Macrophage (M?)-driven inflammation is at the core of autoimmune diseases including rheumatoid arthritis (RA), signifying imbalance between circuits that turn inflammatory genes on and off. The `on' state is conferred by transcription factors such as NF?B that activate basal transcription machinery and chromatin at pro- inflammatory enhancers and promoters. An equally robust `off' signal is conveyed by the glucocorticoid (GC) receptor (GR) which, upon ligand binding, tethers to NF?B and represses its targets. Notably, these genes form distinct classes: those for which RNA polymerase (Pol) II recruitment and transcription initiation are rate- limiting, and those whose promoters are pre-loaded by initiated but paused Pol II and pause-enforcing factors, NELF and DSIF. Pause-release requires the positive elongation factor, P-TEFb, a kinase that phosphorylates Pol II, NELF and DSIF leading to NELF release and productive elongation. Importantly, assembly of the Pol II- NELF-DSIF `paused complex' is tightly regulated by another kinase, CDK7. To date, the role of post-initiation control of transcription in immunity and disease is unknown. Our preliminary data in M? reveal transcriptome- wide Pol II pausing and unexpectedly dynamic behavior of NELF upon inflammatory stimulation. NELF deletion dramatically shifted the balance of pro- and anti-inflammatory mediators and blunted inflammatory responses over time. The pausing complex is also a critical target of GC - the cornerstone of anti-inflammatory therapies. Although basal machinery and chromatin were once considered too general to be `druggable', pioneering work in cancer research uncovered the benefits of targeting epigenetic regulators and, more recently, CDK7. In RA, the transformation of the synovial lining into aggressively proliferating cartilage- and bone-invading tissue is often viewed as malignant. Like tumor cells, synovial M? enter a state of transcriptional dependency with a few super-enhancers dominating the inflammatory transcriptome. These data point to CDK7 inhibition as a novel promising avenue to treat RA as a monotherapy or in combination with GC. Our objective is to dissect the specific contribution of post-initiation control to inflammation - in M? and in mouse models of RA. Our central hypothesis is that pausing plays an essential `permissive' role in M?-driven inflammation by constraining anti- inflammatory gene expression. We further propose that genetic and pharmacologic manipulation of Pol II pausing will yield important insights into the pathogenesis of inflammatory diseases such as RA, with potential to be translated into advances in patient care.
Our Specific Aims are to: 1. Dissect the role of the pause- release checkpoint in inflammatory gene induction in M?; 2. Evaluate early elongation and higher-order chromatin interactions in M? as targets for GR; 3. Assess the utility of manipulating elongation control in vivo using inflammatory arthritis models. The successful completion of this project will: i) reveal the contribution of Pol II pausing to inflammatory M? activation; ii) help refine the anti-inflammatory actions of GC; iii) uncover the potential of new therapeutic strategies targeting the early elongation checkpoint in preclinical models of RA.

Public Health Relevance

Genes responsible for inflammation are tightly controlled by an enzyme RNA Polymerase II, that binds the beginning of each gene, pauses, and then transcribes genes to complete RNAs that ultimately give rise to proteins. Our recent studies show that in immune cells called macrophages, Polymerase `pause-release' is a critical step that determines how well most inflammatory genes are transcribed. Our proposal aims to understand the mechanisms of `pause-release' and the utility of chemicals that target pausing as future anti-inflammatory drugs for autoimmune diseases such as rheumatoid arthritis.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Peyman, John A
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Hospital for Special Surgery
New York
United States
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