Depletion of c-Rel from cytokine gene promoters is required for chromatin reassembly and termination of gene responses to T cell activation.

The role of the Nuclear Factor κB (NF-κB) transcription factor family in T cell function has been well described. The c-Rel family member is of particular importance in initiating T cell responses to antigen and regulating activation of inflammatory cytokine genes, including the Interleukin-2 (IL-2) and Granulocyte macrophage colony stimulating factor (GM-CSF) genes. c-Rel is required for chromatin remodeling of these gene promoters, which involves depletion of histones from the promoters in response to T cell activating signals. These chromatin remodeling events precede transcriptional activation of the genes. The subsequent down-regulation of cytokine gene expression is important in the termination of an immune response and here we examine this process at the murine GM-CSF and IL-2 genes. We show that the cytokine mRNA levels rapidly return to basal levels following stimulus removal and this is associated with reassembly of histones onto the promoter. Histone reassembly at the GM-CSF and IL-2 promoters occurs concomitantly with depletion of RelA, c-Rel and RNA polymerase II from the promoters. Furthermore we show that transcriptional down-regulation and chromatin reassembly is dependent on depletion of c-Rel from the nucleus, and that this is regulated by the nuclear translocation of the NF-κB inhibitor, IκBα. The nuclear activation of c-Rel therefore not only regulates the initiation of GM-CSF and IL-2 gene activation in response to T cell activation, but also the termination of these gene responses following the removal of the activating signal

Transcriptional down-regulation of the GM-CSF gene is dependent on the nuclear depletion of c-Rel.

<p>(A) Nuclear extracts from EL-4 T cells either left unstimulated (NS), stimulated with PI for 4 h (PI), the stimulus withdrawn for 4 h (WD) or the stimulus withdrawn in the presence of pentoxifylline (PTX) or lithium chloride (LiCl), were subjected to western analysis with the indicated antibodies. (B) GM-CSF mRNA levels relative to GAPDH were determined in cells treated as indicated. mRNA levels are shown relative to the 4 h PI sample which was set at 100%. The mean and standard error of three independent experiments is shown. (C–D) Nuclear extracts from EL-4 T cells either left unstimulated (NS), stimulated with PI for 4 h (PI), the stimulus withdrawn (WD) for 4 h, or the stimulus withdrawn in the presence of lithium chloride (LiCl) or leptomycin B (LMB) (C) or BAY 11-7082 (BAYi) or MG132 (D) were subjected to western analysis with the indicated antibodies. (E–F) GM-CSF mRNA levels relative to GAPDH were determined in cells treated as indicated. The mean and standard deviation of two independent experiments is shown.</p

GM-CSF transcriptional down-regulation and promoter resetting is associated with nuclear depletion of c-Rel and accumulation of IκBα.

<p>(A–B) GM-CSF mRNA levels, relative to GAPDH (A) and promoter accessibility to MNase (B) was determined in unstimulated EL-4 T cells (NS), cells stimulated with PI for 4 h (PI) and cells in which the stimulus was withdrawn (WD) for the indicated times, or withdrawn for the indicated times in the presence of cycloheximide (CHX), as indicated. mRNA levels are shown in (A) relative to PI treated sample which was set at 100%. The mean and standard error of three independent experiments is shown. (C) Nuclear extracts of EL-4 T cells treated as indicated were subjected to western analysis with the indicated antibodies. (D) c-Rel occupancy at the GM-CSF promoter was determined by ChIP analysis in EL-4 T cells treated as indicated. c-Rel occupancy was normalised to levels in unstimulated cells (NS). The mean and standard error of four independent experiments is shown. (E) Nuclear extracts from EL-4 T cell treated as indicated were subjected to western analysis with the indicated antibodies. (F) IκBα occupancy at the GM-CSF promoter was determined by ChIP analysis of EL-4 T cells stimulated with PI for 4 h and then the stimulus withdrawn for 20 h in the absence or presence of CHX, as indicated. IκBα occupancy was normalized to levels in cells in which stimulus was withdrawn in the absence of CHX. The mean and standard error of three independent experiments is shown.</p

NF-κB and RNA Polymerase II occupy the active GM-CSF promoter.

<p>(A) c-Rel (B) RelA and (C) RNA polymerase II occupancy was determined at the GM-CSF promoter by ChIP analysis of unstimulated EL-4 T cells (NS), cells stimulated for 4 h with PI (PI) and cells in which the stimulus was withdrawn (WD) for the indicated times. Occupancy levels are shown relative to the inactive rhodopsin promoter. The mean and standard error of three independent experiments is shown.</p

GM-CSF transcriptional down-regulation and promoter chromatin resetting is independent of the cell cycle.

<p>(A–B) Asynchronised and synchronised EL-4 T cells were left unstimulated (NS) or stimulated with PI for 4 h (PI) then the stimulus withdrawn (WD) for the indicated times. GM-CSF mRNA levels relative to GAPDH were then determined by RT-qPCR (A) and promoter accessibility to MNase was determined by CHART-PCR (B). The mean and standard error of three independent experiments is shown. (C) GM-CSF mRNA levels relative to GAPDH were determined in EL-4 T cells stimulated with PI for 4 h (PI) and then the stimulus withdrawn (WD) for 4 h or 20 h, with or without nocodazole (NOC), as indicated. GM-CSF mRNA levels are shown relative to the PI sample which was set at 100%. (D) GM-CSF promoter accessibility to MNase was determined in EL-4 T cells treated as indicated. The mean and standard error of five independent experiments is shown (C–D).</p

Histones are reassembled at the GM-CSF promoter following stimulus withdrawal.

<p>(A) Time course and sampling strategy used for the experiments. Samples were taken from unstimulated (NS) EL-4 T cells, from cells simulated for 4 h with PI (PI), and from cells 20 h and 44 h after withdrawal of the stimulus (20 h WD and 44 h WD). (B) Schematic depicting the position of GM-CSF primer sets used in PCR amplifications. (C–D) GM-CSF mRNA levels relative to GAPDH were determined by RT-qPCR (C) and promoter accessibility to MNase was determined by CHART-PCR (D), in EL-4 T cells treated as indicated. (E–F) Histone H3 (E) and acetylated H3 (F) levels were determined by ChIP analysis at the indicated genomic regions as shown in (B), in EL-4 T cells treated as indicated. (G) The ratio of acetylated H3 to total H3 as determined in (E) and (F) is depicted. (H) EL-4 T cells were either left unstimulated (NS) or treated for 4 h with PI, then the stimulus withdrawn for 20 h (20 h WD) and 44 h (44 h WD). GM-CSF mRNA levels were then determined by RT-qPCR following stimulation with PI for the indicated times. The mean and standard error of three independent experiments is shown in each case (C–H).</p

NF-κB and RNA Polymerase II are associated with the active IL-2 promoter.

<p>(A) IL-2 mRNA levels relative to GAPDH were determined in EL-4 T cells either left untreated or treated with PI for 4 h (PI), then the stimulus withdrawn (WD) for the indicated times. (B) IL-2 promoter accessibility was determined by CHART-PCR in cells treated as in (A). (C–E) c-Rel (C), RelA (D) and RNA polymerase II (E) occupancy was determined at the IL-2 promoter by ChIP in cells treated as indicated. Occupancy levels are shown relative to the inactive rhodopsin promoter. The mean and standard error of three independent experiments (A–D) or mean and standard deviation of two experiments (E) is shown.</p