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Integrator mediates the biogenesis of enhancer RNAs

2015-08-26 20:32:54

NATURE; 26 August 2015: DOI:10.1038/nature14906


Fan Lai, Alessandro Gardini, Anda Zhang & Ramin Shiekhattar



Abstract


Integrator is a multi-subunit complex stably associated with the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). 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. Here we examine 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 in metazoans. Integrator is recruited to enhancers and super-enhancers in a stimulus-dependent manner. Functional depletion of Integrator subunits diminishes the signal-dependent induction of enhancer RNAs (eRNAs) and abrogates stimulus-induced enhancer–promoter chromatin looping. Global nuclear run-on and RNAPII profiling reveals a role for Integrator in 3'-end cleavage of eRNA primary transcripts leading to transcriptional termination. In the absence of Integrator, eRNAs remain bound to RNAPII and their primary transcripts accumulate. Notably, 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.



Introduction


To assess the role for Integrator in the biogenesis of eRNAs, we examined the signal-dependent recruitment of Integrator complex to enhancer sites. HeLa cells were starved of serum for 48 h, after which they were stimulated with epidermal growth factor (EGF) to induce immediate early genes (IEGs). We identified 2,029 enhancers based on their occupancy by RNAPII, CBP/p300 and containing acetylated histone H3 lysine 27 (H3K27ac) chromatin modification. We found that while assessing steady-state levels of eRNAs provided a measure of EGF-induced eRNAs, we obtained a better read-out of eRNAs after sequencing of the chromatin-enriched RNA fractions (ChromRNA-seq). We focused on 91 enhancers that displayed EGFinduced eRNAs in the proximity of EGF-responsive genes following 20min of induction. Notably, the chromatin surrounding these enhancers displayed the H3K27ac modification instarved cells,and following EGF stimulation there was a small increase in H3K27ac levels. To assess the polyadenylation state of eRNAs, total RNA was enriched for polyadenylated and non-polyadenylated fractions and was subjected to high-throughput sequencing. Similar to previous reports, EGF-induced enhancers displayed bi-directional eRNAs that were predominantly not polyadenylated.



We next analysed Integrator occupancy at these enhancers by using antibodies against the INTS11 subunit of the Integrator complex before and after EGF stimulation. While these enhancers were occupied by a detectable amount of Integrator before EGF induction,addition of EGF resulted in a further recruitment of Integrator complex. RNAPII displayed a similar pattern of stimulus-dependent chromatin residence. The stimulus-dependent recruitment of Integrator at enhancers was further confirmed using two additional antibodies against INTS1 and INTS9 subunits of the Integrator complex. These results demonstrated the stimulus-dependent recruitment of the Integrator complex at EGF responsive enhancers. To examine the functional importance of Integrator at enhancers and its role in the biogenesis of eRNAs, we developed HeLa clones expressing doxycycline-inducible short hairpin RNAs (shRNAs) against INTS11 and INTS1 subunits of the Integrator complex. Within the time course of these experiments the mature levels of small nuclear RNAs(snRNAs) were not perturbed (data not shown). Twenty minutes of EGF stimulation resulted in the induction of bi-directional eRNAs similar to previous reports. Depletion of INTS11 diminished the eRNA induction after EGF stimulation. The fold induction of eRNAs at all EGF-induced enhancers decreased significantly. We also observed a significant decrease in the transcriptional induction of EGF-responsive protein-coding genes in the proximity of these EGF-induced enhancers. Interestingly, there was a subtle increase (statistically not significant) in H3K27 acetylation at enhancers following EGF stimulation, which was reduced after Integrator depletion.



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