Jmjd3 contributes to the control of gene expression in LPS-activated macrophages

Abstract

Jmjd3, a JmjC family histone demethylase, is induced by the transcription factor NF-kB in response to microbial stimuli. Jmjd3 erases H3K27me3, a histone mark associated with transcriptional repression and involved in lineage determination. However, the specific contribution of Jmjd3 induction and H3K27me3 demethylation to inflammatory gene expression remains unknown. Using chromatin immunoprecipitation-sequencing we found that Jmjd3 is preferentially recruited to transcription start sites characterized by high levels of H3K4me3, a marker of gene activity, and RNA polymerase II (Pol_II). Moreover, 70% of lipopolysaccharide (LPS)-inducible genes were found to be Jmjd3 targets. Although most Jmjd3 target genes were unaffected by its deletion, a few hundred genes, including inducible inflammatory genes, showed moderately impaired Pol_II recruitment and transcription. Importantly, most Jmjd3 target genes were not associated with detectable levels of H3K27me3, and transcriptional effects of Jmjd3 absence in the window of time analysed were uncoupled from measurable effects on this histone mark. These data show that Jmjd3 fine-tunes the transcriptional output of LPS-activated macrophages in an H3K27 demethylation-independent manner.

Figure 1

Genomic distribution of Jmjd3 in LPS-stimulated macrophages. (A) Jmjd3 induction in primary mouse bone marrow-derived macrophages. A western blot (left) and an RT–qPCR (right) analysis are shown. (B) Genome-wide analysis of Jmjd3 association with TSSs. Macrophages were stimulated with LPS+γIFN for 2 h and distribution of Jmjd3 peaks relative to mapped TSS was determined. (C) Jmjd3 binding to a representative region of mouse chromosome 5. The y axis indicates the number of tags in peaks. (D) A zoomed-in view of the same region shows the association of Jmjd3 with the TSSs of two genes. (E) Kinetics of Jmjd3 recruitment. TSS1 of Arhgef3, which was negative for Jmjd3 in the ChIP-Seq data, was used as a negative control. Guanylate-binding protein 6 (Gbp6) encodes an antiviral GTPase representing one of the most abundant proteins induced by LPS+γIFN. Error bars: s.e.m. from a triplicate experiment. (F) Abrogation of ChIP signals in Jmjd3 knockout macrophages. Anti-Jmjd3 ChIP was carried out in wild type and Jmjd3^−/− foetal liver-derived macrophages.

Figure 2

Jmjd3 association with transcriptionally active and inducible genes. (A) Jmjd3 association with H3K4me3-positive genes. Jmjd3 peaks and H3K4me3 peaks (in unstimulated and LPS-stimulated macrophages) at a representative region of chr 11 are shown. (B) Correlation between H3K4me3 levels and Jmjd3 binding in LPS-stimulated macrophages. H3K4me3 peaks were grouped in bins of decreasing total tag count from left to right. The y axis indicates the per cent of H3K4me3 peaks overlapping Jmjd3 peaks. (C) Association between Jmjd3 and H3K4me3 at representative genes. (D) Correlation between intensity of Jmjd3 binding and high levels of H3K4me3. (E) Correlation between Pol_II level and Jmjd3 binding at 2 h after LPS stimulation. Genes were grouped in bins of decreasing Pol_II intensity from left to right. The y axis shows the per cent of active, RNA Pol_II-positive genes that are associated with Jmjd3.

Figure 3

Jmjd3 binding and H3K27me3. (A) Lack of basal H3K27me3 at Jmjd3-bound genes. Jmjd3 and H3K27me3 ChIP-Seq profiles at two representative regions of chr5 and chr11. (B) Reduction in H3K27me3 is statistically similar at Jmjd3-bound and non-bound regions. The x axis indicates fold changes in H3K27me3 levels in response to LPS stimulation. The y axis shows the fraction of H3K27me3 peaks. (C) Reduction in H3K27me3 at Nos2 and Upp1 after LPS stimulation. The H3K27me3 peak downregulated after LPS is indicated by a shaded box. (D) Reduction in H3K27me3 at Upp1 and Nos2 in LPS-stimulated macrophages reflects nucleosome loss. ChIP–qPCRs were carried out with antibodies against H3K27me3 or total H3. H3K27me3/H3 ratios were calculated by dividing H3K27me3 signals by the signal obtained with the anti-H3 antibody. The data refer to one representative experiment out of four with qualitatively similar results.

Figure 4

Analysis of histone methylation and differentiation in Jmjd3^−/− macrophages. Macrophages derived from the foetal livers of Jmjd3^−/− mice were analysed for Jmjd3 protein and mRNA expression (A) and histone methylation at H3K4 and H3K27 (B). Differentiation and activation markers in Jmjd3^−/− macrophages are shown in (C). Upper panel: mRNA levels of macrophages-restricted LysM and Fms genes. Lower panel: FACS analysis of the macrophage markers CD11b and F4/80, and the activation marker CD86 before and after LPS stimulation.

Figure 5

Jmjd3 contribution to gene expression in LPS-activated macrophages. (A) Summary of the microarray results, indicating the number of genes that are differentially expressed in wt versus Jmjd3^−/− macrophages at various thresholds and their binding to Jmjd3. The position of selected Jmjd3 targets on the heat plot is indicated. (B) mRNA levels for selected differentially expressed Jmjd3 target genes was quantified by qPCR. (C) Analysis of nascent transcripts for the same genes was carried out using two primer sets for each gene (represented as small rectangles numbered 1 and 2 in the gene diagrams). Data are expressed relative to the nascent transcripts of the housekeeping nucleolin gene. RNA Pol_II ChIP (D) and H3K27me3 ChIP (E) in wt and Jmjd3^−/− macrophages. H3K27me3 levels at HoxA11 are shown as a positive ChIP control and used as reference.

Figure 6

RNA_Pol II and nascent transcript analyses show transcriptional effects of Jmjd3 that are not apparent at the mRNA level. (A) Pol_II ChIP was carried out at a panel of validated Jmjd3 target genes. P-values are indicated when statistical significance is reached. (B) Nascent transcript analysis at the IL-6 and Oasl1 genes. Two primer sets corresponding to the 5′ and 3′ of each gene were used, as indicated by black rectangles (#1 and #2).