skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor

Abstract

The polycomb repressive complex 2 (PRC2) histone methyltransferase plays a central role in epigenetic regulation in development and in cancer, and hence to interrogate its role in a specific developmental transition, methods are needed for disrupting function of the complex with high temporal and spatial precision. The catalytic and substrate recognition functions of PRC2 are coupled by binding of the N-terminal helix of the Ezh2 methylase to an extended groove on the EED trimethyl lysine binding subunit. Disrupting PRC2 function can in principle be achieved by blocking this single interaction, but there are few approaches for blocking specific protein–protein interactions in living cells and organisms. Here, in this work, we describe the computational design of proteins that bind to the EZH2 interaction site on EED with subnanomolar affinity in vitro and form tight and specific complexes with EED in living cells. Induction of the EED binding proteins abolishes H3K27 methylation in human embryonic stem cells (hESCs) and at all but the earliest stage blocks self-renewal, pinpointing the first critical repressive H3K27me3 marks in development.

Authors:
ORCiD logo [1];  [1];  [1];  [1];  [2];  [3];  [3];  [1];  [1];  [1];  [1];  [1];  [4];  [1];  [5];  [1];  [1];  [1];  [3];  [6] more »;  [7];  [6]; ORCiD logo [1] « less
  1. Univ. of Washington, Seattle, WA (United States)
  2. Dana Farber Cancer Inst., Boston, MA (United States); Boston Children’s Hospital, MA (United States); Harvard Medical School, Boston, MA (United States)
  3. Univ. of Toronto, ON (Canada)
  4. Univ. of Toronto, ON (Canada); Jinggangshan Univ., Jiangxi Province (People’s Republic of China)
  5. Univ. of Toronto, ON (Canada); Univ. of Science and Technology of China, Hefei (China)
  6. Univ. of Washington, Seattle, WA (United States); Howard Hughes Medical Inst., Seattle, WA (United States)
  7. Dana Farber Cancer Inst., Boston, MA (United States); Boston Children’s Hospital, MA (United States); Harvard Medical School, Boston, MA (United States); Howard Hughes Medical Inst., Boston, MA (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH); NHLBI Progenitor Cell Biology Consortium; WRF Innovation Fellowship Program; University of Washington’s Proteomics Resource Grant; Defense Threat Reduction Agency (DTRA); Claudia Adams Barr Grant; National Institute of General Medical Sciences; USDOE Office of Science (SC); AbbVie; Bayer Pharma AG; Boehringer Ingelheim; Canada Foundation for Innovation; Eshelman Institute for Innovation; the Canadian Institutes of Health Research (CIHR); Genome Canada; Ontario Genomics Institute; Innovative Medicines Initiative; GlaxoSmithKline; Janssen; Merck & Co.; Novartis Pharma AG; Lilly Canada; the Novartis Research Foundation; the Ontario Ministry of Economic Development and Innovation; Pfizer; São Paulo Research Foundation-FAPESP; Takeda; Wellcome Trust
OSTI Identifier:
1430334
Grant/Contract Number:  
R01GM097372; R01GM97372-03S1; R01GM083867; U01HL099997; U01HL099993; P01GM081619; UWPR95794; GM103533; P41 GM103403; AC02-06CH11357; OGI-055; 115766; 092809/Z/10/Z
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 114; Journal Issue: 38; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
ENGLISH
Subject:
60 APPLIED LIFE SCIENCES; polycomb repressive complex; Rosetta protein design; human embryonic stem cell; human early development; epigenetics

Citation Formats

Moody, James D., Levy, Shiri, Mathieu, Julie, Xing, Yalan, Kim, Woojin, Dong, Cheng, Tempel, Wolfram, Robitaille, Aaron M., Dang, Luke T., Ferreccio, Amy, Detraux, Damien, Sidhu, Sonia, Zhu, Licheng, Carter, Lauren, Xu, Chao, Valensisi, Cristina, Wang, Yuliang, Hawkins, R. David, Min, Jinrong, Moon, Randall T., Orkin, Stuart H., Baker, David, and Ruohola-Baker, Hannele. First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor. United States: N. p., 2017. Web. doi:10.1073/pnas.1706907114.
Moody, James D., Levy, Shiri, Mathieu, Julie, Xing, Yalan, Kim, Woojin, Dong, Cheng, Tempel, Wolfram, Robitaille, Aaron M., Dang, Luke T., Ferreccio, Amy, Detraux, Damien, Sidhu, Sonia, Zhu, Licheng, Carter, Lauren, Xu, Chao, Valensisi, Cristina, Wang, Yuliang, Hawkins, R. David, Min, Jinrong, Moon, Randall T., Orkin, Stuart H., Baker, David, & Ruohola-Baker, Hannele. First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor. United States. doi:10.1073/pnas.1706907114.
Moody, James D., Levy, Shiri, Mathieu, Julie, Xing, Yalan, Kim, Woojin, Dong, Cheng, Tempel, Wolfram, Robitaille, Aaron M., Dang, Luke T., Ferreccio, Amy, Detraux, Damien, Sidhu, Sonia, Zhu, Licheng, Carter, Lauren, Xu, Chao, Valensisi, Cristina, Wang, Yuliang, Hawkins, R. David, Min, Jinrong, Moon, Randall T., Orkin, Stuart H., Baker, David, and Ruohola-Baker, Hannele. Fri . "First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor". United States. doi:10.1073/pnas.1706907114. https://www.osti.gov/servlets/purl/1430334.
@article{osti_1430334,
title = {First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor},
author = {Moody, James D. and Levy, Shiri and Mathieu, Julie and Xing, Yalan and Kim, Woojin and Dong, Cheng and Tempel, Wolfram and Robitaille, Aaron M. and Dang, Luke T. and Ferreccio, Amy and Detraux, Damien and Sidhu, Sonia and Zhu, Licheng and Carter, Lauren and Xu, Chao and Valensisi, Cristina and Wang, Yuliang and Hawkins, R. David and Min, Jinrong and Moon, Randall T. and Orkin, Stuart H. and Baker, David and Ruohola-Baker, Hannele},
abstractNote = {The polycomb repressive complex 2 (PRC2) histone methyltransferase plays a central role in epigenetic regulation in development and in cancer, and hence to interrogate its role in a specific developmental transition, methods are needed for disrupting function of the complex with high temporal and spatial precision. The catalytic and substrate recognition functions of PRC2 are coupled by binding of the N-terminal helix of the Ezh2 methylase to an extended groove on the EED trimethyl lysine binding subunit. Disrupting PRC2 function can in principle be achieved by blocking this single interaction, but there are few approaches for blocking specific protein–protein interactions in living cells and organisms. Here, in this work, we describe the computational design of proteins that bind to the EZH2 interaction site on EED with subnanomolar affinity in vitro and form tight and specific complexes with EED in living cells. Induction of the EED binding proteins abolishes H3K27 methylation in human embryonic stem cells (hESCs) and at all but the earliest stage blocks self-renewal, pinpointing the first critical repressive H3K27me3 marks in development.},
doi = {10.1073/pnas.1706907114},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 38,
volume = 114,
place = {United States},
year = {2017},
month = {9}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 11 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Polycomb complexes repress developmental regulators in murine embryonic stem cells
journal, April 2006

  • Boyer, Laurie A.; Plath, Kathrin; Zeitlinger, Julia
  • Nature, Vol. 441, Issue 7091
  • DOI: 10.1038/nature04733

CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs
journal, April 2016

  • Mandegar, Mohammad A.; Huebsch, Nathaniel; Frolov, Ekaterina B.
  • Cell Stem Cell, Vol. 18, Issue 4
  • DOI: 10.1016/j.stem.2016.01.022

ESCs Require PRC2 to Direct the Successful Reprogramming of Differentiated Cells toward Pluripotency
journal, June 2010


EED and KDM6B Coordinate the First Mammalian Cell Lineage Commitment To Ensure Embryo Implantation
journal, May 2013

  • Saha, B.; Home, P.; Ray, S.
  • Molecular and Cellular Biology, Vol. 33, Issue 14
  • DOI: 10.1128/MCB.00069-13

The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition
journal, November 2015

  • Sperber, Henrik; Mathieu, Julie; Wang, Yuliang
  • Nature Cell Biology, Vol. 17, Issue 12
  • DOI: 10.1038/ncb3264

Computational Design of Epitope-Scaffolds Allows Induction of Antibodies Specific for a Poorly Immunogenic HIV Vaccine Epitope
journal, September 2010


Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells
journal, August 2013

  • Yan, Liying; Yang, Mingyu; Guo, Hongshan
  • Nature Structural & Molecular Biology, Vol. 20, Issue 9
  • DOI: 10.1038/nsmb.2660

Targeted disruption of the EZH2–EED complex inhibits EZH2-dependent cancer
journal, August 2013

  • Kim, Woojin; Bird, Gregory H.; Neff, Tobias
  • Nature Chemical Biology, Vol. 9, Issue 10
  • DOI: 10.1038/nchembio.1331

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27)
journal, February 2012

  • McCabe, M. T.; Graves, A. P.; Ganji, G.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 8
  • DOI: 10.1073/pnas.1116418109

m 6 A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation
journal, January 2015

  • Geula, Shay; Moshitch-Moshkovitz, Sharon; Dominissini, Dan
  • Science, Vol. 347, Issue 6225
  • DOI: 10.1126/science.1261417

Structural Basis of EZH2 Recognition by EED
journal, October 2007


Isolating and engineering human antibodies using yeast surface display
journal, July 2006

  • Chao, Ginger; Lau, Wai L.; Hackel, Benjamin J.
  • Nature Protocols, Vol. 1, Issue 2
  • DOI: 10.1038/nprot.2006.94

Resetting Transcription Factor Control Circuitry toward Ground-State Pluripotency in Human
journal, September 2014


Systematic Identification of Culture Conditions for Induction and Maintenance of Naive Human Pluripotency
journal, October 2014


Dynamic stem cell states: naive to primed pluripotency in rodents and humans
journal, February 2016

  • Weinberger, Leehee; Ayyash, Muneef; Novershtern, Noa
  • Nature Reviews Molecular Cell Biology, Vol. 17, Issue 3
  • DOI: 10.1038/nrm.2015.28

Ground State Conditions Induce Rapid Reorganization of Core Pluripotency Factor Binding before Global Epigenetic Reprogramming
journal, October 2015


Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2
journal, April 2013

  • Knutson, S. K.; Warholic, N. M.; Wigle, T. J.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 19
  • DOI: 10.1073/pnas.1303800110

The Murine Polycomb Group Protein Eed Is Required for Global Histone H3 Lysine-27 Methylation
journal, May 2005


Molecular architecture of human polycomb repressive complex 2
journal, October 2012

  • Ciferri, Claudio; Lander, Gabriel C.; Maiolica, Alessio
  • eLife, Vol. 1
  • DOI: 10.7554/eLife.00005

Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis
journal, November 2015


New cell lines from mouse epiblast share defining features with human embryonic stem cells
journal, June 2007

  • Tesar, Paul J.; Chenoweth, Josh G.; Brook, Frances A.
  • Nature, Vol. 448, Issue 7150
  • DOI: 10.1038/nature05972

Identification of the missing pluripotency mediator downstream of leukaemia inhibitory factor
journal, August 2013

  • Martello, Graziano; Bertone, Paul; Smith, Austin
  • The EMBO Journal, Vol. 32, Issue 19
  • DOI: 10.1038/emboj.2013.177

Molecular Criteria for Defining the Naive Human Pluripotent State
journal, October 2016


Distinct features of H3K4me3 and H3K27me3 chromatin domains in pre-implantation embryos
journal, September 2016

  • Liu, Xiaoyu; Wang, Chenfei; Liu, Wenqiang
  • Nature, Vol. 537, Issue 7621
  • DOI: 10.1038/nature19362

Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells
journal, April 2015

  • Grow, Edward J.; Flynn, Ryan A.; Chavez, Shawn L.
  • Nature, Vol. 522, Issue 7555
  • DOI: 10.1038/nature14308

Derivation of novel human ground state naive pluripotent stem cells
journal, October 2013

  • Gafni, Ohad; Weinberger, Leehee; Mansour, Abed AlFatah
  • Nature, Vol. 504, Issue 7479
  • DOI: 10.1038/nature12745

Astemizole Arrests the Proliferation of Cancer Cells by Disrupting the EZH2-EED Interaction of Polycomb Repressive Complex 2
journal, October 2014

  • Kong, Xiangqian; Chen, Limin; Jiao, Lianying
  • Journal of Medicinal Chemistry, Vol. 57, Issue 22
  • DOI: 10.1021/jm501230c

Enrich: software for analysis of protein function by enrichment and depletion of variants
journal, October 2011


Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells
journal, March 2015

  • Liao, Jing; Karnik, Rahul; Gu, Hongcang
  • Nature Genetics, Vol. 47, Issue 5
  • DOI: 10.1038/ng.3258

Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin
journal, January 2010

  • Morin, Ryan D.; Johnson, Nathalie A.; Severson, Tesa M.
  • Nature Genetics, Vol. 42, Issue 2
  • DOI: 10.1038/ng.518

RosettaScripts: A Scripting Language Interface to the Rosetta Macromolecular Modeling Suite
journal, June 2011


Induction of a Human Pluripotent State with Distinct Regulatory Circuitry that Resembles Preimplantation Epiblast
journal, December 2013


Polycomb Repressive Complex 2 Is Dispensable for Maintenance of Embryonic Stem Cell Pluripotency
journal, June 2008


Defining an essential transcription factor program for naive pluripotency
journal, June 2014