학술논문
dCas9 fusion to computer-designed PRC2 inhibitor reveals functional TATA box in distal promoter region.
Document Type
Academic Journal
Author
Levy S; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA 98195, USA.; Somasundaram L; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA 98195, USA.; Raj IX; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA 98195, USA.; Ic-Mex D; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA 98195, USA.; Phal A; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Department of Bioengineering, University of Washington, School of Medicine, Seattle, WA 98105, USA.; Schmidt S; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA 98195, USA.; Ng WI; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA 98195, USA.; Mar D; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Department of Medicine, Division of Allergy and Infectious Disease, University of Washington, Seattle, WA 98195, USA.; Decarreau J; Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA 98195, USA; Institute for Protein Design, University of Washington, Seattle, WA 98195, USA; Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.; Moss N; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Division of Medical Genetics, Department of Medicine, University of Washington, School of Medicine, Seattle, WA 98195, USA; Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA 98195, USA.; Alghadeer A; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Department of Oral Health Sciences, University of Washington, School of Dentistry, Seattle, WA 98109, USA; Department of Biomedical Dental Sciences, Imam Abdulrahman Bin Faisal University, College of Dentistry, Dammam 31441, Saudi Arabia.; Honkanen H; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA 98195, USA.; Sarthy J; Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Cancer and Blood Disorder Center, Seattle Children's Hospital, Seattle, WA 98105, USA.; Vitanza A; The Ben Towne Center for Childhood Cancer Research, Seattle Children's Research Institute, Seattle, WA, USA; Division of Pediatric Hematology/Oncology, Department of Pediatrics, University of Washington, Seattle, WA, USA.; Hawkins RD; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Division of Medical Genetics, Department of Medicine, University of Washington, School of Medicine, Seattle, WA 98195, USA; Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA 98195, USA.; Mathieu J; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Department of Comparative Medicine, University of Washington, Seattle, WA 98195, USA.; Wang Y; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Paul G. Allen School of Computer Science & Engineering, University of Washington, Seattle, WA 98195, USA.; Baker D; Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA 98195, USA; Institute for Protein Design, University of Washington, Seattle, WA 98195, USA; Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.; Bomsztyk K; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Department of Medicine, Division of Allergy and Infectious Disease, University of Washington, Seattle, WA 98195, USA.; Ruohola-Baker H; Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA 98109, USA; Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA 98195, USA; Department of Bioengineering, University of Washington, School of Medicine, Seattle, WA 98105, USA; Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA 98195, USA; Department of Oral Health Sciences, University of Washington, School of Dentistry, Seattle, WA 98109, USA. Electronic address: hannele@u.washington.edu.
Source
Publisher: Cell Press Country of Publication: United States NLM ID: 101573691 Publication Model: Print Cited Medium: Internet ISSN: 2211-1247 (Electronic) NLM ISO Abbreviation: Cell Rep Subsets: MEDLINE
Subject
Language
English
Abstract
Bifurcation of cellular fates, a critical process in development, requires histone 3 lysine 27 methylation (H3K27me3) marks propagated by the polycomb repressive complex 2 (PRC2). However, precise chromatin loci of functional H3K27me3 marks are not yet known. Here, we identify critical PRC2 functional sites at high resolution. We fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9) to allow for PRC2 inhibition at a precise locus using gRNA. Targeting EBdCas9 to four different genes (TBX18, p16, CDX2, and GATA3) results in precise H3K27me3 and EZH2 reduction, gene activation, and functional outcomes in the cell cycle (p16) or trophoblast transdifferentiation (CDX2 and GATA3). In the case of TBX18, we identify a PRC2-controlled, functional TATA box >500 bp upstream of the TBX18 transcription start site (TSS) using EBdCas9. Deletion of this TATA box eliminates EBdCas9-dependent TATA binding protein (TBP) recruitment and transcriptional activation. EBdCas9 technology may provide a broadly applicable tool for epigenomic control of gene regulation.
Competing Interests: Declaration of interests S.L., H.R.-B., and D.B. are co-inventors on US patent application no. 17/434,832. S.L. is a founder and stockholder at Histone Therapeutics Corp., a company that aims to develop inventions described in this manuscript. K.B. is a co-founder, board member, and equity holder of Matchstick Technologies, Inc., and the developer and maker of the PIXUL instrument.
(Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
Competing Interests: Declaration of interests S.L., H.R.-B., and D.B. are co-inventors on US patent application no. 17/434,832. S.L. is a founder and stockholder at Histone Therapeutics Corp., a company that aims to develop inventions described in this manuscript. K.B. is a co-founder, board member, and equity holder of Matchstick Technologies, Inc., and the developer and maker of the PIXUL instrument.
(Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)