NFKB1 Promoter DNA from nt+402 to nt+99 Is Hypomethylated in Different Human Immune Cells

Abstract

Sepsis, with a persistently high 90-day mortality of about 46%, is the third most frequent cause of death in intensive care units worldwide. Further understanding of the inflammatory signaling pathways occurring in sepsis is important for new efficient treatment options. Key regulator of the inflammatory response is the transcription factor NFκB. As we have recently shown, the -94 Ins/Del NFKB1 promoter polymorphism influences sepsis mortality. However, a molecular explanation is still missing. Thus, promoter activity might be varying depending on the NFKB1 genotype, explaining the genotype dependent mortality from sepsis, and one likely mechanism is the degree of promoter methylation. Therefore, we tested the hypothesis that NFκB mRNA expression is regulated by promoter methylation in human cell lines and primary immune cell cultures. First, we examined the methylation of the NFKB1 promoter in U937, REH and HL-60 cells. In the promoter region of nt+99/+229 methylation in all analyzed cell lines was below 1%. Following incubation with bacterial cell wall components, no significant changes in the frequency of promoter methylation in U937 and REH cells were measured and the methylation frequency was under 1%. However, NFκB1 mRNA expression was two-fold increased in U937 cells after 24 h incubation with LPS. By contrast, demethylation by 5-Aza-2′-deoxycytidine incubation enhanced NFκB1 expression significantly. In addition, we analyzed NFKB1 promoter methylation in primary cells from healthy volunteers depending on the NFKB1–94 Ins/Del genotype. Methylation in the promoter region from nt+402 to nt+99 was below 1%. Genotype dependent differences occurred in neutrophil cells, where DD-genotype was significantly more methylated compared to II genotype at nt+284/+402. Besides in the promoter region from nt-227/-8 in ID-genotypes methylation of neutrophils was significantly decreased compared to lymphocytes and in II-genotypes methylation in neutrophils was significantly decreased compared to lymphocytes and monocytes. In addition, CHART-PCR showed that the hypomethylated promoter regions are highly accessible. Therefore we assume that the demethylated regions are very important for NFKB1 promoter activity.

Effects of milk product intake on thigh muscle strength and NFKB gene methylation during home-based interval walking training in older women: A randomized, controlled pilot study

“…The minimum sample size for detecting the group differences in the percent increase in NFKB1 and NFKB2 methylation at α = 0.05 (two-sided) and (1-β) = 0.8, determined using an unpaired t-test, was 4 and 6 for each group, respectively, which is smaller than the sample size in the present study. Because methylation of promoter regions is associated with transcriptional suppression of the corresponding gene, enhanced methylation in NFKB1 and NFKB2 for the HD group suggests reduced NFKB1 and NFKB2 gene expression [ 33 , 34 ].…”

TET1 Knockdown Inhibits Porphyromonas gingivalis LPS/IFN-γ-Induced M1 Macrophage Polarization through the NF-κB Pathway in THP-1 Cells

“…The NF-κB signaling pathway plays an important role in the inflammatory response and macrophage polarization. A growing body of evidence showed that the NF-κB signaling pathway can be regulated by DNA methylation [47,48]. In germinal center B cells, elevated DNMT1 expression causes hypomethylation of the promoter of the nuclear factor kappa B kinase subunit epsilon ( IKBKE ) inhibitor, which is one of the NF-κB pathway signaling components that induces IKB phosphorylation [47].…”

Study on the Immune Escape Mechanism of Acute Myeloid Leukemia With DNMT3A Mutation

“…They play an important role in the inflammatory response and macrophage polarization. A growing body of evidence showed that the two signaling pathways can both be regulated through DNA methylation ( 35 , 36 ).…”