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

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

NFκB expression in U937 cells 24 h after stimulation with 1 μg/ml LPS or 10 μg/ml LTA (n = 4).

<p>NFκB expression in U937 cells 24 h after stimulation with 1 μg/ml LPS or 10 μg/ml LTA (n = 4).</p

Methylation of the NFKB1 promoter in three different cell lines.

<p>Three different promoter regions were analyzed: nt+284/+402, nt+99/+229, nt-227/-8 (n = 3). A methylated (M-DNA) and unmethylated (U-DNA) was used as control.</p

methylation of the NFKB1 promoter in different immune cells depending on the NFKB1–94 (Ins/Del) genotype (n = 12).

<p>Three different promoter regions were analyzed. NFKB1 promoter was unmethylated (<1%) from nt+402/+99. From nt-227 to -8 the methylation was about 5% to 10%.</p

chromatin accessibility (CHART) of different regions of the NFKB1 promoter.

<p>Chromatin from different cell lines was incubated with MNase (+) or left untreated (-) and DNA was extracted. PCR of different NFKB1 promoter regions as performed.</p

Overview of literature of regulation by methylation of important transcription factors and inflammatory genes.

<p>Overview of literature of regulation by methylation of important transcription factors and inflammatory genes.</p

Graphic view showing primers and sequence features such as GC percent, CpG islands, and CpG site derived from MethPrimer [10] nucleotide numbers base on transcription initiation site [16] a) graphic view of the NFKB1 Promoter sequence from nt-800 to nt+600; CpG dinucleotides are indicated as bars b) graphical view of primers utilized for CHART-PCR, c) graphical view of primers utilized for methylation specific PCR.

<p>Graphic view showing primers and sequence features such as GC percent, CpG islands, and CpG site derived from MethPrimer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156702#pone.0156702.ref010" target="_blank">10</a>] nucleotide numbers base on transcription initiation site [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156702#pone.0156702.ref016" target="_blank">16</a>] a) graphic view of the NFKB1 Promoter sequence from nt-800 to nt+600; CpG dinucleotides are indicated as bars b) graphical view of primers utilized for CHART-PCR, c) graphical view of primers utilized for methylation specific PCR.</p

standard curve derived from the CT values measured for the nt+284/402 NFKB1 promoter region.

<p>The CT values were measured in a dilution series with defined portions of methylated and unmethylated DNA (red values). A standard curve was calculated from these values (blue values).</p

Methylation of the +99/+229 NFKB1 promoter region of different blood cells isolated from healthy volunteers.

<p>Cells were isolated from fresh EDTA blood. They were then stimulated with LPS (1 μg/ml) or left unstimulated and DNA was extracted for methylation analysis (n = 3).</p

Primer pairs for CHART-PCR and Methylation-specific PCR.

<p>Primer pairs for CHART-PCR and Methylation-specific PCR.</p