Figure 4 LPS induces early histone H3 methylation and acetylation changes at the promoter region of IL-8 gene. Chromatin from HT-29 cells was harvested at the indicated time points after LPS exposure. (A) Schematic representation of IL-8 promoter region, as in Figure 3. Positions of the primers used for ChIP analyses are shown. Presented are the results of ChIP analyses using anti-acetyl-H3 Combretastatin A4 datasheet (B) anti-dimethyl-H3K4 (C), anti-dimethyl-H3K9
(D) and anti-trimethyl-K27 (E) antibodies. DNA sequences recovered after the indicated times of LPS treatment were quantified by real-time PCR using the primers indicated above. Average% input ± S.D from 4 independent experiments are plotted. *, p < 0.01; **, p < 0.05; n.s.= not significant, compared to control cells. Very interestingly, H3K27me3 levels were initially very low but then increased substantially starting at 6 hours and remained high 24 hours after LPS stimulation (Figure 4E). H3K27 trimethylation is catalyzed by Polycomb group (PcG) protein complexes [21, 22], which have been shown to be involved in cytokines genes
reprogramming occurring in both epithelial and macrophage cells in response to bacterial products and inflammation-related stimuli [23, 24]. It is also worth to note that when all the above ChIP assays were performed in unprimed HT-29 cells (not pre-treated with interferon-γ) we did not detect significant changes in histone H3 methylation MK0683 nmr state during the same time course (data not shown) suggesting that the observed chromatin modifications are dependent on the MD2/TLR4 pathway. However, because it
is well known that even “”pure”" LPS preparations may be contaminated with lipoproteins, we cannot definitively exclude that the observed chromatin modifications could be influenced by TLR2 www.selleckchem.com/products/DAPT-GSI-IX.html signaling. Taken together our data indicate that while changes of H3-acetyl, H3K4me2 and H3K9me2 state in the IL-8 promoter region occur rapidly, transiently and correspond to transcription activation, the changes of H3K27me3 levels PAK5 occur at a later time and are long lasting. Finally it should be considered that a strong mark of gene repression, such as H3K27me3, could predispose to a more repressed state of IL-8 gene and, thus, could render the gene less responsive to further LPS stimulation. Moreover, H3K27me3 is also related to DNA hypermethylation that has been shown to occur in intestinal cancer at PcG target genes. In particular, it has been recently demonstrated that hypermethylation of PcG target genes in intestinal cancer is mediated by inflammation [24]. Thus, although our data indicate that DNA methylation is not directly involved in LPS response, such phenomenon may occur later, after prolonged exposure to LPS, as a consequence of PcG proteins recruitment at IL-8 gene.