DSS1 is required for proper Integrator-PP2A function

Abstract

Integrator-PP2A (INTAC) is a highly modular complex orchestrating the transition of paused RNA polymerase II into productive elongation or promoter-proximal premature termination, with its loss resulting in transcription dysregulation and genome instability. Here, we identify human DSS1-a flexible 70-residue protein found in multiple functionally diverse complexes including the 26S proteasome-as an integral subunit of the INTAC backbone. Structural analysis of DSS1-INTAC, both alone and in association with paused polymerase, demonstrates intimate interactions between DSS1 and the INTAC backbone. We identify tryptophan 39 of DSS1 as being critical for interacting with INTAC and find that its mutation disrupts DSS1's interaction with INTAC, while maintaining DSS1's interaction with the proteasome. This substitution not only impairs INTAC-dependent transcriptional regulation, but also reveals that INTAC is DSS1's major chromatin-bound form. Together, our findings reveal a role for DSS1 in supporting the structure and regulatory functions of INTAC.

Fig. 1. DSS1 is an integral component of the INTAC backbone module.

a The workflow of DSS1 purification and mass spectrometry (MS) using Flp-In T-Rex system in HEK293 cells. b Proteins identified by MS were ranked by the enrichment in purification of DSS1 versus vector. INTAC subunits are labeled. c STRING protein-protein interaction network of DSS1 interactome combined with the proteomics data of DSS1. Red lines indicate the interactions identified in our DSS1 MS data. d Schematic of the generation of human DSS1–Flag–dTAG DLD-1 cells. e Co-IP analysis of Flag (DSS1) followed by western blotting in DSS1–Flag–dTAG cells. Data represent three biological independent experiments with similar results. Source data are provided as a Source Data File. f Co-IP analysis of endogenous INTS3 and INTS5 in DSS1–Flag–dTAG cells. Data represent three biological independent experiments with similar results. Source data are provided as a Source Data File. g, h Overall cryo-EM map (g) and the molecular model (h) of the DSS1–INTAC complex in two different views, with subunit surfaces/models colored as schematic module (right of (g)). Notably, the cryo-EM map is a composite map derived from multibody refinement. i A close-up view of DSS1 (magenta) in DSS1–INTAC was shown. DSS1 is represented as sticks, surrounded by a transparent density map, indicating its nice fitting.

Fig. 2. Similar genomic distribution of DSS1 and INTAC.

a DSS1 degradation induced by time-course dTAG treatment in DSS1–Flag–dTAG cells. β-tubulin serves as loading control. Data represent three biological independent experiments with similar results. Source data are provided as a Source Data File. b Representative images showing the localization of endogenous DSS1 (green) along with the DAPI signal (blue) after 24-h DMSO or dTAG treatment in DSS1–Flag–dTAG cells. Data represent three biological independent experiments with similar results. c Quantification of mean immunofluorescence (IF) intensity of DSS1 in nuclei and cytoplasm of cells. (DMSO/nuclei: n = 110; dTAG/nuclei: n = 83; DMSO/cytoplasm, n = 111; dTAG/cytoplasm, n = 63). AU means arbitrary units. Outliners were excluded by ROUT method, Q = 10 %. P values were calculated using two-tailed unpaired t-tests. (Both the p < 1e-15). Source data are provided as a Source Data file. d Flag (DSS1) CUT&Tag signals over 10 kb regions centered on Flag (DSS1) peaks in DSS1–Flag–dTAG cells treated with DMSO or dTAG for 24 h. e Representative track examples showing DSS1 occupancy in DSS1–Flag–dTAG cells treated with DMSO or dTAG for 24 h. f Pie chart showing DSS1-associated genomic regions. g Metaplot of DSS1’s average occupancy at each genomic element. h Occupancy of DSS1, SSB1, INTS3, and INTS5 over 6 kb regions centered on the TSS of DSS1 target gene promoters. i Representative track examples showing the occupancy of DSS1, SSB1, INTS3, and INTS5.

Fig. 3. The DSS1–INTAC interaction is essential for the optimal chromatin recruitment of DSS1.

a Detailed structural view of the interaction sites between INTAC and DSS1. Residue W39 stacks with the backbone of the helix 22 of INTS7, indicated by the bidirectional arrow. b Co-IP analysis of HA (DSS1) followed by western blotting. PSMD4 and PSMD14 are 26S proteasome subunits. The experiment was repeated independently 3 times with similar results. Source data are provided as a Source Data File. c Overexpression of DSS1-WT, DSS1-W39R, or vector in DSS1–Flag–dTAG cells treated with DMSO or dTAG, followed by western blotting of DSS1. β-tubulin serves as loading control. Data represent three biological independent experiments with similar results. Source data are provided as a Source Data File. d Western blotting of Flag (DSS1) in DSS1-WT, DSS1-W39R, or vector overexpressing DSS1–Flag–dTAG cells with time-course dTAG treatment. β-tubulin serves as loading control. Data represent three biological independent experiments with similar results. Source data are provided as a Source Data File. e The quantified and normalized band intensities in (d). Exponential one phase decay model was utilized to fit nonlinear regression curve for Flag (DSS1) band intensities. Samples derive from the same experiment and that gels/blots in (d) were processed in parallel. Source data are provided as a Source Data file. f Representative images showing the localization of overexpressed DSS1-WT or DSS1-W39R (red) along with the DAPI signal (blue) after endogenous DSS1 degradation in DSS1–Flag–dTAG cells. Data represent two biological independent experiments. g Quantification of mean IF intensity of HA (DSS1) in nuclei and cytoplasm of DSS1–Flag–dTAG cells with DSS1-WT or DSS1-W39R. (WT/nuclei: n = 91; WT/cytoplasm: n = 90; W39R/nuclei, n = 82; W39R/cytoplasm, n = 82). AU means arbitrary units. Outliners were excluded by ROUT method, Q = 10 %. Multiple comparison after One-way-ANOVA was used to calculate p value. (In DSS1-WT, p = 4.53e-13, in DSS1-W39R, p = 2.42e-3). Source data are provided as a Source Data file. h, i Metaplot (h) and heatmaps (i) showing HA (DSS1) CUT&Tag signals over 10 kb regions centered on the TSS of target genes in dTAG-treated DSS1–Flag–dTAG cells expressing DSS1-WT or DSS1-W39R.

Fig. 4. Structure of DSS1–INTAC–PEC.

a, b Cryo-EM map (a) and molecular model (b) of DSS1-containing INTAC–PEC shown in two different views. Notably, the cryo-EM map is a composite map derived from multibody refinement. c Structural model showed a close-up view of the mRNA (yellow) inserted into the catalytic center of INTS11 (orange). d Structural model showing the conformation of DSS1 in DSS1–INTAC (Gray) and DSS1–INTAC–PEC (INTS7: green, DSS1: magenta).

Fig. 5. DSS1 regulates INTAC integrity and its function in transcription.

a Western blotting of INTAC subunits in DSS1-WT or DSS1-W39R expressing DSS1–Flag–dTAG cells with dTAG treatment. β-tubulin serves as loading control. Samples derive from the same experiment and that blots were processed in parallel to be suitable for subsequent quantification in (b). Data represent three biological independent experiments with similar results. Source data are provided as a Source Data File. b Quantification of the biological independent western blotting result in (a). Data are presented as mean values with SD. (INTS3, INTS4, INTS5, INTS7: n = 3, INTS9: n = 4). P values were calculated using two-tailed unpaired t-tests. Source data are provided as a Source Data file. c Heatmaps showing INTS3 ChIP-Rx signals over 10 kb regions centered on the TSS of target genes after endogenous DSS1 degradation in DSS1–Flag–dTAG cells with overexpression of DSS1-WT or DSS1-W39R. d Representative track examples of INTS3 occupancy in dTAG treated DSS1–Flag–dTAG cells with overexpression of DSS1-WT or DSS1-W39R. e Analysis of Perturb-seq showing the correlation of gene expression changes in cells lacking INTAC subunits. f Metagenes showing Pol II ChIP-Rx signals in DSS1–Flag–dTAG cells overexpressing DSS1-WT or DSS1-W39R. g, h Metagenes showing Pol II ChIP-Rx signals in INTS8-dTAG + ΔN (g) and INTS11-dTAG cells (h) treated with DMSO or dTAG.