Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1

Laura D. Gallego; Medini Ghodgaonkar Steger; Anton A. Polyansky; Tobias Schubert; Bojan Zagrovic; Ning Zheng; Tim Clausen; Franz Herzog; Alwin Köhler

doi:10.1073/pnas.1606863113

Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1

Laura D. Gallego, Medini Ghodgaonkar Steger, Anton A. Polyansky, Tobias Schubert, Bojan Zagrovic, Ning Zheng, Tim Clausen, Franz Herzog, Alwin Köhler

Research output: Contribution to journal › Article › peer-review

Abstract

Cotranscriptional ubiquitination of histone H2B is key to gene regulation. The yeast E3 ubiquitin ligase Bre1 (human RNF20/40) pairs with the E2 ubiquitin conjugating enzyme Rad6 to monoubiquitinate H2B at Lys123. How this single lysine residue on the nucleosome core particle (NCP) is targeted by the Rad6-Bre1 machinery is unknown. Using chemical cross-linking and mass spectrometry, we identified the functional interfaces of Rad6, Bre1, and NCPs in a defined in vitro system. The Bre1 RING domain cross-links exclusively with distinct regions of histone H2B and H2A, indicating a spatial alignment of Bre1 with the NCP acidic patch. By docking onto the NCP surface in this distinct orientation, Bre1 positions the Rad6 active site directly over H2B Lys123. The Spt-Ada-Gcn5 acetyltransferase (SAGA) H2B deubiquitinase module competes with Bre1 for binding to the NCP acidic patch, indicating regulatory control. Our study reveals a mechanism that ensures site-specific NCP ubiquitination and fine-tuning of opposing enzymatic activities.

Original language	English
Pages (from-to)	10553-10558
Number of pages	6
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	113
Issue number	38
DOIs	https://doi.org/10.1073/pnas.1606863113
Publication status	Published - 20 Sept 2016
Externally published	Yes

Keywords

Bre1-Rad6
Cross-linking mass spectrometry
Nucleosome
RING E3 ligase
Ubiquitin

Research fields

Chromatin
Chemical Crosslinking
Mass spectrometry
Structural Proteomics

IMC Research Focuses

Medical biotechnology

ÖFOS 2012 - Austrian Fields of Study

106037 Proteomics
106041 Structural biology
106044 Systems biology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1073/pnas.1606863113

Cite this

Gallego, L. D., Steger, M. G., Polyansky, A. A., Schubert, T., Zagrovic, B., Zheng, N., Clausen, T., Herzog, F., & Köhler, A. (2016). Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1. Proceedings of the National Academy of Sciences of the United States of America, 113(38), 10553-10558. https://doi.org/10.1073/pnas.1606863113

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title = "Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1",

abstract = "Cotranscriptional ubiquitination of histone H2B is key to gene regulation. The yeast E3 ubiquitin ligase Bre1 (human RNF20/40) pairs with the E2 ubiquitin conjugating enzyme Rad6 to monoubiquitinate H2B at Lys123. How this single lysine residue on the nucleosome core particle (NCP) is targeted by the Rad6-Bre1 machinery is unknown. Using chemical cross-linking and mass spectrometry, we identified the functional interfaces of Rad6, Bre1, and NCPs in a defined in vitro system. The Bre1 RING domain cross-links exclusively with distinct regions of histone H2B and H2A, indicating a spatial alignment of Bre1 with the NCP acidic patch. By docking onto the NCP surface in this distinct orientation, Bre1 positions the Rad6 active site directly over H2B Lys123. The Spt-Ada-Gcn5 acetyltransferase (SAGA) H2B deubiquitinase module competes with Bre1 for binding to the NCP acidic patch, indicating regulatory control. Our study reveals a mechanism that ensures site-specific NCP ubiquitination and fine-tuning of opposing enzymatic activities.",

keywords = "Bre1-Rad6, Cross-linking mass spectrometry, Nucleosome, RING E3 ligase, Ubiquitin",

author = "Gallego, {Laura D.} and Steger, {Medini Ghodgaonkar} and Polyansky, {Anton A.} and Tobias Schubert and Bojan Zagrovic and Ning Zheng and Tim Clausen and Franz Herzog and Alwin K{\"o}hler",

note = "Funding Information: We thank D. Finley and G. Warren for comments on the manuscript, and E. Turco and A. Kahraman for help in the early stages of the project. L.D.G. is recipient of a DOC Fellowship of the Austrian Academy of Sciences; B.Z. is funded by European Research Council (ERC) Grant 279408, PROTINT; N.Z. is funded by the Howard Hughes Medical Institute and National Institutes of Health Grant R01-CA107134; Institute of Molecular Pathology is funded by Boehringer Ingelheim; F.H. is supported by ERC Grant MolStruKT StG 638218 and by the German Research Foundation (GRK 1721); and A.K. is funded by ERC Grant 281354, NPC GENEXPRESS, START Grant FWF, Y557-B11 from the Austrian Science Fund, and Doctoral Program RNA Biology W1207-B09. Publisher Copyright: {\textcopyright} 2016, National Academy of Sciences. All rights reserved.",

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doi = "10.1073/pnas.1606863113",

language = "English",

volume = "113",

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Gallego, LD, Steger, MG, Polyansky, AA, Schubert, T, Zagrovic, B, Zheng, N, Clausen, T, Herzog, F & Köhler, A 2016, 'Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1', Proceedings of the National Academy of Sciences of the United States of America, vol. 113, no. 38, pp. 10553-10558. https://doi.org/10.1073/pnas.1606863113

Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1. / Gallego, Laura D.; Steger, Medini Ghodgaonkar; Polyansky, Anton A. et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 113, No. 38, 20.09.2016, p. 10553-10558.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1

AU - Gallego, Laura D.

AU - Steger, Medini Ghodgaonkar

AU - Polyansky, Anton A.

AU - Schubert, Tobias

AU - Zagrovic, Bojan

AU - Zheng, Ning

AU - Clausen, Tim

AU - Herzog, Franz

AU - Köhler, Alwin

N1 - Funding Information: We thank D. Finley and G. Warren for comments on the manuscript, and E. Turco and A. Kahraman for help in the early stages of the project. L.D.G. is recipient of a DOC Fellowship of the Austrian Academy of Sciences; B.Z. is funded by European Research Council (ERC) Grant 279408, PROTINT; N.Z. is funded by the Howard Hughes Medical Institute and National Institutes of Health Grant R01-CA107134; Institute of Molecular Pathology is funded by Boehringer Ingelheim; F.H. is supported by ERC Grant MolStruKT StG 638218 and by the German Research Foundation (GRK 1721); and A.K. is funded by ERC Grant 281354, NPC GENEXPRESS, START Grant FWF, Y557-B11 from the Austrian Science Fund, and Doctoral Program RNA Biology W1207-B09. Publisher Copyright: © 2016, National Academy of Sciences. All rights reserved.

PY - 2016/9/20

Y1 - 2016/9/20

N2 - Cotranscriptional ubiquitination of histone H2B is key to gene regulation. The yeast E3 ubiquitin ligase Bre1 (human RNF20/40) pairs with the E2 ubiquitin conjugating enzyme Rad6 to monoubiquitinate H2B at Lys123. How this single lysine residue on the nucleosome core particle (NCP) is targeted by the Rad6-Bre1 machinery is unknown. Using chemical cross-linking and mass spectrometry, we identified the functional interfaces of Rad6, Bre1, and NCPs in a defined in vitro system. The Bre1 RING domain cross-links exclusively with distinct regions of histone H2B and H2A, indicating a spatial alignment of Bre1 with the NCP acidic patch. By docking onto the NCP surface in this distinct orientation, Bre1 positions the Rad6 active site directly over H2B Lys123. The Spt-Ada-Gcn5 acetyltransferase (SAGA) H2B deubiquitinase module competes with Bre1 for binding to the NCP acidic patch, indicating regulatory control. Our study reveals a mechanism that ensures site-specific NCP ubiquitination and fine-tuning of opposing enzymatic activities.

AB - Cotranscriptional ubiquitination of histone H2B is key to gene regulation. The yeast E3 ubiquitin ligase Bre1 (human RNF20/40) pairs with the E2 ubiquitin conjugating enzyme Rad6 to monoubiquitinate H2B at Lys123. How this single lysine residue on the nucleosome core particle (NCP) is targeted by the Rad6-Bre1 machinery is unknown. Using chemical cross-linking and mass spectrometry, we identified the functional interfaces of Rad6, Bre1, and NCPs in a defined in vitro system. The Bre1 RING domain cross-links exclusively with distinct regions of histone H2B and H2A, indicating a spatial alignment of Bre1 with the NCP acidic patch. By docking onto the NCP surface in this distinct orientation, Bre1 positions the Rad6 active site directly over H2B Lys123. The Spt-Ada-Gcn5 acetyltransferase (SAGA) H2B deubiquitinase module competes with Bre1 for binding to the NCP acidic patch, indicating regulatory control. Our study reveals a mechanism that ensures site-specific NCP ubiquitination and fine-tuning of opposing enzymatic activities.

KW - Bre1-Rad6

KW - Cross-linking mass spectrometry

KW - Nucleosome

KW - RING E3 ligase

KW - Ubiquitin

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DO - 10.1073/pnas.1606863113

M3 - Article

C2 - 27601672

AN - SCOPUS:84988664609

SN - 0027-8424

VL - 113

SP - 10553

EP - 10558

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

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ER -

Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1

Abstract

Keywords

Research fields

IMC Research Focuses

ÖFOS 2012 - Austrian Fields of Study

UN SDGs

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