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. 2006 Jul 15;177(2):1282-8.
doi: 10.4049/jimmunol.177.2.1282.

Dynamic and transient remodeling of the macrophage IL-10 promoter during transcription

Xia Zhang  1 , Justin P Edwards, David M Mosser
Affiliations

Dynamic and transient remodeling of the macrophage IL-10 promoter during transcription

Xia Zhang et al. J Immunol. .

Abstract

To gain insight into the molecular mechanism(s) whereby macrophages produce large amounts of IL-10, we analyzed IL-10 gene expression and temporally correlated it with modifications to chromatin associated with the IL-10 promoter. In resting cells, which make essentially no cytokines, the IL-10 promoter is associated with histones containing little or no detectable modifications. Macrophages stimulated in the presence of immune complexes begin to produce high levels of IL-10 pre-mRNA transcripts within minutes of stimulation. Coincident with this transcription was a rapid and dynamic phosphorylation of histone H3 at specific sites in the IL-10 promoter. Histone phosphorylation was closely followed by the binding of transcription factors to the IL-10 promoter. Blocking the activation of ERK prevented histone phosphorylation and transcription factor binding to the IL-10 promoter. In contrast to histone phosphorylation, the peak of histone acetylation at this promoter did not occur until after transcription had peaked. Inhibition of histone deactylase did not alter IL-10 gene expression, suggesting that phosphorylation but not acetylation was the proximal event responsible for IL-10 transcription. Our findings reveal a rapid and well-orchestrated series of events in which ERK activation causes a rapid and transient phosphorylation of histone H3 at specific regions of the IL-10 promoter, resulting in a transient exposure of the IL-10 promoter to the transcription factors that bind there. This exposure is essential for the efficient induction of IL-10 gene expression in macrophages. To our knowledge, this represents a unique way in which the expression of a cytokine gene is regulated in macrophages.

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Figures

FIGURE 1
FIGURE 1
DNA fragmentation and ChIP. A, Schematic illustration representing the nucleosomes along the promoter region of the IL-10 gene and the primer pairs used to amplify each segment of the IL-10 promoter. B, The primers used to amplify specific regions of the IL-10 promoter are listed.
FIGURE 2
FIGURE 2
Histone H3 phosphorylation of Ser10 in nucleosomes associated with the promoter region of IL-10. A, BMMϕ were stimulated with IC plus 10 ng/ml LPS (IC/LPS) for 0, 15, 30, 45, 90, and 180 min. Cross-linked chromatin fragments were immunoprecipitated with anti-phosphorylated histone H3 at Ser10 Ab. The DNA was purified and examined for the presence of IL-10 promoter sequences corresponding to nucleosome −2 by QRT-PCR. The data were normalized to inputs at each time point and plotted graphically as fold changes relative to the data at 0 min. B, Immunoprecipitated DNA from the 30-min ChIP assay described in A was amplified with primers specific to each of the 12 nucleosomes by QRT-PCR. One representative experiment from three independent experiments is presented. C, ChIP assay to amplify segments of the IL-10, IL-12 (p35 and 40), and TNF-α promoters following immunoprecipitations with Ab to phosphorylated (■) or acetylated (□) histone H3. Levels were normalized to amplified TdT segment that was arbitrarily set as 1.
FIGURE 3
FIGURE 3
Histone H3 acetylation of Lys14 on nucleosomes associated with the promoter region of IL-10. A, BMMϕ were stimulated with IC plus LPS (10 ng/ml) for 0, 10, 15, 30, 45, 60, 90, and 120 min. Cross-linked chromatin fragments were immunoprecipitated with an Ab to acetylated histone H3 at Lys14. The DNA was purified and analyzed for the presence of IL-10 promoter sequences corresponding to nucleosomes −2 (●) by QRT-PCR. The data were normalized to inputs at each time point and plotted graphically as fold changes relative to the data at 0 min. B, The recovered DNA from the 60-min ChIP assay described in A was amplified with primers specific to each of the 12 nucleosomes by QRT-PCR. One representative from two independent experiments is presented.
FIGURE 4
FIGURE 4
Recruitment of the Sp1 transcription factor to the IL-10 promoter. A, BMMϕ were stimulated with IC plus LPS (10 ng/ml) for 0, 15, 30, 45, 60, 90, 120, and 180 min. Cross-linked chromatin fragments were immunoprecipitated with anti-Sp1 Ab. The DNA was isolated and examined for the presence of IL-10 promoter sequences corresponding to nucleosome −2 by QRT-PCR. The data were normalized to inputs at each time point and plotted graphically as fold changes relative to the data at 0 min. B, The recovered DNA from the 45-min ChIP assay described in A was amplified with primers specific to each of the 12 nucleosomes by QRT-PCR. One representative from two independent experiments is presented.
FIGURE 5
FIGURE 5
IL-10 gene transcription and mRNA accumulation in macrophages. BMMϕ were stimulated with IC plus LPS (10 ng/ml) for 0, 20, 40, 60, and 120 min. RNA isolated from the cytoplasm and nucleus was purified and treated with RNase-free DNase I. Cytoplasmic and nuclear RNA were reverse transcribed to cDNA using oligo(dT)20 primer and random hexamers, respectively. The generated cDNA was then subjected to QRT-PCR analysis. The IL-10 RNA levels were presented as arbitrary units that were derived from average normalization values of each sample at each time point by corresponding GAPDH. The IL-10 mRNA level at zero time point was arbitrarily set as 1. Results presented are one representative from two independent experiments run in triplicate.
FIGURE 6
FIGURE 6
The kinetics of histone H3 phosphorylation, Sp1 recruitment, and histone H3 acetylation on nucleosome −2. BMMϕ were stimulated with IC plus LPS (10 ng/ml) for 0, 10, 15, 30, 45, 60, 90, and 120 min. Cross-linked chromatin fragments were immunoprecipitated with Ab to phosphorylated histone H3 at Ser10, Sp1 Ab, and Ab to acetylated histone H3 at Lys14, respectively. The DNA was purified and analyzed for the presence of IL-10 promoter sequences corresponding to nucleosome −2 by QRT-PCR. The data were normalized to inputs at each time point and plotted graphically as fold changes relative to the data at 0 min.
FIGURE 7
FIGURE 7
Cytokine changes following histone acetylation. TSA was added to BMMϕ for 1 h, and the cells were then washed with warmed medium before stimulation with 10 ng/ml LPS plus IC. A, After LPS plus IC stimulation for 6 h, the supernatants were collected and IL-10 and IL-12 were measured by ELISA. Results shown are one representative from two independent experiments conducted in triplicate (mean ± SD). B, BMMϕ were stimulated with IC plus LPS (10 ng/ml) for 0, 15, 30, 60, and 90 min. RNA isolation, cDNA synthesis, and QRT-PCR analysis were described above. The IL-10 mRNA levels were presented as arbitrary units that were derived from average normalization values of each sample at each time point by corresponding GAPDH. The IL-10 mRNA level at zero time point was arbitrarily set as 1. C, Different doses of TSA were added to BMMϕ for 1 h and whole-cell lysates were used for Western blotting analysis of histone H3 acetylation as detected by anti-acetyl histone H3 Ab.
FIGURE 8
FIGURE 8
Inhibition of histone modifications by blocking ERK. BMMϕ were pretreated with PD98059 (15 μM) (□) or drug vehicle (■) for 1 h and then stimulated with IC plus LPS (10 ng/ml). Cross-linked chromatin fragments were immunoprecipitated with Ab to phosphorylated histone H3 at Ser10 or Ab to Sp1. The DNA was purified and analyzed for the presence of IL-10 promoter sequences corresponding to nucleosome −2 by QRT-PCR. The data were plotted graphically as the percentage change relative to drug vehicle (IC/LPS) at 30 min poststimulation for histone H3 phosphorylation, and at 45 min poststimulation for Sp1 recruitment. One representative from two independent experiments is presented.
FIGURE 9
FIGURE 9
Enhanced chromatin accessibility following ERK activation. A, BMMϕ were stimulated with IC plus LPS (10 ng/ml) for 0, 15, 30, 60, and 120 min. Nuclei were isolated at each time point poststimulation and treated with or without MNase. DNA was purified and analyzed for the presence of sequences corresponding to nucleosomes 2 and 12 by QRT-PCR. The data were graphically plotted as percentage of MNase accessibility relative to the samples of stimulation at zero time point. B, BMMϕ were pretreated with PD98059 (15 μM) (□) or drug vehicle control (■) for 1 h and then stimulated with IC plus LPS (10 ng/ml) for 60 min. Nuclei were isolated from each group and treated with or without DNase I followed by extraction of genomic DNA. The presence of sequences corresponding to nucleosome −2 was examined by QRT-PCR. The data were expressed as percentage of DNase I accessibility relative to undigested genomic DNA sample and graphically plotted for each treated group. One representative from two independent experiments is presented. Each experiment was run in triplicate and shown as mean ± SD.
FIGURE 10
FIGURE 10
A schematic diagram of regulation of IL-10 gene expression in macrophages treated with IC in the presence of TLR ligands or other inflammatory signals.
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