Mammalian genomes are pervasively transcribed outside mapped protein-coding genes. One class of extragenic transcription products is represented by long non-coding RNAs (lncRNAs), some of which result from Pol_II transcription of bona-fide RNA genes. Whether all lncRNAs described insofar are products of RNA genes, however, is still unclear. Here we have characterized transcription sites located outside protein-coding genes in a highly regulated response, macrophage activation by endotoxin. Using chromatin signatures, we could unambiguously classify extragenic Pol_II binding sites as belonging to either canonical RNA genes or transcribed enhancers. Unexpectedly, 70% of extragenic Pol_II peaks were associated with genomic regions with a canonical chromatin signature of enhancers. Enhancer-associated extragenic transcription was frequently adjacent to inducible inflammatory genes, was regulated in response to endotoxin stimulation, and generated very low abundance transcripts. Moreover, transcribed enhancers were under purifying selection and contained binding sites for inflammatory transcription factors, thus suggesting their functionality. These data demonstrate that a large fraction of extragenic Pol_II transcription sites can be ascribed to cis-regulatory genomic regions. Discrimination between lncRNAs generated by canonical RNA genes and products of transcribed enhancers will provide a framework for experimental approaches to lncRNAs and help complete the annotation of mammalian genomes. Mammalian genomes contain vast intergenic regions that are extensively transcribed and generate various types of short and long non-coding RNAs (ncRNAs). Although in some cases specific functions have been assigned to intergenic transcripts, the functional significance of this transcriptional output remains largely unknown, and the possibility exists that part of this transcription reflects noise generated by random collisions of the transcriptional machinery with the genome to generate meaningless transcription. In this study we used chromatin signatures to characterize extragenic transcription sites targeted by RNA Polymerase II (RNA Pol II) in a highly regulated response—endotoxin activation of macrophages. We found that a significant portion of extragenic transcription sites are associated with the chromatin signature characteristic of enhancers. Consistent with their chromatin signature, we found that these extragenic transcription sites are under purifying selection and contain binding sites for inflammatory transcription factors, as well as for PU.1, a hematopoietic transcription factor that marks enhancers in macrophages. Moreover, much of this extragenic transcription is regulated by stimulation. We also identified hundreds of transcribed regions with a signature of canonical RNA genes. Our data indicate that extragenic transcription sites can be efficiently classified using chromatin signatures, which will be relevant for functional annotation of mammalian genomes.

Cells of the monocyte–macrophage lineage play a central role in the orchestration and resolution of inflammation. Plasticity is a hallmark of mononuclear phagocytes, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic or M1 and alternative or M2. NF-κB is a key regulator of inflammation and resolution, and its activation is subject to multiple levels of regulation, including inhibitory, which finely tune macrophage functions. Here we identify the p50 subunit of NF-κB as a key regulator of M2-driven inflammatory reactions in vitro and in vivo. p50 NF-κB inhibits NF-κB–driven, M1-polarizing, IFN-β production. Accordingly, p50-deficient mice show exacerbated M1-driven inflammation and defective capacity to mount allergy and helminth-driven M2-polarized inflammatory reactions. Thus, NF-κB p50 is a key component in the orchestration of M2-driven inflammatory reactions.

Estrogen is an immunoregulatory agent, in that hormone deprivation increases while 17β-estradiol (E₂) administration blocks the inflammatory response; however, the underlying mechanism is still unknown. The transcription factor p65/relA, a member of the nuclear factor κB (NF-κB) family, plays a major role in inflammation and drives the expression of proinflammatory mediators. Here we report a novel mechanism of action of E₂ in inflammation. We observe that in macrophages E₂ blocks lipopolysaccharide-induced DNA binding and transcriptional activity of p65 by preventing its nuclear translocation. This effect is selectively activated in macrophages to prevent p65 activation by inflammatory agents and extends to other members of the NF-κB family, including c-Rel and p50. We observe that E₂ activates a rapid and persistent response that involves the activation of phosphatidylinositol 3-kinase, without requiring de novo protein synthesis or modifying Iκ-Bα degradation and mitogen-activated protein kinase activation. Using a time course experiment and the microtubule-disrupting agent nocodazole, we observe that the hormone inhibits p65 intracellular transport to the nucleus. This activity is selectively mediated by estrogen receptor alpha (ERα) and not ERβ and is not shared by conventional anti-inflammatory drugs. These results unravel a novel and unique mechanism for E₂ anti-inflammatory activity, which may be useful for identifying more selective ligands for the prevention of the inflammatory response.