Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Facilitate Transcription Termination

Abstract

Efficient release of promoter-proximally paused RNA Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused RNA Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. Here we report that Integrator subunit 8 (IntS8) is critical for transcription repression and required for association with protein phosphatase 2A (PP2A). We find that Integrator-bound PP2A dephosphorylates the RNA Pol II C-terminal domain and Spt5, preventing the transition to productive elongation. Thus, blocking PP2A association with Integrator stimulates pause release and gene activity. These results reveal a second catalytic function associated with Integrator-mediated transcription termination and indicate that control of productive elongation involves active competition between transcriptional kinases and phosphatases.

Keywords: Integrator; paused RNA Pol II; phosphatase; transcription regulation; transcription termination.

Figure 1.. Depletion of IntS8 causes widespread activation of genes bound by Integrator

(A) DL1 cells were treated with dsRNA targeting IntS8 or ß-galactosidase (Control) for 60 h and RNA isolated for total RNA-seq (n=3 per condition). Normalized counts and fold changes are shown for mRNA genes with no alteration in splicing patterns under these conditions (N=9303). Affected genes show a fold-change > 1.5 and adjusted P value <0.0001. (B) Comparison of fold changes in RNA-seq signals between cells depleted of IntS8 and IntS9. Pearson correlations are shown for genes upregulated (red) or downregulated (green) upon depletion of IntS8. (C) Composite metagene analysis of IntS1 ChIP-seq reads around promoters of mRNA genes upregulated (N=1009) or unchanged (N=8022) upon IntS8-depletion. Data are shown as average reads per gene in 25-bp bins. (D) Promoter-proximal read counts for IntS1 ChIP-seq were summed around snRNAs (N=31), randomly selected regions (N=5000) or promoters upregulated or unchanged by IntS8-depletion, as in C. Violin plots depict the range of values, with median indicated by a line. P value calculated using a Mann-Whitney test.

Figure 2.. PR65/PP2Ac require a discrete motif within IntS8 to associate with Integrator

(A) Heatmap derived from IP LC-MS analysis of FLAG immunoprecipitations from S2 cells expressing the indicated FLAG-tagged proteins. Heatmaps reflect normalized spectral counts observed from analysis of samples performed in triplicate. Control cells lack any exogenously expressed FLAG-tagged protein. (B) Western blot analysis of input nuclear extracts (left) and immunoprecipitations (right) from S2 cells stably expressing FLAG-tagged proteins as indicated. Immunoprecipitations conducted using anti-FLAG affinity resin were normalized to FLAG signals in each immunoprecipitation. (C) Yeast two-hybrid analysis where PR65 was fused to the Gal4 DNA binding domain and 14 Integrator subunits were individually expressed as fusions with the Gal4 activation domain. Empty vector is the negative control. Permissive plates lack leucine and tryptophan (-L/-W) to allow growth of plasmid-containing yeast whereas selective plates also lack histidine to screen for interaction (-L/-W/-H). Yeast are plated in five-fold serial dilutions. (D) Alignment of the N-terminus of IntS8 from multiple species. Scanning mutations are depicted where four consecutive amino acids are converted to alanine. Note the numbering is relative to Homo sapiens INTS8. (E) Results of yeast two-hybrid analysis where either PR65 or IntS5 are fused to the Gal4 DNA binding domain and either wild-type or alanine mutant IntS8 constructs are fused to the Gal4 activation domain. (F) Western blot analysis of nuclear extracts (left) and immunoprecipitations (right) from stable cell lines expressing FLAG-IntS8-WT or FLAG-IntS8-WFEF/A.

Figure 3.. The interaction between IntS8 and PP2A is critical for proper gene expression profiles

(A and B) DL1 cells were depleted of IntS8 using 60 h treatment with dsRNA. RNA was harvested for RNA-seq (n=3 per condition). Where indicated, IntS8-depleted cells were rescued using a transgene expressing wild-type IntS8 (WT) or IntS8 with mutations that disrupt its interaction with PP2A (WFEF/A). To specifically deplete endogenous IntS8 from rescue samples, silent mutations were introduced into the transgene to prevent loss by RNAi (region highlighted in orange). Representative Western blot is shown, indicating IntS8 protein levels in each condition. Tubulin was used as a loading control. (C) SP1029 (CG11956) locus showing an upregulated gene whose expression is rescued by WT IntS8, but not IntS8-WFEF/A. RNA-seq tracks are shown in control cells and each of the treatments. IntS1 ChIP-seq is from Elrod et al., 2019. (D) Heatmap representation of RNA-seq fold changes in IntS8-depleted cells, as compared to cells rescued with WT or WFEF/A mutant IntS8. Genes shown are those upregulated by IntS8 depletion (N=649), ranked by fold change. (E) Fold change in RNA-seq signal upon IntS8 depletion at genes upregulated by IntS8 (N=649). Changes in RNA-seq levels as compared to the control cells are shown following IntS8-depletion, and with rescue by WT or WFEF/A mutant IntS8. Violin plots show range of values, with a line indicating median.

Figure 4.. Integrator-mediated attenuation at coding and non-coding loci requires the IntS8-PP2A interaction

(A) PRO-seq tracks are shown at the 5’ end of the upregulated oaf gene, with TSS designated by arrow. (B) Heatmap representations of PRO-seq reads from indicated samples, treated as in Figure 3. The location of TSSs is indicated by an arrow. Genes that are upregulated upon IntS8 depletion in RNA-seq are shown (N=649), ranked from most to least upregulated. Color bar at bottom indicates range of read counts per 10-nt bin. (C) Violin plots depict the fold change in gene body PRO-seq reads (+500 to +2500 nt from TSS) for upregulated (N=649) or unchanged genes (N=7,182) genes. Plots show the range of values, with a line indicating median. P values are from Kruskal-Wallis test, with Dunn’s multiple comparisons test performed against control. *** indicates P<0.0001; n.s. indicates P>0.05. (D) The difference in PRO-seq signal at upregulated genes between IntS8-depleted samples and control samples is shown. Data are shown in 5-nt bins, smoothed over 2 nearest neighbors. (E) Average distribution of PRO-seq signal is shown at enhancers previously shown to be targeted by Integrator (defined in Elrod et al., 2019). Data represent N=228 Integrator-target eRNAs in 25-nt bins.

Figure 5.. The Pol II CTD and Spt5 are in vitro and in vivo substrates of Integrator-PP2A

(A) Schematics of synthetic peptides used for in vitro phosphatase assays. (B) Results of malachite green assay where peptides shown in panel A were incubated with Integrator-PP2A purified with anti-FLAG affinity resin from nuclear extracts expressing FLAG-PR65. Control is from S2 cells not expressing a FLAG-tagged protein. Representative colorimetric results are shown in the upper panel. Results from triplicate assays are quantified as picomoles of orthophosphate produced above negative control. (C) Results of malachite green assay using Integrator-PP2A purified from cells expressing FLAG-IntS8-WT or FLAG-IntS8-WFEF/A mutant. The substrate used is CTD-Ser7P (n=3). (D) Dose curves of Integrator-PP2A incubated with CTD-Ser7P and increasing amounts of PP2A inhibitors Calyculin A or Okadaic Acid (n=3). (E) Western blot analysis of lysates from cells either mock-depleted or depleted of IntS8. Antibodies shown probe either endogenous proteins or are phospho-specific, as indicated. (F) Western blot analysis of lysates from control or IntS8-depleted cells. Where indicated, cells were induced to express IntS8-WT or IntS8-WFEF/A mutant.

Figure 6.. Assembly and function of Integrator-PP2A is conserved in human cells

(A) Western blot analysis of nuclear extracts (left) and immunoprecipitations (right) from 293T cells harboring FLAG epitope tags within human Integrator genes generated using CRISPR/Cas9 genome-editing. Immunoprecipitations were conducted using anti-FLAG affinity resin and were normalized to FLAG signals in the immunoprecipitation. Control extracts were derived from 293T cells lacking any exogenous FLAG-tagged protein. (B) Western blot analysis of nuclear extracts (left) and immunoprecipitations from either 293T control nuclear extract, or nuclear extracts derived from stable cell lines expressing either FLAG-hINTS8-WT or FLAG-hINTS8-WFEF/A mutation. (C) Western blot analysis of cell lysates derived from 293T cells transfected with either control siRNA or INTS8-targeting siRNA. Lysates were probed with a series of antibodies labeled on the right side of the blots. Note that hSpt5-Ser666 is the equivalent residue of Drosophila Spt5-Ser707, and hSp55-Thr806 is the equivalent of fly Spt5-Thr847. (D) Results of malachite green assay using a CTD-Ser7P peptide substrate represented as pmoles released above control. Integrator-PP2A was purified from 293T nuclear extracts containing either FLAG-hINTS8-WT or FLAG-hINTS8-WFEF/A.

Figure 7.. Function of INTS8 is conserved in human cells

(A) Western blot analysis of cell lysates derived from 293T cells transfected with control or INTS8 targeting siRNA. Lysates were harvested after 48 h and probed with antibodies against INTS8 or GAPDH as a loading control. (B) 293T cells were transfected with control or INTS8 targeting siRNA. After 48 h, RNA was harvested for total RNA-seq (n=3). Normalized counts are shown for mRNA genes (N=12059). Affected genes are those with a fold-change > 2 and adjusted p-value <0.001. (C) RNA-seq tracks are shown for ARC and GADD45B, genes upregulated upon INTS8 depletion. (D) Schematic of the ARC and GADD45B reporter systems, wherein promoter and 5’ UTRs were cloned upstream of GFP. Top: In wild type cells, Integrator-PP2A drives Pol II termination and prevents GFP expression. Bottom: Upon loss of Integrator-PP2A association through INTS8 depletion or expression of INTS8-WFEF/A, Pol II productively elongates through the GFP ORF, yielding GFP expression. (E) Western blot analysis of 293T cells transfected with the GFP reporters, along with control or IntS8 targeting siRNA, or IntS8 targeting siRNA plus RNAi-resistant INTS8-WT or INTS8-WFEF/A mutant cDNA.