Muons penetrate much further than X-rays, they do essentially zero damage, and they are provided for free by the cosmos.
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Edited by Brigid L. M. Hogan, Duke University Medical Center, Durham, NC, and approved August 1, 2017 (received for review April 25, 2017)
Significance
We describe an approach to blocking protein–protein interactions in living cells and use it to probe the earliest stages of epigenetic regulation in stem cell differentiation. We describe a computationally designed protein that tightly binds EED and disrupts PRC2 function in both cancer and stem cells. Expression of the binder at different stem cell stages identifies the first critical repressive H3K27me3 mark in embryonic development.
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.
- polycomb repressive complex
- Rosetta protein design
- human embryonic stem cell
- human early development
- epigenetics
Footnotes
↵1J.D.M., S.L., J. Mathieu., Y.X., and W.K. contributed equally to this work.
↵2Present address: Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602.
- ↵3To whom correspondence may be addressed. Email: dabaker{at}uw.edu or hannele{at}u.washington.edu.
Author contributions: J.D.M., S.L., J. Mathieu, Y.X., W.K., J. Min, R.T.M., S.H.O., D.B., and H.R.-B. designed research; J.D.M., S.L., J. Mathieu, Y.X., W.K., C.D., W.T., A.M.R., L.T.D., A.F., D.D., S.S., L.Z., L.C., C.X., C.V., Y.W., and R.D.H. performed research; J.D.M. contributed new reagents/analytic tools; J.D.M., S.L., J. Mathieu, Y.X., W.K., C.D., W.T., A.M.R., L.T.D., A.F., D.D., S.S., L.Z., C.X., C.V., Y.W., R.D.H., J. Min, R.T.M., S.H.O., D.B., and H.R.-B. analyzed data; and J.D.M., S.L., J. Mathieu, Y.X., W.K., S.H.O., D.B., and H.R.-B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The crystallographic coordinates have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID code 5WP3). The sequence data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. GSE102702 ).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1706907114/-/DCSupplemental.
Designed protein reveals 1st hESC repressive marks
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