Integrator regulates transcriptional initiation and pause release following activation

Abstract

In unicellular organisms, initiation is the rate-limiting step in transcription; in metazoan organisms, the transition from initiation to productive elongation is also important. Here, we show that the RNA polymerase II (RNAPII)-associated multiprotein complex, Integrator, plays a critical role in both initiation and the release of paused RNAPII at immediate early genes (IEGs) following transcriptional activation by epidermal growth factor (EGF) in human cells. Integrator is recruited to the IEGs in a signal-dependent manner and is required to engage and recruit the super elongation complex (SEC) to EGF-responsive genes to allow release of paused RNAPII and productive transcription elongation.

Figure 1. Integrator is recruited to immediate early genes following EGF induction

(A) ChIP-seq tracks of RNAPII in HeLa cells before and after 20 minutes of EGF stimulation. Serum starvation (48h) causes accumulation of paused RNAPII at the TSS of immediate early genes such as CCNL1 and FOS. EGF stimulation releases RNAPII from the proximal promoter into the gene body. ChIP-seq tracks are visualized in a BigWig format and aligned to the hg19 assembly of the UCSC Genome Browser. (B) Average profile of RNAPII across 76 EGF-responsive genes, mean density is calculated as the average read number normalized to sequencing depth (total bin number: 240, from −1kb before the TSS to +3kb after the TES). (C) Fold recruitment of RNAPII after EGF stimulation. Fold increase is calculated as the ratio of the average read distribution before and after EGF (the average gene locus was divided in 35 bins from −500 upstream the TSS to 1kb downstream the TES). (D) Traveling ratio of RNAPII before and after EGF stimulation at 76 EGF-responsive genes. The ratio is calculated as log10 of read density at TSS/read density over the gene body. (E) qChIP analysis of Integrator recruitment at the TSS and 3′ end of JUN, FOS and NR4A1 genes. Three Integrator subunits (INTS1, INTS9 and INTS11) were examined during the time-course of EGF activation (0, 20, 40 and 60 minutes) in wild type HeLa cells. Averages of three experiments is shown. (F) INTS11 is recruited to FOS, JUN, NR4A1 and CCNL1 in an EGF-dependent manner in wild type HeLa cells. ChIP-seq tracks of INTS11 before and 20 minutes following EGF stimulation show recruitment of INTS11 at the TSS and gene body of these four immediate early genes. Before EGF treatment cells were starved for 48h in low serum. (G) Average density profile of INTS11 across 76 EGF-responsive genes. The entire gene body is profiled, with the addition of 1kb upstream of the transcription start site (TSS) and 3kb downstream of the transcription end site (TES). Density profiles are normalized to sequencing depth. (See also Figure S1 and Table S1)

Figure 2. Integrator is essential for EGF responsiveness

(A) Heatmap of the 116 top microarray probes (corresponding to 103 unique genes) upregulated by EGF in normal conditions (CTRL shRNA, t=30min over t=0). The color scale represents the modified log2 ratio (“sweep” function R, scaled by row) between the induced and the basal state. The color variation accounts for the difference of induction across the 2 conditions (Red=augmented induction, Green=decreased induction). The average of three independent experiments is shown. (B) Boxplot of the fold activation (expressed as log2) of the 116 most responsive microarray probes after EGF induction (*, p<0.01; Wilcoxon test). (C) EGF responsiveness at NR4A1, JUN, FOS and CCNL1 is dramatically decreased in the absence of INTS11. RNA-Seq analysis was performed in a cell clone expressing tet-inducible shRNAs targeting INTS11, doxycycline (DOX) was added to the culture medium for 72h to deplete INTS11 protein level. RNA-Seq tracks are aligned to the UCSC hg19 human genome. (D) Expression analysis of 76 EGF-responsive genes in a HeLa inducible shRNA clone by RNA-seq. A robust EGF-mediated activation of EGF genes (mean: 4.13 and 5.25, p=0.005) is impaired in the absence of INTS11 (mean: 3.95 and 4.52, p=0.15). The Boxplot represents the distribution of log2(FPKM) values for the top 76 genes induced by EGF in the control (log2FoldChange>0.4) and occupied by Integrator (see Fig. 1G). (E) Average density of RNA-Seq reads at 76 EGF responsive genes. Data were normalized to sequencing depth. (See also Figure S2 and Table S1)

Figure 3. Integrator regulates initiation as well as pause release following EGF activation

(A) Global Run-On (GRO-Seq) and RNAPII ChIP-Seq data were obtained from cells expressing doxycycline (DOX) -inducible shRNAs against INTS11, after EGF stimulation. JUN, CCNL1, and FOS profiles show a dramatic decrease in the amount of nascent RNA reads mapping to the gene body and 3′ end, whilst the peaks of RNA at the 5′ is proportionally increased. NR4A1 shows a moderate decrease across the gene body but a consistent accumulation of reads at the TSS. Concomitantly, INTS11 depletion dramatically reduces RNAPII occupancy at the body of all genes. All tracks represent read density normalized to sequencing depth. (B) Average density of GRO-Seq reads at 76 EGF responsive genes. Mean densities were normalized to sequencing depth. The entire gene locus is displayed with additional 1kb upstream the TSS and 3kb downstream the TES. The right panel shows a control analysis performed on 76 transcriptionally active genes non responsive to EGF (Table S1). These genes were randomly chosen among the most active genes in HeLa cells according to RNAPII occupancy. (C) Average density of RNAPII ChIP-Seq reads at 76 EGF responsive genes. Mean densities for all genes were normalized to sequencing depth. The right panel shows a control profile of RNAPII at 76 control genes. (D) Residual occupancy of RNAPII after INTS11 depletion in EGF stimulated cells. The percentage is calculated from the ratio of the average read distribution with or without DOX (the average gene locus was divided in 35 bins from −500 upstream the TSS to 1kb downstream the TES). Depletion of Integrator affects elongating RNAPII in the gene body and 3′ end to a greater extent than initiating RNAPII. (E) Traveling Ratio of RNAPII in the presence or absence of INTS11. TR increases at nearly all EGF-responsive genes in the absence of INTS11, indicating accumulation of non-processive paused RNAPII. (See also Figure S3)

Figure 4. Integrator is required for SEC recruitment

(A) Recruitment of AFF4 and ELL2 components of the SEC complex is severely impaired in the absence of Integrator. ChIP-Seq data were obtained from a cell clone expressing tet-inducible shRNAs targeting INTS11, before and after EGF stimulation. JUN and FOS profiles show a dramatic decrease in the amount of SEC recruited (see Fig. S4A for NR4A1 and CCNL1 profiles). Y axis represents read density normalized to sequencing depth. (B) Average density of AFF4 and ELL2 ChIP-Seq reads at 76 EGF responsive genes. Distribution are shown for INTS11 knock-down and its control, before (dashed lines) and after (solid lines) induction with EGF. Density profiles were normalized to total read number. (See also Figure S4)

Figure 5. Integrator functionally and physically interacts with SEC

(A) INTS11 knockdown prevents CDK9 recruitment at IEG promoters in response to EGF stimulation. CDK9 occupancy was measured by qChIP after INTS11 knockdown and EGF induction. Data are the average of three independent experiments (*, p<0.15; **, p<0.05, t-test). (B) Co-immunoprecipitation of overexpressed Myc-CDK9 and Flag-Integrator subunits in HEK293T cells shows CDK9 interaction with INTS1, INTS4 and INTS11. (C) Endogenous Integrator and CDK9 co-precipitate in HEK293T nuclear extracts, immunoprecipitations were performed with INTS1, INTS4, INTS11 and two different CDK9 antibodies. The negative elongation factor, NELFA, was used as a negative control. (D) Co-immunoprecipitation of SEC and Integrator using antibodies against endogenous CDK9 and AFF4. During the course of the IP, nuclear extract of HEK293T was incubated with 50 ug/ml Ethidium Bromide to disrupt indirect protein interactions mediated by DNA (upper panels) and with RQ1 DNase or RNase T1/RNase H to disrupt indirect protein interactions (lower panel).

Figure 6. Integrator is required for Heat Shock activation in Drosophila

(A) Position of the qPCR amplicons along the Drosophila melanogaster HSP70Aa gene. (B) qChIP analysis of RNA Polymerase II (unmodified CTD) localization on the HSP70 gene before and after heat shock (S2 cells, 5 min at 37C). (C) qChIP analysis of IntS9 localization on the HSP70 gene before and after heat shock (S2 cells, 5 min at 37C). (D) qChIP analysis of IntS12 localization on the HSP70 gene before and after heat shock (S2 cells, 5 min at 37C). (E) Immunoblot analysis of Integrator subunits after treatment of S2 cells with dsRNA against each Integrator subunit, dsRNA targeting the E. coli LacZ gene is used as a negative control. (F) Induction of HSP70 transcription by 20 min heat shock at 37C after knockdown of different Integrator subunits and LacZ as a control. The fold induction is compared with room temperature expression levels after normalization using the RPS17 gene, a control housekeeping gene (average of three independent experiments). (See also Figure S5)

Figure 7. Integrator’s role in EGF-mediated gene activation

We propose a model for the role of the Integrator complex in elongation and pause release. During serum starvation, an immediate early gene reduces its transcriptional activity as paused polymerase is accumulated at the TSS. Upon EGF stimulation, a signaling cascade results in a sequence-specific binding of a combination of transcription factors at the promoter of IEGs. Following activation, Integrator is directed to EGF target genes through the C-terminal domain of RPB1 resulting in the recruitment of the SEC complex leading to eviction of negative elongation factors and inducing of productive transcriptional elongation.