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Proc Natl Acad Sci U S A. 2017 Sep 19;114(38):10125-10130. doi: 10.1073/pnas.1706907114. Epub 2017 Sep 1.

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

Moody JD1,2, Levy S3,4, Mathieu J3,4, Xing Y3,4, Kim W5,6,7, Dong C8, Tempel W8, Robitaille AM4,9, Dang LT1,2, Ferreccio A3,4, Detraux D3,4, Sidhu S3,4, Zhu L8,10, Carter L2, Xu C8,11, Valensisi C4,12, Wang Y4,13, Hawkins RD4,12, Min J8,14, Moon RT4,9,15, Orkin SH5,6,7,16,17, Baker D18,3,15, Ruohola-Baker H19,4.

Author information

1
Department of Molecular and Cellular Biology, University of Washington, Seattle, WA 98195.
2
Institute for Protein Design, University of Washington, Seattle, WA 98195.
3
Department of Biochemistry, University of Washington, Seattle, WA 98195.
4
Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98109.
5
Department of Pediatric Oncology, Dana Farber Cancer Institute, Boston, MA 02215.
6
Department of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115.
7
Harvard Medical School, Boston, MA 02115.
8
Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada M5G 1L7.
9
Department of Pharmacology, University of Washington, Seattle, WA 98105.
10
School of Life Sciences in Jinggangshan University, Jiangxi Province, People's Republic of China.
11
School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, People's Republic of China.
12
Genome Sciences, University of Washington, Seattle, WA 98195.
13
Paul G. Allen School of Computer Science & Engineering, University of Washington, Seattle, WA 98195.
14
Department of Physiology, University of Toronto, Toronto, ON, Canada M5S 1A8.
15
Howard Hughes Medical institute, Seattle, WA 98195.
16
Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115.
17
Howard Hughes Medical institute, Boston, MA 02115.
18
Institute for Protein Design, University of Washington, Seattle, WA 98195; dabaker@uw.edu hannele@u.washington.edu.
19
Department of Biochemistry, University of Washington, Seattle, WA 98195; dabaker@uw.edu hannele@u.washington.edu.

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, 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.

KEYWORDS:

Rosetta protein design; epigenetics; human early development; human embryonic stem cell; polycomb repressive complex

PMID:
28864533
PMCID:
PMC5617284
DOI:
10.1073/pnas.1706907114
[Indexed for MEDLINE]
Free PMC Article

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