Changes in nucleosome occupancy upon LPS induction at a putative distal enhancer and promoter of IL1A, kinetics of nucleosome removal, and changes in histone modifications.

<p>(A and B), Nucleosome occupancy at a putative enhancer 10 kb upstream of the TSS of IL1A was determined in BMDMs prior to (blue bars and lines) and upon 1.5 h (yellow) or 3 h (red) induction with 1 μg/ml LPS as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093971#pone-0093971-g001" target="_blank">Figure 1</a>. ConSite predicted consensus sites for PU.1, C/EBP, IRF, AP1 and NFκB are indicated. (C and D), Nucleosome occupancy at the promoter of IL1A was determined as described in panel (A) in a region surrounding the TSS of IL1A. The TSS (black bar) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093971#pone.0093971-Carninci1" target="_blank">[35]</a> and a putative TATA-box (blue bar) is indicated in panel (C). (E), Kinetics of nucleosome removal at IL1A and IL12B. BMDMs were induced with LPS for 15′, 30′, 60′ and 90′, and nucleosome occupancy was analyzed as described in (A). Nucleosome removal at three locations in the distal enhancer of IL12B (red lines) and in the enhancer of IL1A (blue lines) is shown. The data is displayed as the fold change in nucleosome occupancy over the levels found before induction at each location. The absence of changes in nucleosome occupancy at the TSS of IL12B (black line) and of IL1A (gray line) is shown for comparison. (F), ChIP experiments with antibodies against H3 (dark blue bars), H2A.Z (light blue), H3K4me1 (green), H3K4me3 (yellow) and H3K27ac (red) were performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093971#s2" target="_blank">Materials and Methods</a>. For these experiments cross-linked chromatin was lightly digested with MNase before incubation with the respective antibodies to increase resolution of the ChIP signal and the data was normalized to a region in the ORF of RPL4. Changes upon LPS induction in histone binding and histone modifications at the enhancers and promoters of IL12B and IL1A as well as at a control region in the GAPDH pseudo gene are shown as fold over levels found before induction. For H3K27ac the changes 1.5 h after LPS induction, and for all other histone variants and modifications the changes after 3 h of induction are shown. The error bars show the SEM of at least 3 independent experiments. Statistical significance of the changes in H3K4me3 and H3K27ac upon LPS induction compared to levels found prior to induction determined by <i>Student's</i> T-tests is indicated (*P<0.05; **P<0.01).