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Disordered C-terminal domain drives spatiotemporal confinement of RNAPII to enhance search for chromatin targets

Nature Cell Biology volume 26, pages 581–592 (2024)Cite this article

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Abstract

Efficient gene expression requires RNA polymerase II (RNAPII) to find chromatin targets precisely in space and time. How RNAPII manages this complex diffusive search in three-dimensional nuclear space remains largely unknown. The disordered carboxy-terminal domain (CTD) of RNAPII, which is essential for recruiting transcription-associated proteins, forms phase-separated droplets in vitro, hinting at a potential role in modulating RNAPII dynamics. In the present study, we use single-molecule tracking and spatiotemporal mapping in living yeast to show that the CTD is required for confining RNAPII diffusion within a subnuclear region enriched for active genes, but without apparent phase separation into condensates. Both Mediator and global chromatin organization are required for sustaining RNAPII confinement. Remarkably, truncating the CTD disrupts RNAPII spatial confinement, prolongs target search, diminishes chromatin binding, impairs pre-initiation complex formation and reduces transcription bursting. The present study illuminates the pivotal role of the CTD in driving spatiotemporal confinement of RNAPII for efficient gene expression.

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Fig. 1: Spatiotemporal mapping of transcription machinery in living yeast nucleus.
Fig. 2: CTD governs spatiotemporal dynamics of RNAPII.
Fig. 3: The interplay of CTD, chromatin organization and Mediator shapes RNAPII anisotropic diffusion.
Fig. 4: No substantial RNAPII clustering in yeast nucleoplasm.
Fig. 5: CTD facilitates RNAPII target search kinetics.
Fig. 6: CTD truncation impairs PIC formation.
Fig. 7: CTD length controls burst size and frequency of HSP82 gene transcription.

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Data availability

Raw single-molecule trajectory coordinates are accessible at Zenodo (https://doi.org/10.5281/zenodo.10570246 (ref. 93)). All other data supporting the findings of the present study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

Customized code can be found at GitHub (https://github.com/yhinling/Ling-et-al-2024 (ref. 94)).

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Acknowledgements

We thank S. Cho and G. Ling for assistance in image processing; L. Lavis for providing Janelia Fluor dyes; K. Xie for IUPRED3 data mining; X. Tang and S. Liu for computational assistance; N. Jones, V.Schoonderwoert and C. Almonte for microscopy technical support; K. Yuen and G. Mizuguchi for plasmids and yeast strains; C. Kaplan and J. Yao for valuable information on strain-sensitivity to MPA; A. Hansen, A. Musacchio, C. Wong, I. Cissé and J. M. Kim for helpful discussions; and members of the Wu laboratory for comments. The present study was supported by the National Institutes of Health (grant nos. GM132290 and R35GM149291 to C.W.) and the Croucher Foundation (Y.H.L.).

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Author notes

  1. These authors contributed equally: Ziyang Ye, Chloe Liang, Chuofan Yu, Giho Park.

Authors and Affiliations

  1. Department of Biology, Johns Hopkins University, Baltimore, MD, USA

    Yick Hin Ling, Ziyang Ye, Chloe Liang, Chuofan Yu, Giho Park & Carl Wu

  2. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Jeffry L. Corden & Carl Wu

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Contributions

Y.H.L., J.L.C. and C.W. designed the experiments. Y.H.L. performed the experiments and analysed the data. Z.Y. performed SMT on Hsf1-Halo in the WT background. G.P. contributed to the initial trial of spatiotemporal mapping. Z.Y., C.L. and C.Y. assisted in experiments. Y.H.L., J.L.C. and C.W. wrote the manuscript.

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Correspondence to Carl Wu.

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Nature Cell Biology thanks Hiroshi Kimura, Yan Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Single-molecule tracking in living yeast.

a, ER and nucleolus shown by Elo3-GFP and Gar1-GFP. Scale bar, 1.0 μm. b, Coarse-grained representation of yeast interphase chromosomes, physically constrained by telomere (TEL)-nuclear envelope and centromere-spindle pole body (CEN-SPB) tethering. c, Overlay of RPB1Rpo21-HaloJF552 trajectories on the nucleus relative to ER and nucleolar GFP markers. Rainbow colours indicate the first appearance of each trajectory. Scale bar, 1 μm. d, Single-molecule trajectories of RPB1Rpo21-HaloJF552 (free or bound) with diffusion coefficients. Scale bar, 0.2 μm. e, Protein of interested (POI) fused with HaloTag, labeled with JF552-HaloTag ligand (JF552-HTL) (top). Initial laser exposure of JF552 resulted in strong ‘nuclear glow’, followed by shelving in dark state and stochastic reactivation, leading to single-molecule detection. Nucleus circled in red. Scale bar, 1.0 μm. (bottom). f, Strong 555 nm laser exposure for 5 min does not result in noticeable cell death or growth arrest. Scale bar, 4.0 μm. g, ER and nucleolar GFP markers for cell cycle stage identification. Scale bar, 1.0 μm. h, Diffusion coefficient histograms of various nuclear proteins (n: number of trajectories; mean value ± s.d.). i, Assessing the mean diffusion coefficients of the bound population reveals no substantial difference between histones (H2B, H3 and H2A.Z) and proteins that can move along DNA (for example RNAPII, RNAPIII, Paf1), while nucleolar RNAPI, and proteins that are physically tethered (Cse4 and Sir4) have notably lower values (n = 100 resamplings; mean value ± s.d.). Source numerical data are available in source data.

Source data

Extended Data Fig. 2 Multiparameter classification of trajectories.

a, Unsupervised vbSPT overfits SMT data of RNAPII (n = 100 resamplings; mean value ± s.d.). b, Diffusion coefficient histogram for fast and slow states from 2-state HMM using vbSPT. A minor fraction of very slowly diffusing trajectories was misclassified as fast-state (dashed circle) (n: number of trajectories; mean value ± s.d.). c, Unsupervised multiparameter classification of RNAPII trajectories with UMAP dimensionality reduction and GMM clustering. d, Diffusion coefficient (D) distribution for the classified clusters (n: number of trajectories; mean value ± s.d.). e,f, similar to c and d, but with multiparameter classification semi-supervised by 2-state HMM. d,f, Compared to b, Fast-class diffusion coefficient distribution showed less tailing (dashed circle). g, 10 parameters used for classification and their distributions in identified classes (n = 3733 trajectories for class 1 and n = 2607 trajectories for class 2; median (line), IQR (box), 10–90 percentile (whiskers) and outliers (crosses); two-tailed unpaired t-test, *** P < 0. 000000001). Source numerical data are available in source data.

Source data

Extended Data Fig. 3 Spatiotemporal mapping reveals dynamics of nucleosome and nuclear landmark proteins.

a, Nuclei in G1 cells with minimal drift selected for spatiotemporal mapping. Scale bar: 1.0 μm. b, Localization density calculated using 150 nm detection radius from bins separated by 10 nm. c, 150 nm detection radius and bin distance of 10 nm provide optimal coverage and resolution for the density on the bound map of RNAPII. Scale bar: 0.5 μm (n = 15 cells; Error bar: centroid of nucleolus ± s.d.). d, 3D simulation of the yeast interphase genome based on 3 C data37. e, Top mRNA genes simulation maps based on gene expression data in minimal medium38. Left panel is reuse of Fig. 1d top panel (n: number of genes). f, Freely diffusing NLSx2-Halo map. Scale bar: 0.5 μm (Error bar: centroid of nucleolus ± s.d.; n: number of nuclei). g, Bound Cse4 (left) and Sir4 (right) maps (Error bar: centroid of nucleolus ± s.d.; n: number of nuclei). Sir4 bound map showed dense regions proximal and distal to the centromere, mirroring the telomere clusters of short and long chromosomes35,37,95. h, Bound H2B diffusion coefficient map. A 300 nm detection radius was used. Scale bar: 0.5 μm (Error bar: centroid of nucleolus ± s.d.; n: number of nuclei). Telomere and centromere movements are confined due to attachment to the nuclear envelope and spindle pole body, respectively96. Likewise, the local diffusion coefficients of chromatin-bound H2B reveal lower histone dynamics near centromeres and telomeres (Extended Data Fig. 1h, i).

Extended Data Fig. 4 Spatiotemporal dynamics of RNAPII, RNAPI and RNAPIII.

a, Fraction of free and bound in nucleoplasm (NPL) and nucleolus (NOL) (n = 100 resamplings; mean value ± s.d.). b, Distribution (area normalized) of free RNAPII, RNAPI and RNAPIII in nucleoplasm and nucleolus (n = 100 resamplings; mean value ± s.d.). c, Simulation of Nucleoplasm/Nucleolus distribution. d, Diffusion coefficients and molecular weights of free RNAPII, RNAPI and RNAPIII in nucleoplasm (n = 100 resamplings; mean value ± s.d.). e,f IUPRED3 disorder scores for the largest subunit of e, RNAPI (Rpa190) and f, RNAPIII (Rpo31). Residues with predicted scores above 0.5 considered disordered. g, Dfree of RNAPII and CTD mutants in nucleoplasm (n = 100 resamplings; mean value ± s.d.). Source numerical data are available in source data.

Source data

Extended Data Fig. 5 CTD length affects RNAPII subcellular distribution and expression.

a, Diffusion coefficient histogram for RNAPII with varying CTD lengths (n: number of trajectories; mean value ± s.d.). b-c, b, nuclear/cytoplasmic ratio and c, relative nuclear intensity of WT RNAPII and CTD mutants (median (line), IQR (box), 10–90 percentile (whiskers) and outliers (crosses), n = 95 nuclei for CTD0, 52 nuclei for CTD8, 69 nuclei for CTD9, 64 nuclei for CTD10, 112 nuclei for CTD15, 108 nuclei for CTD20, 69 nuclei for CTD26, 73 nuclei for CTD52, 91 nuclei for CTD78, 102 nuclei for CTD104). CTD truncation led to increased cytoplasmic and nucleolar distribution, and overexpression of RNAPII. d, Amount of bound RNAPII normalized to nuclear fluorescence intensity (n = 100 resamplings; mean value ± s.d.). The normalized value is an approximation, as the relationship between nuclear fluorescence intensity and RNAPII levels may not be strictly linear. e, Fluorescence signal of RPB1Rpo21-AID*-miRFP670nano3 (red) before and after RNAPII degradation by auxin. Nucleolar and ER markers in green. Scale bar: 1.0 μm. f, Western blot showing time course degradation of RPB1Rpo21-AID* by auxin, detected using CTD antibody (top), and Ponceau S staining on total protein (bottom). g,h, Cells expressing ectopic RPB1Rpo21-Halo in an RPB1Rpo21 AID background show minimal changes in the g, bound fraction (n = 100 resamplings; mean value ± s.d.; two-tailed unpaired t-test, * P = 0.015275) and h, f180/0 of RNAPII after auxin treatment, compared to those in WT expressing RPB1Rpo21-Halo (n = 100 resamplings; mean value ± s.d.). i, Spot assay for strains with prd5Δ, GFP markers (GGEGp), RPB1Rpo21-nCDT26, or RPB1Rpo21-nCDT26-Halo showing identical growth. j, Spot assay demonstrating impaired cell growth for RPB1Rpo21 or MED14Rgr1 AID degron. All AID* constructs contain miRFP670nano3 as a fluorescence marker. k, Spot assay for strains with HaloTag fusions to various PIC component subunits to assess MPA sensitivity. Ssl2-Halo (TFIIH), Halo-Ssl2 (TFIIH), Tfa2-Halo (TFIIE), Tfg2-Halo (TFIIF) exhibited MPA sensitivity. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 6 CTD length impacts free RNAPII anisotropy.

a, Brownian simulations indicate substantial apparent anisotropy for mean displacement longer than 350 nm (n = 100 resamplings; mean value ± s.d.). b, Apparent anisotropy due to nuclear confinement in small yeast nucleus. c, CTD length increase leads to an elevation in f180/0 trend (n = 100 resamplings; mean value ± s.d.). Source numerical data are available in source data.

Source data

Extended Data Fig. 7 Investigating the role of Mediator in spatiotemporal confinement of RNAPII.

a, Bound Mediator map (left). Free RNAPII-CTD26 map showing confined trajectories (right). Scale bar: 0.5 μm. (Error bar: centroid of nucleolus ± s.d.; n: number of nuclei). b, Free RNAPII-CTD26 map in MED14Rgr1 AID. Scale bar: 0.5 μm (Error bar: centroid of nucleolus ± s.d.; n: number of nuclei). c-d, c, Dfree in nucleoplasm (n = 100 resamplings; mean value ± s.d.; two-tailed unpaired t-test, ns P = 0.510991) and d, Nucleoplasm/Nucleolus of RNPAII-CTD26 in MED14Rgr1 AID (n = 100 resamplings; mean value ± s.d.; two-tailed unpaired t-test, *** P = 0.000008). e-g, e, Mediator bound fraction (n = 100 resamplings; mean value ± s.d.; two-tailed unpaired t-test, ** P = 0.006771), f, RNAPII bound fraction (n = 100 resamplings; mean value ± s.d.; two-tailed unpaired t-test, ns P = 0.157495) and g, f180/0 for RNAPII-CTD9 in CTD26 background (n = 100 resamplings; mean value ± s.d.). h,i, h, Relative bound fraction (n = 100 resamplings; mean value ± s.d.; two-tailed unpaired t-test, * P = 0.012696, ** P = 0.001329) and i, f180/0 for RNAPII-CTD26 and RNAPII-CTD9 in WT and ssn3Δ (n = 100 resamplings; mean value ± s.d.). Deleting Cdk8SSN3 from the Mediator kinase module enhances interaction between the Mediator and RNAPII97,98,99 without changing free RNAPII confinement. j,k, j, Relative bound fraction (n = 100 resamplings; mean value ± s.d.; two-tailed unpaired t-test, * P ≤ 0.05 and *** P ≤ 0.001. Comparison between before and after auxin treatment: P = 0.000014 for CTD26 and P = 0.0222222 for CTD9. Comparison between CTD26 and CTD26 after auxin treatment: P = 0.034745) and k, f180/0 (n = 100 resamplings; mean value ± s.d.) for RNAPII-CTD26 and RNAPII-CTD9 in MED14Rgr1 AID. l–n, l, Survival probability of H2B-corrected residence times (n = 10,000 resamplings; mean value ± s.d.), m, stably bound fractions (n = 10,000 resamplings; mean value ± s.d.; two-tailed unpaired t-test, ** P = 0.002245) and n, mean residence times (n = 10,000 resamplings; mean value ± s.d.; two-tailed unpaired t-test, * P = 0.019928) for RNAPII-CTD9 in MED14Rgr1 AID. Source numerical data are available in source data.

Source data

Extended Data Fig. 8 Fast and slow tracking mode of single-molecule tracking.

a, Fast tracking with high laser power and short frame rate (10 ms/frame) reveals diffusive dynamics of free (red) and bound (blue) molecules. b, Single-step disappearance of chromatin-bound RNAPII in fast tracking. c, Slow tracking with low laser power and long frame rate (250 ms/frame) reveals residence time of bound molecules (blue). d, Single-step disappearance of chromatin-bound RNAPII in slow tracking. b-d, Here we term ‘disappearance’ as we cannot definitively differentiate between the molecule diffusing out of the focal plane or photobleaching within the living cell. e, CDF of bound displacement of RNAPII, H2B, H3 and H2A.Z to determine the rmax for trajectory linking in slow tracking100. f, In G1 cells, nucleosomal H2B (representing the stable binding population) was hypothesized to bind for a sufficiently long period, such that during image acquisition, their dissociation was barely observed. Nonetheless, the observed residence times for H2B typically fall within 30 seconds, due to factors such as photobleaching, dye blinking, chromatin/nuclear movements, and focus drift. g, Survival probability of Halo-H2B observed residence times in CTD26 (WT) and CTD9 strains (n: number of trajectories; mean value ± s.d.). h, Decay constant of stably bound Halo-H2B (ksb) in CTD26 (WT) and CTD9 strains (n = 10,000 resamplings; mean value ± s.d.; two-tailed unpaired t-test, ns P = 0.339598). Source numerical data are available in source data.

Source data

Extended Data Fig. 9 CTD length controls HSP82 transcription bursting, with minimal effect on Hsf1 dynamics.

a, b, a, Diffusion coefficient histograms for Hsf1-Halo and b, survival probability of bound Hsf1-Halo under heat shock in CTD26 (WT) (left) and CTD9 (right) strains (n: number of trajectories; mean value ± s.d.). c, d, c, HSP82 ON time and d, OFF time cumulative distribution function (CDF) under heat shock in CTD26 (WT) (left) and CTD9 (right) strains (n: number of ON or OFF events; mean value ± s.d.). Source numerical data are available in source data.

Source data

Extended Data Fig. 10 CTD is necessary but not sufficient for confining protein diffusion.

a, HaloTag (NLSx2-Halo) and Halo-CTD fusions diffusion coefficient histogram by CTD length (n: number of trajectories; mean value ± s.d.). The first plot is reuse of the top panel’s second plot from Extended Data Fig. 1h. b, HaloTag and Halo-CTD fusions bulk wide-field staining using JFX650 in live cell. Scale bar: 1.0 μm. c, Nucleoplasm/Nucleolus ratios for freely diffusing HaloTag and Halo-CTD fusions (n = 100 resamplings; mean value ± s.d.). d, Protein disorder affects hydrodynamic radius (Rh). e, Theoretical relationship between molecule weight and Rh for folded and unfolded peptides (data from Fluidic Analytics). f, g, f, Dfree and estimated Rh of the HaloTag and Halo-CTD fusions in nucleoplasm and nucleolus. Data fitted with Stokes-Einstein equation to calculate g, apparent viscosity (η) of yeast nucleoplasm (NPL) and nucleolus (NOL), respectively. KB: Boltzmann’s constant. T: Temperature in Kelvin; 295.15 K (22 °C) (n = 100 resamplings; mean value ± s.d.). h, f180/0 plot for HaloTag and Halo-CTD fusions (n = 100 resamplings; mean value ± s.d.). Source numerical data are available in source data.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and Figs. 1–5.

Reporting Summary

Peer Review File

Supplementary Video 1

Single-molecule tracking of RNAPII. JF552 signal is in red and ER and nucleolar GFP markers are in green. For demonstration only, because simultaneous GFP marker imaging affects single-molecule signal-to-noise ratio. Scale bar, 2.0 μm, Recorded frame rate: 30 ms per frame. Playback speed: 30 ms per frame.

Supplementary Video 2

Fast tracking of RNAPII-CTD26 (WT) and RNAPII-CTD9. Nucleolus is in grey, nuclear envelope and plasma membrane outlined in solid and dashed white, respectively. Scale bar, 1.0 μm. Colour scale represents mean instantaneous velocity of trajectory (µm s−1). Recorded frame rate: 10 ms per frame. Playback speed: 40 ms per frame.

Supplementary Video 3

Anisotropic trajectory of free RNAPII. Scale bar, 0.2 μm. Recorded frame rate: 10 ms per frame. Playback speed: 50 ms per frame.

Supplementary Video 4

Sir4-HaloJFX650 in WT and esc1Δ yku70Δ strain. Scale bar, 1.0 μm. Recorded frame rate: 50 ms per frame. Playback speed: 50 ms per frame.

Supplementary Video 5

Slow tracking kymograph of chromatin-bound RNAPII. Recorded frame rate: 250 ms per frame. Playback speed: 100 ms per frame.

Supplementary Video 6

HSP82 transcription bursting in WT under non-HS condition. In addition to a strong signal at nuclear TS, cytoplasmic mRNAs are visible from 10:40 to 19:20. Scale bar, 1.0 μm. Recorded frame rate: 20 s per frame. Playback speed: 100 ms per frame.

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Ling, Y.H., Ye, Z., Liang, C. et al. Disordered C-terminal domain drives spatiotemporal confinement of RNAPII to enhance search for chromatin targets. Nat Cell Biol 26, 581–592 (2024). https://doi.org/10.1038/s41556-024-01382-2

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