PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC
Appel L-M, Franke V, Bruno M, Grishkovskaya I, Kasiliauskaite A, Kaufmann T, Schoeberl UE, Puchinger MG, Kostrhon S, Ebenwaldner C, Sebesta M, Beltzung E, Mechtler K, Lin G, Vlasova A, Leeb M, Pavri R, Stark A, Akalin A, Stefl R, Bernecky C, Djinovic-Carugo K, Slade D. 2021. PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC. Nature Communications. 12(1), 6078.
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Author
Appel, Lisa-Marie;
Franke, Vedran;
Bruno, Melania;
Grishkovskaya, Irina;
Kasiliauskaite, Aiste;
Kaufmann, Tanja;
Schoeberl, Ursula E.;
Puchinger, Martin G.;
Kostrhon, Sebastian;
Ebenwaldner, Carmen;
Sebesta, Marek;
Beltzung, Etienne
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All
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Abstract
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.
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Publishing Year
Date Published
2021-10-19
Journal Title
Nature Communications
Publisher
Springer Nature
Acknowledgement
D.S. thanks Claudine Kraft, Renée Schroeder, Verena Jantsch, Franz Klein and Peter Schlögelhofer for support. We thank Anita Testa Salmazo for help with purifying Pol II; Matthias Geyer and Robert Düster for sharing DYRK1A kinase; Felix Hartmann and Clemens Plaschka for help with mass photometry; Goran Kokic for design of the arrest assay sequences; Petra van der Lelij for help with generating mESC KO; Maximilian Freilinger for help with the purification of mEGFP-CTD; Stefan Ameres, Nina Fasching and Brian Reichholf for advice on SLAM-seq and for sharing reagents; Laura Gallego Valle for advice regarding LLPS assays; Krzysztof Chylinski for advice regarding CRISPR/Cas9 methodology; VBCF Protein Technologies facility for purifying PHF3 and providing gRNAs and Cas9; VBCF NGS facility for sequencing; Monoclonal antibody facility at the Helmholtz center for Pol II antibodies; Friedrich Propst and Elzbieta Kowalska for advice and for sharing materials; Egon Ogris for sharing materials; Martin Eilers for recommending a ChIP-grade TFIIS antibody; Susanne Opravil, Otto Hudecz, Markus Hartl and Natascha Hartl for mass spectrometry analysis; staff of the X-ray beamlines at the ESRF in Grenoble for their excellent support; Christa Bücker, Anton Meinhart, Clemens Plaschka and members of the Slade lab for critical comments on the manuscript; Life Science Editors for editing assistance. M.B. and D.S. acknowledge support by the FWF-funded DK ‘Chromosome Dynamics’. T.K. is a recipient of the DOC fellowship from the Austrian Academy of Sciences. U.S. is supported by the L’Oreal for Women in Science Austria Fellowship and the Austrian Science Fund (FWF T 795-B30). M.L is supported by the Vienna Science and Technology Fund (WWTF, VRG14-006). R.S. is supported by the Czech Science Foundation (15-17670 S and 21-24460 S), Ministry of Education, Youths and Sports of the Czech Republic (CEITEC 2020 project (LQ1601)), and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant agreement no. 649030); this publication reflects only the author’s view and the Research Executive Agency is not responsible for any use that may be made of the information it contains. M.S. is supported by the Czech Science Foundation (GJ20-21581Y). K.D.C. research is supported by the Austrian Science Fund (FWF) Projects I525 and I1593, P22276, P19060, and W1221, Federal Ministry of Economy, Family and Youth through the initiative ‘Laura Bassi Centres of Expertise’, funding from the Centre of Optimized Structural Studies No. 253275, the Wellcome Trust Collaborative Award (201543/Z/16), COST action BM1405 Non-globular proteins - from sequence to structure, function and application in molecular physiopathology (NGP-NET), the Vienna Science and Technology Fund (WWTF LS17-008), and by the University of Vienna. This project was funded by the MFPL start-up grant, the Vienna Science and Technology Fund (WWTF LS14-001), and the Austrian Science Fund (P31546-B28 and W1258 “DK: Integrative Structural Biology”) to D.S.
Volume
12
Issue
1
Article Number
6078
eISSN
IST-REx-ID
Cite this
Appel L-M, Franke V, Bruno M, et al. PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC. Nature Communications. 2021;12(1). doi:10.1038/s41467-021-26360-2
Appel, L.-M., Franke, V., Bruno, M., Grishkovskaya, I., Kasiliauskaite, A., Kaufmann, T., … Slade, D. (2021). PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC. Nature Communications. Springer Nature. https://doi.org/10.1038/s41467-021-26360-2
Appel, Lisa-Marie, Vedran Franke, Melania Bruno, Irina Grishkovskaya, Aiste Kasiliauskaite, Tanja Kaufmann, Ursula E. Schoeberl, et al. “PHF3 Regulates Neuronal Gene Expression through the Pol II CTD Reader Domain SPOC.” Nature Communications. Springer Nature, 2021. https://doi.org/10.1038/s41467-021-26360-2.
L.-M. Appel et al., “PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC,” Nature Communications, vol. 12, no. 1. Springer Nature, 2021.
Appel L-M, Franke V, Bruno M, Grishkovskaya I, Kasiliauskaite A, Kaufmann T, Schoeberl UE, Puchinger MG, Kostrhon S, Ebenwaldner C, Sebesta M, Beltzung E, Mechtler K, Lin G, Vlasova A, Leeb M, Pavri R, Stark A, Akalin A, Stefl R, Bernecky C, Djinovic-Carugo K, Slade D. 2021. PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC. Nature Communications. 12(1), 6078.
Appel, Lisa-Marie, et al. “PHF3 Regulates Neuronal Gene Expression through the Pol II CTD Reader Domain SPOC.” Nature Communications, vol. 12, no. 1, 6078, Springer Nature, 2021, doi:10.1038/s41467-021-26360-2.
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