Thus, although Bcl-6/p65 sites account for just 8% of the NF-B p65 cistrome (Fig. and activation via epigenetically Docosahexaenoic Acid methyl ester markedcis-regulatory elements. Keywords:Bcl-6, NF-B, macrophage, inflammation, cistrome, ChIP-seq Toll-like receptors (TLRs) sense conserved pathogen-associated molecular patterns that rapidly activate the immune system to induce inflammation (Takeuchi and Akira 2010). The transcription factor NF-B is a common mediator for TLR signaling and is critical for TLR-elicited gene expression programs controlling immune function as well as normal development and tissue homeostasis (Hayden and Ghosh 2004). Growing evidence indicates that TLRs recognize not only microbial products, but also endogenous molecules derived from damaged cells. Underscoring this notion, noninfectious diseases including atherosclerosis, type 2 diabetes mellitus, neurodegeneration, and certain cancers have now been linked to TLRNF-B signaling and chronic inflammation (Frantz et al. 2007;Medzhitov 2010). Thus, deciphering the TLRNF-B transcriptional network and how it can be controlled is broadly important for understanding immunity as well as disease. The macrophage is a benign immune sentinel that expresses TLRs and, in response to a range of signals, becomes a principal cellular effector of both acute and chronic inflammation. The tremendous complexity of the TLR-induced inflammatory response, particularly at the level of gene control, has suggested that modular networks demarcate functional pathways and control innate immunity (Medzhitov and Horng 2009). However, the genomic architecture of such networks and the mechanisms by which they are regulated to prevent excessive inflammatory responses are poorly understood. One potential mediator of macrophage quiescence is the sequence-specific transcriptional repressor B-cell SIRT4 lymphoma 6 (BCL-6) (Chang et al. 1996;Dhordain et al. 1997;Huynh and Bardwell 1998;Huynh et al. 2000;Lemercier et al. 2002;Parekh et al. 2007;Mendez et al. 2008). Although best known for its role in B-cell development and non-Hodgkin’s lymphomas (Dent et al. 1997;Ye et al. 1997), we and others have Docosahexaenoic Acid methyl ester shown that BCL-6 can bind nuclear receptors and their corepressors, which have been implicated in macrophage modulation of inflammation (Lee Docosahexaenoic Acid methyl ester et al. 2003;Ogawa et al. 2004;Ghisletti et al. 2009). Moreover, Bcl-6-deficient mice develop lethal neonatal pulmonary vasculitis as well as myocarditis (Dent et al. 1997;Ye et al. 1997) and dysregulated expression of a small number of chemokines and cytokines (Toney et al. 2000;Takeda et al. 2003;Yu et al. 2005;Mondal et al. 2010). These collective observations raised the possibility Docosahexaenoic Acid methyl ester that Bcl-6 might play a broader role in macrophage quiescence and the termination of the inflammatory response. In this study, we provide genetic and genomic evidence that Bcl-6, acting through a limited number of binding sites, broadly constrains the inflammatory response through cistromic antagonism of a TLRNF-B subnetwork. == Results and Discussion == == Bcl-6 controls one-third of the Tlr4 transcriptome == To begin, we performed genome-wide expression analysis to define the Bcl-6-regulated gene network in quiescent and lipopolysaccharide (LPS)-stimulated Bcl-6+/+(wild-type) and Docosahexaenoic Acid methyl ester Bcl-6/(knockout) bone marrow-derived macrophages (BMDMs). Unexpectedly, a large number of mRNAs (>2500 genes) were altered by the absence of Bcl-6 (Fig. 1A), and >500 could be functionally categorized as involved in inflammation (35%); differentiation, apoptosis, and cancer (24%); cell signaling (12%); metabolism (16%); or other assorted pathways (13%) (Fig. 1B; Supplemental Table 1). Treatment with the inflammatory trigger LPS resulted in vast changes in the expression of >3500 genes in either wild-type or knockout cells (Fig. 1A; Supplemental Fig. 1A). Remarkably, comparison of these gene networks revealed that more than a third of the LPS-elicited transcriptome is also controlled by Bcl-6 (Fig. 1A), and, for >60% of these coregulated genes, loss of Bcl-6 mimicked LPS stimulation. Analysis of Bcl-6- and LPS-coregulated genes found that nearly half of those classified were inflammatory (Fig. 1C; Supplemental Table 2). Using quantitative PCR (qPCR), we interrogated many of these targets in wild-type and knockout BMDMs over a 6-h time course of LPS exposure, demonstrating that loss of Bcl-6 produces a broad and often striking hypersensitive response (Fig. 1D; Supplemental Fig. 1B). Among the many dramatically coregulated genes were an array of C-C and C-X-C chemokines involved in inflammatory cell recruitment (Charo and Ransohoff 2006), including Ccl-2, Ccl-3, Ccl-4, and Ccl-7, as well as Cxcl-2, Cxcl-11, and Cxcl-14; growth factors such as Csf-1, which drives leukocyte chemotaxis, differentiation, and proliferation (Pixley and Stanley 2004); acute phase cytokines (e.g., Il-1 and Il-1); and anti-inflammatory modulators, including Ppar and Dusp1 (Jeffrey et al. 2007). Many of these genes showed striking changes in basal expression that.