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. 2021 Nov 26;374(6571):1113-1121.
doi: 10.1126/science.abe2913. Epub 2021 Nov 25.

A ubiquitous disordered protein interaction module orchestrates transcription elongation

Katerina Cermakova #  1   2 , Jonas Demeulemeester #  3 , Vanda Lux #  2 , Monika Nedomova  2 , Seth R Goldman  4 , Eric A Smith  1 , Pavel Srb  2 , Rozalie Hexnerova  2 , Milan Fabry  5 , Marcela Madlikova  2 , Magdalena Horejsi  5 , Jan De Rijck  3 , Zeger Debyser  3 , Karen Adelman  4 , H Courtney Hodges  1   6   7 , Vaclav Veverka  2   8
Affiliations

A ubiquitous disordered protein interaction module orchestrates transcription elongation

Katerina Cermakova et al. Science. .

Abstract

During eukaryotic transcription elongation, RNA polymerase II (RNAP2) is regulated by a chorus of factors. Here, we identified a common binary interaction module consisting of TFIIS N-terminal domains (TNDs) and natively unstructured TND-interacting motifs (TIMs). This module was conserved among the elongation machinery and linked complexes including transcription factor TFIIS, Mediator, super elongation complex, elongin, IWS1, SPT6, PP1-PNUTS phosphatase, H3K36me3 readers, and other factors. Using nuclear magnetic resonance, live-cell microscopy, and mass spectrometry, we revealed the structural basis for these interactions and found that TND-TIM sequences were necessary and sufficient to induce strong and specific colocalization in the crowded nuclear environment. Disruption of a single TIM in IWS1 induced robust changes in gene expression and RNAP2 elongation dynamics, which underscores the functional importance of TND-TIM surfaces for transcription elongation.

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Conflict of interest statement

Competing interests: KA is a consultant for Syros Pharmaceuticals, and is a stockholder and scientific advisory board member of CAMP4 Therapeutics.

Figures

Fig. 1:
Fig. 1:. TFIIS N-terminal domain (TND) is a conserved scaffold enriched among transcription elongation factors.
(A) Magnitude of enrichment of structural folds among transcription initiation, elongation and termination factors (see also Data S1); *** P < 0.001. (B) Domains enriched among transcription elongation factors are ranked based on significance (adjusted P-values). Top hits are highlighted and labeled. (C) Enrichment of TND modules in different nuclear processes, gene expression, and processes involving different RNA polymerases (RNAP). (D) Interaction network of TND-containing proteins with proteins known to participate in transcription elongation from RNAP2 promoters (defined by GO:0006368). Edges represent experimental evidence-based interaction data from the STRING database. Interactions involving TND-containing proteins are highlighted in pink and TND-containing proteins are highlighted in bold. Full labels are provided in Fig. S1. (E) Solution structures of different TNDs determined by NMR spectroscopy. (F) Root-mean-square deviation (RMSD) and sequence identity between each TND pair. (G) Structural multiple sequence alignment generated by PDBeFold. Shading indicates the degree of conservation and fold stabilizing residues are marked by a triangle. The mean fractional solvent accessible surface area (SASA) is also indicated for each position.
Fig. 2:
Fig. 2:. TNDs are selectively recognized by TND-interacting motifs (TIMs) conserved in transcription regulators.
(A) Multiple sequence alignments illustrating conserved TND-recognizing TIMs. (B) Interaction network of proteins containing a TND module or TIM. Edges represent interaction data from the STRING database and are weighted by the confidence score calculated by STRING. TIM-containing proteins are highlighted in bold. (C) Comparison of 2D 15N/1H SOFAST-HMQC NMR spectra of ELOA TND alone (yellow) or with an increasing amount of MED13 TIM (gradient of blue). (D) Quantification of chemical shift perturbation (CSP) in backbone amide signals of the ELOA TND upon titration with MED13 TIM from panel (C). (E) Determination of the dissociation constant (KD) for the ELOA-TND:MED13-TIM complex formation by NMR. Error bars represent the error of the fit for the most perturbed residues (n = 10). (F) Heatmap of the mean CSP of the 10% most affected residues for each TND:TIM binary interaction. (G) Interaction network reconstructed from TND:TIM binary interactions as determined by NMR. Line darkness indicates the strength of each pairwise interaction based on mean CSP values presented in panel (F). (H) Quantification of CSP for each TND:TIM combination at a 1:4 ratio. (I) Examples of CSP values from panel (H) highlighted on the surfaces of different TNDs.
Fig. 3:
Fig. 3:. TND and TIM surfaces are necessary and sufficient for the association of elongation factors in living cells.
(A) Fluorescent two-hybrid (F2H)-based protein-protein interaction assay and construct design. TND-containing proteins are fused to TagRFP and TIM-containing factors to EGFP-LacI. (B) Fluorescence images of U2OS 2–6-3 cells expressing IWS1-TIM(1–3)-EGFP-LacI bait and a TagRFP-TND or control. See also Fig. S11. (C) Images of cells expressing TagRFP-LEDGF-TND and EGFP-LacI fused to WT or F488A/F491A TIM(1–3) IWS1. (D) Quantification of enrichment at fluorescent foci (n = 100) from panel (C); *** P < 0.001. (E) IWS1 mutants affecting TIMs employed in this study. Helical propensity determined by TALOS+ is indicated. (F) Single-cell interaction scores for EGFP-LacI control, WT or mutant (M1, 2 and 3) IWS1-TIM-EGFP-LacI fusion with TagRFP-TNDs (n = 81 each). See also Fig. S12 and S13. (G) LEDGF and TFIIS protein structures with residues important for interaction with TIMs labeled. (H) Single-cell interaction score for full-length WT and M1 mutant IWS1 fused to EGFP-LacI or control with TagRFP-TFIIS WT or RK-AA mutant. See also Fig. S14. (I) Enrichment of proteins in the IWS1 interactome measured by pull-down mass spectrometry. (J) Volcano plot of differentially enriched proteomic interaction partners in M123 FLAG-IWS1 compared to WT. (K) Western blot analysis of co-IP of WT and M123 FLAG-IWS1 protein variants. (L) Volcano plot of differentially enriched proteomic interaction partners in M3 FLAG-IWS1 compared to WT. (M) Fold change enrichment of selected proteins from mass spectrometry study presented in panels (J) and (L). **** P < 0.0001, *** P < 0.001, ** P < 0.01; Student’s t-test.
Fig. 4:
Fig. 4:. IWS1 is a central hub organizing transcription regulators.
(A) Solution structures of TFIIS, PPP1R10 and LEDGF TND in complex with IWS1 TIMs. Residues contributing to the interaction with each TND are indicated, as are TIM1, 2 and 3 interfaces mutated in Fig. 3F. (B) Competition curves of TFIIS-IWS1 (left) or ELOA-IWS1 (right) with unlabelled ELOA, TFIIS or LEDGF TND domain measured via NMR. (C) Competition curves of FLAG-IWS1 bound to GST-LEDGF TND (left) or GST-HRP2 TND (right) with increasing amount of untagged HRP2 or LEDGF TND domain using AlphaScreen assay. (D) Schematic of competitive interactions of IWS1. (E) Quantification of chemical shift perturbation (CSP) in backbone amide signals of the IWS1 TIMs upon addition of unlabeled TFIIS-TND, PPP1R10-TND and LEDGF-TND individually and simultaneously measured in 3D-HNCO experiments. IWS1 residues selectively responding to addition of TFIIS-TND are highlighted in green, PPP1R10 in yellow and LEDGF in violet. (F) Sum of CSPs for IWS1 residues selectively responding to addition of TFIIS, PPP1R10 and LEDGF TNDs for conditions presented in panel (E). (G) Comparison of the interaction surface area, number of hydrogen bonds and salt bridges at the PPP1R10:TIM2, LEDGF:TIM2 and LEDGF:TIM3 interfaces. (H) Detail of the IWS1-PPP1R10-LEDGF interface. Residues important to support the interaction with each TND are presented as sticks and labeled. (I) Hybrid structure of the IWS1-SPT6-TFIIS-PPP1R10-LEDGF quinary complex.
Fig. 5:
Fig. 5:. Mutation of IWS1 TIMs alters elongation dynamics of RNAP2.
(A) IWS1 expression levels in different cell lines with deep deletion, amplification or diploid cells from cBioPortal. (B) Western blot analysis of IWS1 expression in IMR-32 and KELLY cells. Cell lines with endogenous expression (endo) and cells re-expressing IWS1 wild type (WT) and mutant (M3) were analyzed. (C) Construct design for IWS1 re-expression in KELLY cells. (D) Changes in mRNA levels of KELLY cells expressing IWS1 mutant M3 compared to WT IWS1 determined by RNA-seq. Selected differentially expressed transcripts are labeled. (E) Genome-wide distribution of IWS1 WT and M3 as determined by ChIP-seq. (F) Example browser tracks of IWS1 WT and M3 localization at gene bodies. (G) Correlation of RNA-seq expression changes induced by IWS1 M3 compared to WT, with IWS1 M3 ChIP-seq density. (H) Metagene plot of average IWS1 WT and M3 ChIP-seq density at gene bodies. Average ChIP-seq signal at promoter proximal region presented in inset. (I) Density of IWS1 M3 ChIP-seq signal at decreased, increased, and unaffected genes based on RNA-seq data from panel (D). (J) Metagene plot comparing difference in average PRO-seq signal between IWS1 M3 and WT at decreased, increased, and unaffected genes. The difference in PRO-seq signal near promoters is presented in inset. Solid lines are running means. (K) Example PRO-seq browser tracks at a decreased gene. Additional examples are provided in Fig. S19D���F. (L) Transcript-level changes in pausing index between WT and M3 IWS1 determined by PRO-seq.
Fig. 6:
Fig. 6:. Schematic of the TND-TIM module-driven interaction network.
All TND-TIM interactions are represented by grey lines, previously unknown TND-TIM interaction interfaces in the elongation machinery are highlighted in pink.
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