Gene-specific mechanisms dictate glucocorticoid receptor-mediated repression of inflammatory response genes in macrophages [ChIP-seq]

The Glucocorticoid Receptor (GR) potently represses macrophage-elicited inflammation, however, the underlying mechanisms remain obscure. Our genome-wide analysis reveals that pro-inflammatory paused genes, activated via global negative elongation factor (NELF) dissociation and RNA Polymerase (Pol)2 release from early elongation arrest, and non-paused genes, induced by de novo Pol2 recruitment, are equally susceptible to acute glucocorticoid repression. Moreover, in both cases the dominant mechanism involves rapid GR tethering to p65 at NF-kB binding sites. Yet, specifically at paused genes, GR activation triggers widespread promoter accumulation of NELF, with myeloid cell-specific NELF deletion conferring glucocorticoid resistance. Conversely, at non-paused genes, GR attenuates the recruitment of p300 and histone acetylation, leading to a failure to assemble BRD4 and Mediator at promoters and enhancers, ultimately blocking Pol2 initiation. Thus, GR displays no preference for a specific pro-inflammatory gene class, however, it effects repression by targeting distinct temporal events and components of transcriptional machinery ChIPSeq experiments in primarry mouse bone marrow derived macrophages (BMMF) from the wild-type (C57BL/6 mice (NCI, Charles River Laboratories)) mice treated with LPS,Dex or LPS+Dex in comparision with untreated control to determine genome-wide distribution of RNA polymerase II, Glucocorticoid receptors (GR), transcription factor NFkB, NELFE and BRD4. Please note that 32_p65_Input.bam was used as an input in the Brd4 experiment