Bcl-6 and NF-kappaB cistromes mediate opposing regulation of the innate immune response

Abstract

In the macrophage, toll-like receptors (TLRs) are key sensors that trigger signaling cascades to activate inflammatory programs via the NF-κB gene network. However, the genomic network targeted by TLR/NF-κB activation and the molecular basis by which it is restrained to terminate activation and re-establish quiescence is poorly understood. Here, using chromatin immunoprecipitation sequencing (ChIP-seq), we define the NF-κB cistrome, which is comprised of 31,070 cis-acting binding sites responsive to lipopolysaccharide (LPS)-induced signaling. In addition, we demonstrate that the transcriptional repressor B-cell lymphoma 6 (Bcl-6) regulates nearly a third of the Tlr4-regulated transcriptome, and that 90% of the Bcl-6 cistrome is collapsed following Tlr4 activation. Bcl-6-deficient macrophages are acutely hypersensitive to LPS and, using comparative ChIP-seq analyses, we found that the Bcl-6 and NF-κB cistromes intersect, within nucleosomal distance, at nearly half of Bcl-6-binding sites in stimulated macrophages to promote opposing epigenetic modifications of the local chromatin. These results reveal a genomic strategy for controlling the innate immune response in which repressive and inductive cistromes establish a dynamic balance between macrophage quiescence and activation via epigenetically marked cis-regulatory elements.

Figure 1.

Bcl-6 coregulates the Tlr4-elicited gene expression program. (A) Representation of genes significantly induced or repressed in expression microarrays from wild-type (Bcl-6^+/+) and knockout (Bcl-6^−/−) BMDMs versus genes significantly altered in wild-type BMDMs by exposure to LPS (100 ng/mL) for 6 h. (B,C) Functional categorization of Bcl-6-regulated genes (B) or Bcl-6- and LPS-coregulated genes (C) depicted as percentages of total. (D) qPCR of microarray-identified Bcl-6- and LPS-coregulated genes in wild-type and knockout BMDMs at 0, 2, or 6 h following exposure to LPS (100 ng/mL). The mean relative expression ± SD compared with wild-type BMDMs at baseline (0 h) is listed.

Figure 2.

ChIP-seq reveals extensive colocalization of Bcl-6 with NF-κB. (A) Distribution of Bcl-6 and NF-κB p65-binding sites in BMDMs and their overlap with other cistromes as indicated. (B) Graphical depiction of the number of genes identified with significantly altered expression in Bcl-6^−/− versus Bcl-6^+/+ BMDMs (yellow), the number of genes annotated with Bcl-6-binding sites by ChIP-seq (blue), and the intersections of these gene sets in unstimulated BMDMs. (C) Functional classification of genes identified with binding sites for Bcl-6, NF-κB, or proximal Bcl-6 and NF-κB by ChIP-seq in unstimulated and LPS-stimulated BMDMs. (D) Schematic representing Tlr4-initiated transcriptional programs that are NF-κB p65-controlled (left) and NF-κB p65-independent (right) based on p65 ChIP-seq annotation. (Left) Bcl-6 regulation of Tlr4 responses is concentrated in genes annotated with proximal Bcl-6 and NF-κB sites. (Right) Bcl-6 binding is infrequent in Tlr4 target genes controlled by other transcription factors (TFs).

Figure 3.

ChIP-seq and epigenetic analysis reveals Bcl-6 and NF-κB coregulation of inflammatory genes. (A) ChIP-seq tracks for acetylated H3, monomethylated H3K4 (H3K4me1), and RNA polymerase II (RNA pol II) in unstimulated BMDMs as well as p300, Pu.1, NF−κB p65, and Bcl-6 in unstimulated or LPS-stimulated (100 ng/mL for 3 h) BMDMs along the Il-1 gene cluster. For factors sequenced with and without exposure to LPS, track heights were normalized to the number of aligned reads. (B,C) ChIP qPCR of acetylated H3 normalized to H3 (B), and Hdac3 enrichment relative to input chromatin in wild-type and knockout BMDMs with or without exposure to LPS (100 ng/mL for 3 h) at Bcl-6/p65-binding sites (C). The annotated gene name and position of the Bcl-6-binding site relative to the transcription start site is listed. 36b4 is a negative control DNA region lacking a Bcl-6- or p65-binding site. Values are expressed as means ± SD. Statistical testing using one-way ANOVA with Tukey's multiple comparison tests. (+) P < 0.05; (#) P < 0.01; (*) P < 0.001. Significant differences are noted for acetylation (B) or Hdac3 enrichment (C) relative to unstimulated wild-type BMDMs, unless otherwise indicated with brackets.

Figure 4.

Model of macrophage Bcl-6 in inflammatory control. Bcl-6 restrains inflammation through transcriptional repression via histone deacetylation at inflammatory gene enhancers, in close proximity to sites inducibly bound by NF-κB and the histone acetyltransferase p300 following Tlr4 stimulation. In the setting of TLR signals, loss of Bcl-6 and its corepressor, Hdac3, results in unopposed NF-κB transcriptional activation and hyperinflammatory responses.