Extended Data Fig. 6: Implementation of an epigenome editing system for site-specific 5mC deposition at candidate SWRs.
From: DNA methylation shapes the Polycomb landscape during the exit from naive pluripotency
a. Schema showing inducible constructs integrated into the Rosa26 locus in ESCs. One allele contains a construct composed of the TetOn array promoter for Doxycycline-dependent activation; DD: FKBP12-derived destabilizing domain, stabilized by the addition of the Shield-1 ligand; dCas9: catalytically dead Cas9, where two point mutations abrogate nucleolytic activity; 10xGCN4: an array of ten GCN4 epitopes (dCas9-SunTag). The homologous allele encodes a TetOn promoter, a destabilizing DD domain, a single-chain antibody (scFv) that recognizes the GCN4 epitope, green fluorescent protein (GFP) and the catalytic domain of DNMT3A (3ACD). Control cells contain an identical construct that encodes a catalytically inactive form of DNMT3A (d3ACD). A separate plasmid containing single guide RNAs (sgRNAs) was randomly integrated by piggyBac transposition. Resistance genes driven by an independent promoter are indicated. b. Western blot validating the inducible dCas9-SunTag/scFv-GFP-3ACD cell line. The two proteins (dCas9-SunTag and scFv-GFP-3ACD) encoded by the two inducible constructs are not detected in the absence of Dox, detected at intermediate levels after Dox induction, and robust expression after the addition of both Dox and Shield-1. c. UCSC Genome Browser screenshots of candidate SWR regions adjacent to the genes: Zdbf2, Celsr2, Atp4a, Arsi, Pga5, Pdyn, and Krt80. The target site for 5mC editing using 3ACD and TET1 is shown; gRNAs are indicated in red (forward strand) or blue (reverse strand). H3K27me3 CUT&Tag in TKO EpiLCs is included. The location of H3K27me3 CUT&RUN quantitative PCR and BS-pyro amplicons are also shown. CpG density, CpG islands (CGIs) and Refseq gene annotations have been added for reference. Coordinates: Zdbf2 chr1:63,246,946-63,275,173; Celsr2 chr3:108,401,129-108,424,638; Atp4a: chr7:30,703,888-30,733,844; Arsi chr18:60,899,912-60,924,326; Pga5 chr19:10,660,312-10,683,383; Pdyn chr2:129,683,316-129,707,017; Krt80 chr15:101,366,622-101,390,127.
