Phase separation of taz compartmentalizes the transcription machinery to promote gene expression

ABSTRACT TAZ promotes growth, development and tumorigenesis by regulating the expression of target genes. However, the manner in which TAZ orchestrates the transcriptional responses is

poorly defined. Here we demonstrate that TAZ forms nuclear condensates through liquid–liquid phase separation to compartmentalize its DNA-binding cofactor TEAD4, coactivators BRD4 and MED1,

and the transcription elongation factor CDK9 for transcription. TAZ forms phase-separated droplets in vitro and liquid-like nuclear condensates in vivo, and this ability is negatively

regulated by Hippo signalling through LATS-mediated phosphorylation and is mediated by the coiled-coil (CC) domain. Deletion of the TAZ CC domain or substitution with the YAP CC domain

prevents the phase separation of TAZ and its ability to induce the expression of TAZ-specific target genes. Thus, we identify a mechanism of transcriptional activation by TAZ and demonstrate

that pathway-specific transcription factors also engage the phase-separation mechanism for efficient and specific transcriptional activation. Access through your institution Buy or

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MOTIF IN TRANSCRIPTIONAL MACHINERIES AND FACILITATES THEIR CLUSTERING BY PHASE SEPARATION Article Open access 21 August 2020 NUCLEATED TRANSCRIPTIONAL CONDENSATES AMPLIFY GENE EXPRESSION

Article 14 September 2020 A CHAPERONE-LIKE FUNCTION OF FUS ENSURES TAZ CONDENSATE DYNAMICS AND TRANSCRIPTIONAL ACTIVATION Article 03 January 2024 DATA AVAILABILITY Source data for Figs. 1–4

and 6–8 and Extended Data Figs. 1, 3, 4 and 8 are available online. The RNA-seq data are available in the Gene Expression Omnibus (GEO) with the accession number GSE142474. All other data

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CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank K.-L. Guan and A. Mauviel for providing cDNAs of components of the Hippo pathway and H. Sasaki for the

8xGT-IIC-δ51LucII construct; J. He for technical assistance and Q. Zhu for helpful suggestions, discussions and help with experimental procedures; and D. Schichnes and S. Ruzin at the CNR

biological imaging facility at the University of California, Berkeley for assistance with microscopy. This study was supported by DOD/US Army Medical Research And Materiel Command

W81XWH-15-1-0068 (to K.L. and Q.Z.), a Tel Aviv University-University of California Berkeley collaborative research grant (to Y.I.H. and K.L.), and NIH R01AI41757 (to Q.Z.). Y.I.H. is an

incumbent of the Zalman Weinberg Chair in Cell Biology. Y.L. is supported by the Berkeley Scholars program, and T.W. was supported by the China Scholarship Council. AUTHOR INFORMATION Author

notes * These authors contributed equally: Yi Lu, Tiantian Wu. AUTHORS AND AFFILIATIONS * Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA Yi

Lu, Tiantian Wu, Huasong Lu, Qiang Zhou & Kunxin Luo * State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, China Tiantian Wu * Department

of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Orit Gutman & Yoav I. Henis Authors * Yi Lu View author publications You can also search

for this author inPubMed Google Scholar * Tiantian Wu View author publications You can also search for this author inPubMed Google Scholar * Orit Gutman View author publications You can also

search for this author inPubMed Google Scholar * Huasong Lu View author publications You can also search for this author inPubMed Google Scholar * Qiang Zhou View author publications You

can also search for this author inPubMed Google Scholar * Yoav I. Henis View author publications You can also search for this author inPubMed Google Scholar * Kunxin Luo View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Y.L., T.W. and K.L. designed the research. Y.L. performed in vivo experiments. T.W. performed in vitro

experiments. Y.I.H. designed and O.G. performed FRAP experiments. Y.L., T.W., H.L., Y.I.H., Q.Z. and K.L. analysed data and wrote the paper. K.L. conceived and directed the project. All of

the authors discussed the results and commented on the manuscript. CORRESPONDING AUTHOR Correspondence to Kunxin Luo. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA

EXTENDED DATA FIG. 1 REGULATION OF TAZ DROPLET FORMATION _IN VITRO_ AND NUCLEAR PUNCTA FORMATION _IN VIVO_. A, GFP-TAZ purified from _E. coil_ were analysed by SDS-PAGE and visualized by

Coomassie blue staining. B. 50 μM GFP-TAZ were heated-inactivated (5 min at 95 °C and immediately put on ice for 5 min) or treated with 100 μg/ml Proteinase K for 30 min at 40 °C, and then

subjected to droplet formation assay _in vitro_ in the presence of 500 mM NaCl at room temperature. C, Ectopically expressed GFP-TAZ was expressed at a lower level than endogenous TAZ in

MCF-10A cells as shown by western blotting. GAPDH was used as a loading control. D, Flag-TAZ formed nuclear puncta when transfected into the MCF-10A cells, as detected by immunofluorescence

staining with anti-Flag. Scale bar, 10 μm. Experiments in A–D were repeated independently three times with similar results. Unprocessed blots are provided in Unprocessed Blots Extended Data

Fig. 1. Source data EXTENDED DATA FIG. 2 YAP DOES NOT FORM DROPLETS _IN VITRO_ AND _IN VIVO_ IN THE ABSENCE OF CROWDING AGENTS. A, GFP-YAP purified from _E. coil_ were analyzed by SDS-PAGE

and visualized by Coomassie blue staining. B, GFP-YAP at varying concentrations was subjected to the droplet formation assay at room temperature and in the presence of 500 mM NaCl. C, 50 μM

GFP-YAP was subjected to the droplet formation assay at room temperature in the presence of indicated salt concentrations. D, 50 μM GFP-YAP was subjected to droplet formation in the presence

of 150 mM NaCl at 4 °C or 37 °C. E, Two YAP isoforms, GFP-YAP1–1β or GFP-YAP1–2α, did not form droplets (50 μM protein, 500 mM NaCl and room temperature). aa, amino acids. F, 50 μM GFP-YAP

formed droplets in the presence of 10% PEG-8000, Ficoll or Dextran but not 10% glycerol or sucrose. Droplet formation assay was performed in the presence of 500 mM NaCl at room temperature.

G, 50 μM GFP-YAP did not form droplets in the presence of BSA at varying concentrations. H, GFP-YAP did not form nuclear puncta in both HeLa cells and 293T cells. Scale bars, 10 μm.

Experiments in A–H were repeated independently three times with similar results. EXTENDED DATA FIG. 3 THE CC AND WW DOMAINS ARE REQUIRED FOR TAZ TO FORM NUCLEAR PUNCTA. A, Domain structure

of TAZ and TAZ truncations. The numbers above indicate the position of amino acid residues. B, Bacterially purified GFP-TAZ, ∆TB, ∆WW, ∆CC, and ∆WW+∆CC proteins were analyzed by SDS-PAGE and

detected by Coomasssie blue staining. C, Localization of GFP-TAZ and various mutants in HeLa cells. D, Localization of GFP-TAZ and various TAZ/YAP chimera in HeLa cells. Scale bars, 10 μm.

E, A GST pull-down assay was performed by incubating immobilized GST fusion proteins with lysates of cells expressing HA-tagged WT or mutant TAZ, and the associated TAZ proteins were

detected by western blotting with anti-HA (upper). GST fusion proteins were assessed by western blotting with anti-GST, and HA-TAZ proteins in the cell lysates were measured by western

blotting (lower). Experiments in B–E were repeated independently three times with similar results. Unprocessed blots are provided in Unprocessed Blots Extended Data Fig. 3. Source data

EXTENDED DATA FIG. 4 TAZ CC DOMAIN ENHANCES YAP PHASE SEPARATION IN THE PRESENCE OF PEG. A, Domain structure of YAP chimera. B, Substitution of the YAP CC and WW domains with that of TAZ is

not sufficient to enable YAP to undergo LLPS in MCF10A cells in the absence of PEG. C, Coomasssie blue staining of various recombinant proteins purified from _E. coil_. D, 25 μM bacterially

purified GFP-YAP chimera proteins were subjected to droplet formation assay in the presence of 10% PEG-8000. Quantification of the droplets is on the right. Scale bar, 10 μm. Data shown as

the mean ± s.e.m. Statistical significance was evaluated using One-way ANOVA with Krusk-Wallis test. Droplets in n = 3 fields in each group were quantified. E, The TAZ CC and WW domains

enhanced LLPS by GFP-YAP in transfected MCF10A cells in the presence of PEG as shown by confocal microscopy. Scale bar, 10 μm. Quantification of the percentage of cells that displayed

nuclear puncta is shown on the right. Data shown as the mean ± s.e.m.. _P_ value was determined by unpaired two-tailed Student’s _t_-test. 80 transfected cells in each group were quantified.

n = 3 biologically independent samples. Experiments in B, C, E were repeated independently three times with similar results. Experiments in D were repeated twice with similar results.

Statistical source data for D, E, are provided in Statistical Source Date Extended Data Fig. 4. Source data EXTENDED DATA FIG. 5 HIPPO SIGNALING NEGATIVELY REGULATES TAZ PHASE SEPARATION IN

HELA CELLS. TAZ localization was examined by immunofluorescence staining with anti-TAZ (green) in HeLa cells that have been subjected to the following treatments: A, Serum-starved HeLa cells

were treated with 1 μM LPA or 50 ng/ml EGF for 1 h. B, Serum-starved HeLa cells were seeded on fibronectin-coated coverslips for 10 min or 2 h in serum-free medium. C, HeLa cells were grown

on fibronectin-coated polyacrylamide hydrogels of 1 kPa and 40 kPa stiffness. D, HeLa cells were treated with 1 μg/ml Latrunculin B for 1 h. Alexa Fluor 555-conjugated phalloidin (Red)

staining was performed to detect F-actin in b-d. Scale bar, 10 µm. Experiments in A–D were repeated independently three times with similar results. EXTENDED DATA FIG. 6 LATS2 REGULATES TAZ

LLPS AND RECRUITMENT OF TEAD4 AND BRD4. A, MCF-10A cells transfected with GFP-TAZ-S89A and Flag-LATS2 were subjected to immunofluorescence staining with anti-Flag (Red). Scale bar, 10 µm. B,

MCF-10A cells stably expressing siLATS1/2 were transfected with GFP-TAZ and Flag-TEAD4. TEAD localization at high cell density was detected by immunofluorescence staining with anti-Flag

(Red). Scale bar, 10 μm. C, MCF-10A cells stably expressing siLATS1/2 were transfected with GFP-TAZ. Endogenous BRD4 localization was examined by immunofluorescence staining with anti-BRD4

(Red). Scale bar, 10 μm. All experiments were repeated independently three times with similar results. EXTENDED DATA FIG. 7 TAZ NUCLEAR CONDENSATES DO NOT CO-LOCALIZE WITH THE PML BODIES,

CAJAL BODIES OR NUCLEOLI. The PML nuclear bodies, Cajal Bodies and nucleoli in MCF-10A cells expressing GFP-TAZ (green) were detected by immunofluorescence staining with antibodies targeting

PML, Coilin and Fibrillarin, respectively (red). Scale bar, 10 μm. Experiments were repeated independently three times with similar results. EXTENDED DATA FIG. 8 TAZ MUTANTS LACKING THE CC

DOMAIN STILL BIND TO LAST2 AND TEAD4. A, HA-tagged WT and mutant TAZ were co-transfected into 293T cells with Flag-LATS2. TAZ proteins associated with LATS2 were isolated by

immunoprecipitation with anti-Flag and detected by western blotting with anti-HA antibodies (upper panels). The abundance of these proteins in the cell lysates was assessed by western

blotting (lower panels). GAPDH was used as a loading control. B, Interaction of various TAZ mutants with Flag-TEAD4 was analyzed by co-IP assay as described in A. C, Interaction of various

TAZ/YAP chimera with LATS2 was analyzed by co-IP as described in A. All experiments were repeated independently three times with similar results. Unprocessed blots are provided in

Unprocessed Blots Extended Data Fig. 8. Source data SUPPLEMENTARY INFORMATION REPORTING SUMMARY SOURCE DATA SOURCE DATA FIG. 1 Statistical source data. SOURCE DATA FIG. 2 Statistical source

data. SOURCE DATA FIG. 3 Unprocessed blots. SOURCE DATA FIG. 3 Statistical source data. SOURCE DATA FIG. 4 Unprocessed blots. SOURCE DATA FIG. 4 Statistical source data. SOURCE DATA FIG. 6

Unprocessed blots. SOURCE DATA FIG. 6 Statistical source data. SOURCE DATA FIG. 7 Unprocessed blots. SOURCE DATA FIG. 8 Unprocessed blots. SOURCE DATA FIG. 8 Statistical source data. SOURCE

DATA EXTENDED DATA FIG. 1 Unprocessed blots. SOURCE DATA EXTENDED DATA FIG. 3 Unprocessed blots. SOURCE DATA EXTENDED DATA FIG. 4 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 8

Unprocessed blots. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Lu, Y., Wu, T., Gutman, O. _et al._ Phase separation of TAZ compartmentalizes the

transcription machinery to promote gene expression. _Nat Cell Biol_ 22, 453–464 (2020). https://doi.org/10.1038/s41556-020-0485-0 Download citation * Received: 28 May 2019 * Accepted: 14

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