significantly upregulated at baseline and in particular in response to USS as compared to the -922GG/-786CC genotype construct without the mutation. Furthermore, chromatin accessibility analysis indicated that the region around position -922 is less accessible than that around position -786, pointing towards a more compact chromatin structure around this position, which is potentially influenced by CTCF binding.
Conclusion
The promoter sequence of the NOS3 gene around position -922 in conjunction with the sequence around position -786 and the STAT binding site in-between control expression of the human NOS3 gene in response to USS. CTCF may act as an inhibitory regulator, potentially influencing chromatin remodeling and thereby modulating transcription of the NOS3 gene in response to USS.