Integrator mediates the biogenesis of enhancer RNAs

Abstract

Integrator is a multi-subunit complex stably associated with the carboxy-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 genes1. 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 metazoans2,3,4,5. Integrator is recruited to enhancers and super-enhancers in a stimulus-dependent manner. Functional depletion of Integrator subunits diminishes the signal-dependent induction of 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.

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Figure 1: Integrator mediates induction of eRNAs.
Figure 2: Integrator is required for enhancer–promoter interaction.
Figure 3: Integrator has a role in termination of eRNAs.
Figure 4: Integrator has a global role in enhancer regulation.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

High-throughput data are deposited at the Gene Expression Omnibus (GEO) under accession number GSE68401.

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Acknowledgements

We would like to thank J. M. Marinis and M. A. Lazar for technical support for GRO-seq experiments. We thank D. Hu in A. Shilatifard’s laboratory for performing the SEC ChIP-seq experiments. We thank the Oncogenomics core facility at Sylvester Comprehensive Cancer Center for performing high-throughput sequencing. We also thank Shiekhattar laboratory members and P.-J. Hamard for support and discussions. This work was supported by funds from University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center and grants R01 GM078455 and R01 GM105754 (R.S.) from the National Institute of Health.

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Contributions

F.L. and A.G. are co-first authors. R.S., F.L. and A.G. conceived and designed the overall project. F.L., A.G. and A.Z. performed the experiments. R.S., F.L. and A.G. analysed the data and wrote the paper.

Corresponding author

Correspondence to Ramin Shiekhattar.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Identification eRNAs responsive to EGF.

a, We identified 91 EGF-responsive enhancer regions in HeLa cells. We annotated extragenic RNAPII sites (see Methods) and used the middle of the RNAPII peak as an anchor to display average profiles of p300, H3K27ac and H3K4me1 (data from the ENCODE project). The profiles represent the mean read density of ChIP-seq data. The 91 loci display a typical enhancer signature, with enrichment of p300 and H3K27ac around the TSS and a broader decoration by H3K4me1. b, Profiles of H3K27ac were obtained from ChIP-seq analysis of HeLa cells before and after 20 min of EGF induction. Mean read density was normalized to sequencing depth. c, EGF stimulates bi-directional transcription from 91 enhancer regions. We displayed the mean read density obtained from strand-specific sequencing of the chromatin-bound RNA fraction (ChromRNA-seq). d, e, Normalized read density (RPKM) was calculated from RNA-seq data for 91 eRNAs (d) and 57 neighbouring protein-coding genes (e) that responded to EGF stimulation (FC >1.6) and mapped within 500 kb from an EGF-responsive eRNA. f, Average profiles of ChromRNA-seq data at 91 enhancer loci (mean density of reads, normalized to total read number). g, h, Box plot of 91 eRNAs before and after treatment with EGF shows the average increase of transcription 20 min after stimulation (P < 0.001), matched by an increase in the neighbouring protein-coding genes (P < 0.02). i, NR4A1 is activated by EGF in HeLa cells: RNAPII and INTS11 are recruited to the NR4A1 locus after 20 min of stimulation, with concomitant accumulation of reads from RNA-seq and ChromRNA-seq. A neighbouring eRNA locus also exhibits increased transcription along with RNAPII and INTS11 recruitment. Sequencing tracks are visualized in BigWig format and aligned to the hg19 assembly of the UCSC Genome Browser. Whiskers on the box plots indicate the variability in the datasets.

Extended Data Figure 2 EGF-induced eRNAs are predominantly non-polyadenylated.

a, We examined transcription at three enhancers adjacent to EGF-responsive genes CCNL1, NR4A1 and DUSP1. Total RNA samples were collected before and after EGF induction. Reverse transcription was performed with random hexamer primer or oligo d(T) primer. Each eRNA strand was analysed by real-time PCR with specific primers. Error bars represent ± standard error of the mean (s.e.m., n = 3 biological independent experiments). P < 0.01 by two-sided t-test. b, c, RNA-seq was performed on the polyadenylated and non-polyadenylated fraction of total RNA. RNA-seq tracks were visualized in BigWig format and aligned to the hg19 assembly of the UCSC Genome Browser. CCNL1 and DUSP1 enhancers were displayed (b) along with a polyadenylated control (DUSP1 protein-coding locus) and a non-polyadenylated transcript (snRNA U12) (c). All EGF-induced eRNAs and protein-coding genes (RefSeq hg19) were examined for their average RPKM throughout the entire locus. d, We compared polyadenylation levels of 225 eRNAs and 150 protein-coding genes (2 fold induction upon EGF, RPKM calculated from ChromRNA-seq data previously described). The box plot shows predominance of non-polyadenylated transcripts mapping to eRNA loci, as opposed to transcripts coding for RefSeq genes. Whiskers on the box plot indicate the variability in the datasets.

Extended Data Figure 3 The Integrator complex is recruited to enhancers upon EGF stimulation.

a, qChIP analysis of Integrator occupancy using INTS11, INTS1 and INST9 antibodies at four eRNA loci. Data were collected during a time course of EGF induction in HeLa cells (0, 20, 40 and 60 min). Error bars represent ± standard error of the mean (s.e.m., n = 3 biological independent experiments). P < 0.01 by two-sided t-test. b, Depletion of INST1 and INST11 protein levels in tet-inducible HeLa clones. The arrow indicates the INTS11-specific signal; the asterisk shows a non-specific band. c, Fold change of H3K27 acetylation (0 min/20 min EGF) before (ctrl) and after (dox) depletion of INTS11. Data were calculated from read density of ChIP-seq experiments across EGF-induced enhancers. Depletion of Integrator significantly affects EGF-dependent increase in H3K27ac (P < 0.05). Whiskers on the box plot indicate the variability in the datasets.

Extended Data Figure 4 Depletion of Integrator impairs activation of eRNAs by EGF.

a, b, Activation of eRNAs near DUSP1, CCNL1 and NR4A1 genes were assayed by qRT–PCR in three independent experiments, using INTS11 (a) or INTS1 (b) inducible shRNA clones. Transcription was followed throughout a 20-min time-course experiment. Each eRNA was amplified with two different sets of specific primers to analyse both strands; dashed lines indicate treatment with doxycycline (dox) to induce shRNAs. Data at every time point are reported as fold change (EGF/non-induced). Error bars represent ± s.e.m. (n = 3 biological independent experiments), P < 0.01 by two-sided t-test. c, Schematic representation of ATF3 and its super-enhancer region located 30 kb upstream (top). Snapshots of ChIP-seq and RNA-seq tracks show EGF-dependent recruitment of RNAPII and INTS11 at the ATF3 locus and at several upstream enhancers. Depletion of INTS11 nearly abolished transcription of eRNAs and ATF3 mRNA. d, Real-time RT–PCR analysis of the ATF3 super-enhancer region upon depletion of INTS11. qPCR analysis was performed before and 5, 10, 15, 20 min after EGF treatment with strand-specific primer sets (indicated below the RNA-seq tracks in c). Error bars represent ± s.e.m. (n = 3 biological independent experiments), P < 0.01 by two-sided t-test.

Extended Data Figure 5 Chromatin conformation capture at control loci.

a, 3C analysis of NR4A1 promoter and control sites. The Con1 site lies 74 kb upstream of the NR4A1 protein-coding gene and the Con2 site is located 42 kb downstream of the enhancer site. There are no looping events between either control sites with the NR4A1 promoter region after EGF induction. b, Similarly, no looping events were detected between the promoter of DUSP1 and a downstream control site (Con). All data were averaged from three independent experiments, P < 0.01 by two-sided t-test.

Extended Data Figure 6 Integrator has a role in eRNA termination.

a, Mean density profiles of GRO-seq data at 91 EGF-induced enhancers. Data are presented as strand-specific mean read density, centred at the middle of the RNAPII peak and normalized to sequencing depth. The underlying box plots were used to quantify the enrichment of GRO-seq reads at the 3′ end of both eRNA transcripts (2 kb window, centred 1 kb downstream of the RNAPII peak). b, RNAPII profiling at 91 enhancers after INTS11 depletion shows accumulation of ChIP-seq reads towards the 3′ end. Data are presented as mean read density, centred at the middle of the RNAPII peak and normalized to sequencing depth. Box plots represent the enrichment of RNAPII reads of both eRNA transcripts (2 kb window, centred 1 kb downstream of the RNAPII peak). RNAPII significantly accumulated (P < 0.004) after depletion of INTS11. Whiskers on the box plots indicate the variability in the datasets. c, d, RNAPII travelling ratio at enhancers was measured as the ratio between RNAPII density close to the transcription start site (the surrounding 300 bp) and 3 kb downstream. Given the bi-directional nature of transcription at enhancers, travelling ratio was calculated for both sense (c) and antisense (d) transcripts.

Extended Data Figure 7 Analysis of super elongation complex at enhancers.

a, b, Metagene analysis on 91 eRNA loci shows the effect of EGF stimulation and INTS11 depletion on the recruitment of the ELL2 (a) and AFF4 (b) subunits of the super elongation complex (SEC). SEC was recruited to enhancers upon EGF stimulation. Depletion of Integrator decreases AFF4 and ELL2 recruitment. Data were visualized as mean read density, normalized to sequencing depth, across 8 kb surrounding the centre of enhancers. c, To investigate the role of the negative elongation factor (NELF) in induction of eRNAs, we infected HeLa cells with lentiviral shRNAs against NELFA, NELFE and a control GFP. Quantitative RT–PCR analysis shows the extent of NELF depletion 72 h after infection. Error bars represent ± s.e.m. (n = 3 biological independent experiments), P < 0.01 by two-sided t-test. d, Depletion of two different NELF subunits does not significantly impact activation of EGF-responsive eRNAs. Data represent fold change of induction (EGF/not induced) after 20 min of stimulation and were normalized against GUSB expression. Error bars represent ± s.e.m. (n = 3 biological independent experiments), P < 0.01 by two-sided t-test. e, ChIP-seq analysis of NELFA before and after depletion of INTS11. Metagene analysis shows mean read density (normalized to sequencing depth) across 91 eRNAs. NELF occupancy at enhancers was not affected by depletion of Integrator.

Extended Data Figure 8 Integrator depletion causes accumulation of unprocessed eRNAs and prevents release of RNAPII.

a, Termination of eRNAs was examined with quantitative RT–PCR. Primer pairs were designed to amplify a portion of the enhancer transcript detected in normal condition (t, total) or a longer template further extending into the 3′of the enhancer region (u, unprocessed). qPCR analysis was performed before (ctrl) and after (dox) depletion of INTS11 at three eRNAs (sense and antisense strand), after stimulation with EGF. In the absence of INTS11, we observed accumulation of unprocessed eRNA, suggestive of a termination defect. Error bars represent ± s.e.m. (n = 3 biological independent experiments), P < 0.01 by two-sided t-test. Release of eRNA transcripts from RNA polymerase was investigated by means of RNAPII immunoprecipitation following UV cross-link (UV-RIP). bd, After RNAPII immunoprecipitation, eRNAs near DUSP1, CCNL1 and NR4A1 genes were assayed by qRT–PCR and showed increased association with RNAPII in the absence of Integrator. Each eRNA was detected by two different sets of specific primers (sense and antisense). Error bars represent ± s.e.m. (n = 3 biological independent experiments). *P < 0.01, **P < 0.01, ***P < 0.001 by two-sided t-test. eg, RNAPII UV-RIP analysis was also performed on several eRNAs from the ATF3 super-enhancer. qRT–PCR on the RNA recovered after immunoprecipitation shows increased association between RNAPII and eRNAs in the absence of Integrator. Each eRNA was detected by two different sets of specific primers (sense and antisense). Error bars represent ± s.e.m. (n = 3 three independent experiments). **P < 0.01 by two-sided t-test.

Extended Data Figure 9 Distribution of RNAPII and nascent RNAs across protein-coding genes.

a, Expression level of exogenous INTS11 wild type (WT) and its catalytic mutant (E203Q). Nuclear extracts were subjected to Flag immunoprecipitation and probed with a polyclonal antibody raised against the C terminus of INTS11. b, Heat map of nascent RNA (GRO-seq) and RNAPII ChIP-seq across the 2,000 most active genes in HeLa cells. Gene loci were analysed for their entire gene body, with 3 additional kilobases on both ends. H3K27ac data from ENCODE is shown on the left; genes are ranked according to the intensity of RNAPII signal. Depletion of Integrator does not appear to affect termination at protein-coding genes.

Supplementary information

Supplementary Table 1

This table contains a list of 91 EGF stimulated enhancer RNA loci. (XLSX 70 kb)

Supplementary Table 2

This table contains all the PCR primer sequences and hairpin sequences. (XLSX 49 kb)

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Lai, F., Gardini, A., Zhang, A. et al. Integrator mediates the biogenesis of enhancer RNAs. Nature 525, 399–403 (2015). https://doi.org/10.1038/nature14906

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