Regulatory upstream region (proximal NF-κB binding site and TATA box), Transcriptional start site (arrow) and exon 1 (gray box) are indicated. The relative positions of each CpG site present in the analyzed region and of the primers utilized for amplification are indicated. (B) Methylation degree at CpG sites -83, -7, +73, +119, and +191 on both upper (gray bars)

and lower strand (black bars) was measured in Torin 2 in vivo untreated HT-29, in cells treated 24 hours with LPS and in normal colon mucosa samples by MALDI-TOF analysis. Methylation of sites -83 and Selleckchem Etomoxir -73 on lower strand could not be determined by MALDI analysis (ND). Each experiment was repeated three times on three different samples. Shown are the average values for each indicated CpG site ± SD. LPS-mediated IL-8 gene activation is accompanied by both histone H3 acetylation and methylation changes Then we performed chromatin immunoprecipitation (ChIP) experiments in order to Batimastat purchase investigate whether specific changes in histone modifications occurred at IL-8 promoter during LPS-induced gene activation. First we determined whether IL-8 activation corresponded to increased levels of histones H3 acetylation in the promoter region of IL-8 gene. Cells were incubated with LPS for different times and chromatin was immunoprecipitated with anti acetyl-H3 antibodies; then PCR amplifications were performed

using promoter-specific primers (see Figure 4A and Methods section). We found that upon LPS treatment H3 acetylation state was transiently modulated. The histone H3 was highly acetylated after 30 minutes while the deacetylated state was restored after 6 hours (Figure 4B). Hyper-acetylation of histone H3 is in agreement with expression pattern of the IL-8 Aspartate gene. Then, we determined whether the induction of IL-8 gene was accompanied by modifications of histone methylation state. Antibodies against dimethylated H3K4 (H3K4me2), dimethylated H3K9 (H3K9me2) and trimethylated H3K27 (H3K27me3), were used in

ChIP assays. We found that the levels of H3K4me2 were low in untreated HT-29 cells, significantly increased 1 hour after LPS administration, and gradually returned to basal levels within 24 hours (Figure 4C). Conversely, H3K9me2 showed a significant increase after 30 minutes and then rapidly decreased at 1 hour remaining lower than basal levels after 24 hours (Figure 4D). These results, examined together with the expression data (see Figure 1), are in agreement with the repressive role of H3K9me2 and with the activating role described for H3K4me2 in gene transcription [3, 4, 7]. The sharp increase in H3K9me2 levels observed at 30 minutes time point at IL-8 promoter, despite the transcriptional activated status, could be explained by a possible concomitant demethylation of trimethylated H3K9 and consequent transient accumulation of the dimethylated form.