REORGANIZATION OF THE MACROPHAGE EPIGENOME DURING SUSTAINED STIMULATION

The chromatin of cells whose main function is to sense and react to environmental inputs, such as macrophages and other innate immune cells, undergoes rapid modifications in response to microenvironmental signals and provides general paradigms of how epigenomes are dynamically reorganized in a changing environment. A short exposure of macrophages to endotoxin (lipopolysaccharide, LPS) strongly activates transcription of hundreds of inflammatory genes. Conversely, a sustained stimulation results in a state of hypo-responsiveness to a subsequent microbial stimulation, which is commonly referred to as endotoxin tolerance. We used nascent RNA-seq and ChIP-seq to characterize genes and cis-regulatory regions that are differentially activated in unperturbed, LPS-stimulated and LPS-tolerized primary mouse bone marrow-derived macrophages (BMDM). We characterized promoter and enhancer states by mapping the methylation and acetylation states of associated histones and we identified differentially expressed genes by nascent RNA profiling. We clustered genes into different subsets based on their activity profiles and assigned to them enhancers with correlated dynamic behaviors. A detailed analysis of these datasets allowed us to dissect the mechanisms underlying functional switches in the macrophage gene expression program during sustained inflammation. Consistently with the RNA-seq data, genomic regions associated with transient genes were mainly characterized by transient acetylation of histone H3 lysine 27 (H3K27Ac), a mark of active transcription, while sustained genes were mainly associated with persistent acetylation. Conversely, the promoters of both transient and sustained genes maintained high levels of trimethylation of histone H3 at lysine 4 (H3K4me3) regardless of their transcriptional activity. Moreover, promoters of transient and sustained genes showed a distinct content of transcription factor binding sites. Sustained gene clusters displayed a significant enrichment in binding sites for Interferon Regulatory Factor (IRF) family transcription factors. The overlap between sustained genes and Interferon (IFN)-dependent genes, confirm the dependence of this cluster on the IFN-β regulated feed-forward loop. In particular, IRF7 played a key role in the transcriptional regulation of sustained genes as indicated by the impact of its depletion. Transient gene clusters were mainly regulated by the Early Growth Response (EGR) and Nuclear factor-κB (NF-kB) family transcription factors, which are both downstream effectors of the TLR4 signaling pathway. Biochemical analysis of the key players of this signaling cascade revealed an almost complete exhaustion of the pathway after sustained LPS treatment, which correlated with reduced TRAF6 protein levels. These data suggest that the failure to reactivate those transcription factors that are involved in the transcriptional expression of transient genes is likely due to the hypo-responsive state of the TLR4 signaling pathway in cells exposed to LPS in a sustained manner.