Figure 2. The HUSH complex is responsible for silencing at the Sox2 short allele.

(A) MPP8 western blot in Sox2–1kb reporter (control) and MPP8 knockout (KO) subclones. Membranes were probed with antibodies against MPP8 and β-actin as a loading control.

(B) FACS 2D plots of GFP (x axis) and forward scatter (y axis) in WT mESCs, Sox2–1kb reporter (control), and MPP8 KO clones.

(C) FACS histogram plots of GFP in Sox2–1kb reporter cells transduced with two non-targeting sgRNAs (control) or TASOR-targeting sgRNAs (TASOR KO), 9 days after induction of Cas9 expression.

(D) H3K9me3 ChIP-seq coverage aligned independently to Sox2-GFP (green) or Sox2-BFP (blue) alleles in the Sox2–1kb cell line.

(E) Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) of H3K9me3 in Sox2–1kb reporter. Schematic diagram of the Sox2 locus in the Sox2–1kb reporter and location of primers used for the qPCR (top). Percentage of input recovered for H3K9me3 ChIP in Sox2–1kb reporter control (red) and MPP8 KO clones (blue) with the different primers for qPCR (bottom).

(F) Top: schematic diagram of the MPP8 and TASOR endogenous tagging strategy in WT mESCs (termed “AGH” for AID-GFP-HAx3). Bottom: western blots in WT (control) and endogenously tagged mESCs, probing for MPP8 (left), HA (right) and β-actin as a loading control.

(G) Browser tracks of TASOR, MPP8 and H3K9me3 ChIP-seq in WT mESCs surrounding the Sox2 locus. Highlighted in yellow are the major ChIP-seq pileups for either MPP8 and TASOR (HUSH peaks) or MPP8-only peaks.