Abstract
Studies on macrophage gene expression have historically focused on events leading to RNA polymerase II recruitment and transcription initiation, whereas the contribution of post-initiation steps to macrophage activation remains poorly understood. Here, we report that widespread promoter-proximal RNA polymerase II pausing in resting macrophages is marked by co-localization of the negative elongation factor (NELF) complex and facilitated by PU.1. Upon inflammatory stimulation, over 60% of activated transcriptome is regulated by polymerase pause-release and a transient genome-wide NELF dissociation from chromatin, unexpectedly, independent of CDK9, a presumed NELF kinase. Genetic disruption of NELF in macrophages enhanced transcription of AP-1-encoding Fos and Jun and, consequently, AP-1 targets including Il10. Augmented expression of IL-10, a critical anti-inflammatory cytokine, in turn, attenuated production of pro-inflammatory mediators and, ultimately, macrophage-mediated inflammation in vivo. Together, these findings establish a previously unappreciated role of NELF in constraining transcription of inflammation inhibitors thereby enabling inflammatory macrophage activation.
Highlights
Studies on macrophage gene expression have historically focused on events leading to RNA polymerase II recruitment and transcription initiation, whereas the contribution of postinitiation steps to macrophage activation remains poorly understood
To comprehensively define the global Pol II pausing patterns as related to signal-induced transcription in murine primary bone marrow-derived macrophages (BMDM), we performed Pol II chromatin immunoprecipitation followed by high throughput sequencing (chromatin immunoprecipitation (ChIP)-seq) and precision nuclear run-on sequencing (PROseq)
Paused (PI ≥ 3, group 1) and moderately paused (1.5 ≤ pausing index (PI) < 3, group 2) genes made up 76% of the BMDM transcriptome (Fig. 1c, Supplementary Fig. 1b), whereas non-paused genes made up 24% (PI < 1.5, group 3)
Summary
Studies on macrophage gene expression have historically focused on events leading to RNA polymerase II recruitment and transcription initiation, whereas the contribution of postinitiation steps to macrophage activation remains poorly understood. Studies by us and others have shown how PTEFb loading and transcription elongation are targeted by negative regulators of inflammation including the glucocorticoid receptor and other transcription repressors[21,22,26], underscoring the physiological importance of immune gene regulation during early elongation. These studies mainly focused on specific subsets of genes of interest, whereas the characteristics and a global impact of post-initiation control of transcription to macrophage activation remain to be thoroughly investigated. We describe the consequences of macrophage-specific NELF depletion in vivo thereby establishing a physiological role of NELF in mammalian inflammatory response
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