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.

Jmjd3, a JmjC family histone demethylase, is induced by the transcription factor NF‐kB in response to microbial stimuli. Jmjd3 erases H3K27me3, a histone mark associated with transcriptional repression and involved in lineage determination. However, the specific contribution of Jmjd3 induction and H3K27me3 demethylation to inflammatory gene expression remains unknown. Using chromatin immunoprecipitation‐sequencing we found that Jmjd3 is preferentially recruited to transcription start sites characterized by high levels of H3K4me3, a marker of gene activity, and RNA polymerase II (Pol\_II). Moreover, 70% of lipopolysaccharide (LPS)‐inducible genes were found to be Jmjd3 targets. Although most Jmjd3 target genes were unaffected by its deletion, a few hundred genes, including inducible inflammatory genes, showed moderately impaired Pol\_II recruitment and transcription. Importantly, most Jmjd3 target genes were not associated with detectable levels of H3K27me3, and transcriptional effects of Jmjd3 absence in the window of time analysed were uncoupled from measurable effects on this histone mark. These data show that Jmjd3 fine‐tunes the transcriptional output of LPS‐activated macrophages in an H3K27 demethylation‐independent manner.

Patterns of methylation at lysine 4 and 27 of histone H3 have been associated with states of gene activation and repression that are developmentally regulated and are thought to underlie the establishment of lineage specific gene expression programs. Recent studies have provided fundamental insight into the problem of lineage specification by comparing global changes in chromatin and transcription between ES and neural stem (NS) cells, points respectively of departure and arrival for neural commitment. With these maps of the differentiated state in place, a central task is now to unravel the chromatin dynamics that enables these differentiation transitions. In particular, the observation that lineage-specific genes repressed in ES cells by Polycomb-mediated H3-K27 trimethylation (H3-K27me3) are demethylated and derepressed in differentiated cells posited the existence of a specific H3-K27 demethylase. In order to gain insight into the epigenetic transitions that enable lineage specification, we investigated the early stages of neural commitment using as model system the monolayer differentiation of mouse ES cells into neural stem (NS) cells. Starting from a comprehensive profiling of JmjC-domain genes, we report here that Jmjd3, recently identified as a H3-K27me3 specific demethylase, controls the expression of key regulators and markers of neurogenesis and is required for commitment to the neural lineage. Our results demonstrate the relevance of an enzymatic activity that antagonizes Polycomb regulation and highlight different modalities through which the dynamics of H3-K27me3 is related to transcriptional output. By showing that the H3-K27 demethylase Jmjd3 is required for commitment to the neural lineage and that it resolves the bivalent domain at the Nestin promoter, our work confirms the functional relevance of bivalent domain resolution that had been posited on the basis of the genome-wide correlation between their controlled resolution and differentiation. In addition, our data indicate that the regulation of H3-K27me3 is highly gene- and context- specific, suggesting that the interplay of methyltransferases and demethylases enables the fine-tuning more than the on/off alternation of methylation states.

The importance of post‐transcriptional mechanisms for the regulation of the homoeostasis of the immune system and the response to challenge by microorganisms is becoming increasingly appreciated. We investigated the contribution of microRNAs (miRNAs) to macrophage activation induced by lipopolysaccharide (LPS). We first observed that Dicer knockout in bone marrow‐derived macrophages (BMDMs) increases the LPS‐induced expression of some inflammation mediators. miRNA microarray analysis in BMDMs revealed that LPS significantly induces the expression of a single miRNA, miR‐155, and this induction depends on enhanced miR‐155 maturation from its precursors. The single‐strand RNA‐binding protein KHtype splicing regulatory protein (KSRP) binds to the terminal loop of miR‐155 precursors and promotes their maturation. Both inhibition of miR‐155 and KSRP knockdown enhance the LPS‐induced expression of select inflammation mediators, and the effect of KSRP knockdown is reverted by mature miR‐155. Our studies unveil the existence of an LPS‐dependent post‐transcriptional regulation of miR‐155 biogenesis. Once induced, miR‐155 finely tunes the expression of select inflammation mediators in response to LPS.—Ruggiero, T., Trabucchi, M., De Santa, F., Zupo, S., Harfe, B. D., McManus, M. T., Rosenfeld, M. G., Briata, P., Gherzi, R. LPS induces KH‐type splicing regulatory protein‐dependent processing of microRNA‐155 precursors in macrophages. FASEB J. 23, 2898–2908 (2009). www.fasebj.org