SUMMARY Macrophages respond to inflammatory stimuli by modulating the expression of hundreds of genes in a defined temporal cascade, with diverse transcriptional and post-transcriptional mechanisms contributing to the regulatory network. We examined pro-inflammatory gene regulation in activated macrophages by performing RNA-Seq with fractionated chromatin-associated, nucleoplasmic, and cytoplasmic transcripts. This methodological approach allowed us to separate the synthesis of nascent transcripts from transcript processing and the accumulation of mature mRNAs. In addition to documenting the sub-cellular locations of coding and non-coding transcripts, the results provide a high-resolution view of the relationship between defined promoter and chromatin properties and the temporal regulation of diverse classes of co-expressed genes. The data also reveal a striking accumulation of full-length yet incompletely spliced transcripts in the chromatin fraction, suggesting that splicing often occurs after transcription has been completed, with transcripts retained on the chromatin until fully spliced.

Much has been learned about transcriptional cascades and networks from large-scale systems analyses of high-throughput data sets. However, analysis methods that optimize statistical power through simultaneous evaluation of thousands of ChIP-seq peaks or differentially expressed genes possess substantial limitations in their ability to uncover mechanistic principles of transcriptional control. By examining nascent transcript RNA-seq, ChIP-seq, and binding motif data sets from lipid A-stimulated macrophages with increased attention to the quantitative distribution of signals, we identified unexpected relationships between the in vivo binding properties of inducible transcription factors, motif strength, and transcription. Furthermore, rather than emphasizing common features of large clusters of co-regulated genes, our results highlight the extent to which unique mechanisms regulate individual genes with key biological functions. Our findings demonstrate the mechanistic value of stringent interrogation of well-defined sets of genes as a complement to broader systems analyses of transcriptional cascades and networks.

Profound global loss of DNA methylation is a hallmark of many cancers. One potential consequence of this is the reactivation of transposable elements (TEs) which could stimulate the immune system via cell-intrinsic antiviral responses. Here, we develop REdiscoverTE, a computational method for quantifying genome-wide TE expression in RNA sequencing data. Using The Cancer Genome Atlas database, we observe increased expression of over 400 TE subfamilies, of which 262 appear to result from a proximal loss of DNA methylation. The most recurrent TEs are among the evolutionarily youngest in the genome, predominantly expressed from intergenic loci, and associated with antiviral or DNA damage responses. Treatment of glioblastoma cells with a demethylation agent results in both increased TE expression and de novo presentation of TE-derived peptides on MHC class I molecules. Therapeutic reactivation of tumor-specific TEs may synergize with immunotherapy by inducing inflammation and the display of potentially immunogenic neoantigens.