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S41467-017-00644-Y.Pdf ARTICLE DOI: 10.1038/s41467-017-00644-y OPEN Rapid and reversible epigenome editing by endogenous chromatin regulators Simon M.G. Braun1, Jacob G. Kirkland 1, Emma J. Chory 1,2, Dylan Husmann1, Joseph P. Calarco1 & Gerald R. Crabtree1,3 Understanding the causal link between epigenetic marks and gene regulation remains a central question in chromatin biology. To edit the epigenome we developed the FIRE-Cas9 system for rapid and reversible recruitment of endogenous chromatin regulators to specific genomic loci. We enhanced the dCas9–MS2 anchor for genome targeting with Fkbp/Frb dimerizing fusion proteins to allow chemical-induced proximity of a desired chromatin reg- ulator. We find that mSWI/SNF (BAF) complex recruitment is sufficient to oppose Polycomb within minutes, leading to activation of bivalent gene transcription in mouse embryonic stem cells. Furthermore, Hp1/Suv39h1 heterochromatin complex recruitment to active promoters deposits H3K9me3 domains, resulting in gene silencing that can be reversed upon washout of the chemical dimerizer. This inducible recruitment strategy provides precise kinetic infor- mation to model epigenetic memory and plasticity. It is broadly applicable to mechanistic studies of chromatin in mammalian cells and is particularly suited to the analysis of endo- genous multi-subunit chromatin regulator complexes. 1 Departments of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA. 2 Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA. 3 Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Simon M.G. Braun, and Jacob G. Kirkland contributed equally to this work. Correspondence and requests for materials should be addressed to G.R.C. (email: [email protected]) NATURE COMMUNICATIONS | 8: 560 | DOI: 10.1038/s41467-017-00644-y | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00644-y uring development, epigenetic regulators coordinate gene discoveries. For example, BAF250a and BAF250b, two mutually Dexpression changes that drive stem cell differentiation exclusive mSWI/SNF (BAF) complex subunits have very different into different cell types. Epigenetic regulators modify mutation patterns in human disease. BAF250a is mutated in chromatin compaction and DNA accessibility via multiple many human malignancies3, whereas BAF250b is the most processes including post-translational histone modifications, commonly identified de novo mutated gene in human neurode- DNA methylation, and nucleosome remodeling1. Recent velopmental disorders5, yet neither of these subunits are required human genome sequencing studies have called attention to the for the in vitro activities of the BAF chromatin-remodeling widespread role of chromatin regulators in human disease, often complex. New methods are essential to understand the mechan- identifying unexpected biological roles for known chromatin isms by which these essential epigenetic regulators carry out their – regulators in human development2 4. These studies have revealed distinct biologic roles in different cell types. the highly cell type-specific nature of epigenetic regulation, Understanding the causal link between epigenetic marks and underlining the need for new technologies to study the function gene expression remains a central question in chromatin biology of chromatin regulators in specific cell types, at specific devel- especially as recent advances in epigenome editing techniques are opmental times and in their proper genomic contexts. Methods beginning to shed new light on these processes. The discovery of using in vitro chromatin templates have not reflected these new CRISPR-Cas9 interference, by targeting a catalytically dead ab EF1a dCas9 Blast Chromatin regulator MS2 U6 sgRNA Zeo loops FIRE-Cas9 EF1a MS2 Fkbp Hygro Frb RAP or EF1a MS2 Fkbp Fkbp Hygro dCas9 Fkbp MS2 EF1a HP1cs Frb Puro MS2 or loops EF1a HP1cs Frb Frb Puro sgRNA EF1a MS2 HP1cs Hygro c H3K9me3 ChIP at CXCR4 locus 250 sgRNAs CXCR4 200 –193 –162 –116 150 Control MS2-1xFkbp + HP1-1xFrb 100 MS2-1xFkbp + HP1-2xFrb MS2-2xFkbp + HP1-1xFrb MS2-2xFkbp + HP1-2xFrb 50 Fold change ( RAP / Con) MS2-HP1 0 pr –166 pr +951 d CXCR4 levels (qPCR) 1 sgRNAs CXCR4 0.75 –193 –162 –116 * * 0.5 Control * MS2-1xFkbp + HP1cs-1xFrb MS2-1xFkbp + HP1cs-2xFrb 0.25 * MS2-2xFkbp + HP1cs-1xFrb MS2-2xFkbp + HP1cs-2xFrb Fold change ( RAP / Con) * MS2-HP1cs 0 Fig. 1 Inducible heterochromatin complex recruitment silences target gene expression in HEK 293 cells. a Schematic representation for rapid and reversible recruitment of chromatin regulators to specific genomic loci by FIRE–Cas9. b Lentiviral constructs used for dCas9, sgRNA-MS2, MS2-Fkbp (1x or 2x) and HP1cs-Frb (1x or 2x), or MS2-HP1cs expression in HEK 293 cells. Lentiviral integration is selected for with four unique resistance genes. c H3K9me3 ChIP analysis at the CXCR4 locus (−166 bp and +951 bp from TSS) after 5 days of RAP treatment to induce HP1cs recruitment. Fold changes represent RAP/Control (Con: untreated, no RAP) in respective cell lines except for direct fusion experiment where fold change represents MS2-HP1/MS2-Fkbp recruitment (both untreated, no RAP). n = 3; error bars s.e.m. d CXCR4 expression levels measured by qPCR after 5 days of RAP treatment to recruit HP1cs. n = 3; error bars s.e.m. p < 0.05 2 NATURE COMMUNICATIONS | 8: 560 | DOI: 10.1038/s41467-017-00644-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00644-y ARTICLE a b Heterochromatin complex Oct4-GFP mESC colonies HP1cs RAP dCas9 MS2 Control +RAP sgRNA Oct4 HP1cs recruitment to promoter GFP c 250K 250K 250K Control (no RAP) +RAP +RAP FK506 200K 200K 200K 150K 150K 150K FSC FSC FSC 100K 100K 100K 50K 50K 50K 0.89 99.1 8.61 91.4 2.17 97.8 0 0 0 100 102 104 100 102 104 100 102 104 GFP GFP GFP d 40 H3K9me3 ChIP at Oct4 locus in mESCs sgRNAs Oct4 30 GFP –396 –239 –114 20 Control +RAP 10 +RAP FK506 Fold change (norm. HoxA9) 0 pr –739 pr –130 pr +254 GAPDH Fig. 2 Reversible heterochromatin complex recruitment regulates Oct4 expression in mESCs. a Schematic representation of heterochromatin complex recruitment to the Oct4 locus in reporter Oct4-GFP mESCs. b Microscopy reveals loss of GFP expression in Oct4-GFP mESC colonies 5 days after RAP treatment to induce HP1cs recruitment. Arrows show examples of GFP-negative cells within Oct4-GFP ESC colonies. Scale bar represents 100 µm. c FACS analysis shows reversible repression of Oct4-GFP expression in mESCs. After 5 days of RAP to recruit HP1cs to the Oct4 locus, washout experiments using FK506 restore GFP expression levels after 5 days. Numbers represent % of GFP±cells d H3K9me3 ChIP analysis at the Oct4 locus (−739, −130, and +254 bp from transcriptional start site, TSS) 5 days post RAP treatment and 5 days after washout with FK506. n = 3; error bars s.e.m mutant of Streptococcus pyogenes SpCas9 (dCas9) to block In this study we describe a method, Fkbp/Frb inducible recruit- transcription, has provided a valuable tool for regulating ment for epigenome editing by Cas9 (FIRE–Cas9), which allows gene expression6, 7. Several groups have since fused dCas9 to rapid and reversible recruitment of endogenous chromatin well-characterized repressors and activators (e.g., KRAB and complexes to any genomic locus in almost any cell type. Many of VP64) to modulate gene expression with enhanced silencing and the enzymes that are responsible for writing, erasing, and reading activation capacity8, 9. Furthermore, novel tagging approaches epigenetic marks are present in multi protein complexes that bind have allowed more efficient recruitment of multiple effectors to a chromatin. Previously described methods recruit exogenous single-dCas9 anchor bound to a specific genomic locus10, 11. activators/repressors to turn gene expression on and off in cells Recruitment strategies have also been combined with chemically cultured over several days. In contrast, this endogenous complex inducible approaches to provide temporal control of transcrip- recruitment approach uses induced proximity16 which enables us to tional regulation12, 13. Finally, recent studies have also focused on determine causal links between epigenetic regulators and histone regulatory DNA sequences, via the recruitment of dCas9 fused to modifications within minutes of recruitment. By fusing a single the histone acetyl-transferase p300 or dCas9 fused to the DNA subunit of a chromatin complex with a chemical-induced proximity demethylase Tet1 to activate enhancers14, 15. While these tech- tag, Frb (FKBP-rapamycin-binding domain of mTOR), we can nologies provide new methods for epigenome editing, they focus rapidly recruit intact multi-subunit complexes to a specific genomic on the recruitment of synthetic modulators and lack the temporal sequence upon rapamycin (RAP) treatment as described originally resolution and reversibility required for mechanistic studies of for signaling proteins16.Locusspecificity is obtained via expression epigenetic regulation. of a complementary dimerizer Fkbp (FK506-binding-protein) that NATURE COMMUNICATIONS | 8: 560 | DOI: 10.1038/s41467-017-00644-y | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00644-y ab mSWI/SNF (BAF) Expression levels of bivalent genes (qPCR) recruitment 20 BAF complex SS18 RAP dCas9 10 MS2 sgRNA Bivalent gene Fold change ( RAP / Con) 1 K27me3/K4me3 domain 0 Con Ascl1 Gata4 Nkx2.9 Nfatc1 Pcdh7 Prom1 c 4 BAF 155 ChIP 1.2 H3K27me3 ChIP Con Con 5 min 5 min 1.1 15 min 3 15 min 60 min 60 min 1 2 0.9 1 0.8 Fold change (norm. HoxA9) Fold change (norm. GAPDH) 0 0.7 0 0 500 500 –500 1000 1500 –500 1000 1500 –2000 –1500 –1000 –2000 –1500 –1000 Nkx2.9 Nkx2.9 sgRNAs –152 –68 –25 1.75 H3K4me3 ChIP Con d 5 min 8 Nkx2.9 levels (qPCR) 1.5 15 min 60 min 6 * 1.25 4 1 2 * Fold change (norm. GAPDH) 0 Fold change (norm. GAPDH) Con 5 min 15 min 60 min 4 h 0.75 0 +RAP 500 –500 1000 1500 –2000 –1500 –1000 Fig.
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