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. Author manuscript; available in PMC: 2023 Mar 24.
Published in final edited form as: Nat Cell Biol. 2023 Jan 5;25(1):42–55. doi: 10.1038/s41556-022-01051-2

Fig. 1: CRISPR screen identifies heterochromatin regulators required for heritable Oct3/4 gene silencing.

Fig. 1:

a) Design of the CiA Oct4 dual reporter locus in ESCs. One of the Oct3/4 alleles was modified in ESCs by inserting seven Tet Operator sites (TetO) flanked by a GFP and a BFP reporter gene on either side. GFP expression is under control of the Oct3/4 promoter whereas a PGK promoter drives BFP expression. The genomic screen shot (below) shows histone modifications and RNA expression at the Oct3/4 locus in wild-type ESCs. b) Scheme of the experimental design. TetR facilitates reversible HP1 tethering to TetO binding sites to establish heterochromatin and silence both GFP and BFP reporters. Doxycycline (Dox) addition releases TetR binding to distinguish heritable maintenance of chromatin modifications and gene silencing in the absence of the sequence-specific stimulus. c) Flow cytometry histograms of wild-type and Dnmt1 KO CiA Oct4 dual reporter ESCs show GFP expression before TetR-FLAG-HP1 tethering, in the presence of TetR-FLAG-HP1 and after four days of Dox-dependent release of TetR-FLAG-HP1. Percentages indicate fraction of GFP-negative cells. d) ChIP-qPCR shows relative enrichment of TetR-HP1 (FLAG) and histone modifications surrounding TetO before TetR-FLAG-HP1 tethering, in the presence of TetR-FLAG-HP1 and after four days of Dox-dependent release of TetR-FLAG-HP1. n = 2 independent biological replicates. e) Scheme of CRISPR screen design. MOI refers to multiplicity of infection. Volcano plot shows enrichment (log fold change GFP-pos. sorted vs unsorted cells) and corresponding significance (−log10 MAGeCK significance score) of genes in CRISPR screen (n = mean of three independent experiments). f) Flow cytometry histograms show GFP expression of two independent Zfp462 −/− CiA Oct4 dual reporter cell lines in the presence of TetR-FLAG-HP1 and after four days of Dox-dependent release of TetR-FLAG-HP1. Percentages indicate fraction of GFP-positive cells.