While the long non-coding RNAs (ncRNAs) constitute a large portion of the mammalian transcriptome, their biological functions has remained elusive. A few long ncRNAs that have been studied in any detail silence gene expression in processes such as X-inactivation and imprinting. We used a GENCODE annotation of the human genome to characterize over a thousand long ncRNAs that are expressed in multiple cell lines. Unexpectedly, we found an enhancer-like function for a set of these long ncRNAs in human cell lines. Depletion of a number of ncRNAs led to increased expression of their neighboring protein-coding genes, including the master regulator of hematopoiesis, SCL (also called TAL1), Snai1 and Snai2. Using heterologous transcription assays we demonstrated a requirement for the ncRNAs in mediating such enhancement of gene expression. These results reveal an unanticipated role for a class of long ncRNAs in activation of critical regulators of development and differentiation.

Integrator is a multi-subunit complex stably associated with the C-terminal domain (CTD) of RNA polymerase II (RNAPII) 1. Integrator is endowed with a core catalytic RNA endonuclease activity, which is required for the 3′-end processing of non-polyadenylated RNAPII-dependent uridylate-rich small nuclear RNA genes (UsnRNAs) 1. Here, we examined the requirement of Integrator in the biogenesis of transcripts derived from distal regulatory elements (enhancers) involved in tissue- and temporal-specific regulation of gene expression 2–5. Integrator is recruited to enhancers and super-enhancers in a stimulus-dependent manner. Functional depletion of Integrator subunits diminishes the signal-dependent induction of eRNAs and abrogates the stimulus-induced enhancer-promoter chromatin looping. Global nuclear run-on and RNAPII profiling reveals a role for Integrator in 3′-end cleavage of eRNAs primary transcripts leading to transcriptional termination. In the absence of Integrator, eRNAs remain bound to RNAPII and their primary transcripts accumulates. Importantly, the induction of eRNAs and gene expression responsiveness requires the catalytic activity of Integrator complex. We propose a role for Integrator in biogenesis of eRNAs and enhancer function in metazoans.

SUMMARY The control of promoter-proximal pausing and the release of RNA polymerase II (Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify Pol II-associated Factor 1 (PAF1) to possess an evolutionarily conserved function in metazoans in the regulation of promoter-proximal pausing. Reduction in PAF1 levels leads to an increased release of paused Pol II into gene bodies at thousands of genes. PAF1 depletion results in increased nascent and mature transcripts and increased levels of phosphorylation of Pol II’s C-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase Super Elongation Complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing a novel function for PAF1 as a regulator of promoter-proximal pausing by Pol II.