Fig. 4: The essential function of MPP8 requires the formation of a stable HUSH core complex but is independent of its detectable chromatin binding.

a Schematic overview of the LC-MS/MS workflow for interactome analysis. b–d LC-MS/MS of Mpp8^mAID cells expressing either ectopically double flag-tagged MPP8^wt, MPP8^1–729, or MPP8^112–858 as well as OsTIR1 treated with 500 µM IAA (12 h). Parental Mpp8^mAID cells serve as background control (n = 3 biologically independent samples for parental cells, MPP8^wt and MPP8^1–729, n = 2 for MPP8^112–858). HUSH complex members are color-highlighted. e Competition-based proliferation assays indicated Cas9-expressing cell lines. An sgRNA targeting the core essential gene Nat10 served as positive control while a non-targeting sgRNA (NegControl) served as negative control. An sgRNA against Nanog served as mESC-specific positive control. The percentage of GFP+ cells was normalized to the day 0 measurement and the measurement of NegControl at the respective day (mESCs: n = 2 independent experiments, MEFs: n = 1). f, g Representative genome browser tracks showing: (f) MPP8 ChIP-seq in Mpp8^mAID; OsTIR1 cells ± 500 µM IAA (16 h); FLAG ChIP-seq of endogenously FLAG-tagged MPP8, PPHLN1 and TASOR proteins in 2i/LIF-cultered mESCs (parental untagged mESCs served as empty control) or overexpressed FLAG-MORC2A in serum/LIF-cultured mESCs (input served as control) (data taken from Ref. ³²); (g) MPP8 ChIP-seq in indicated Mpp8^mAID; OsTIR1 cell lines ± 500 µM IAA (16 h), FLAG ChIP-seq in indicated Mpp8^mAID; OsTIR1 cell lines + 500 µM IAA (16 h) (E14 cells served as empty control) (f, g) H3K9me3 ChIP-seq signal in Mpp8^mAID; OsTIR1 cells; DNA methylation profiles in ESCs grown under serum/LIF or 2i/LIF culture conditions (0 = unmethylated, 1 = fully methylated CpG sites; data taken from Ref. ³⁵). Repeatmasker track showing the location of relevant LINE1 elements is indicated at the bottom. h, i Aggregate plot comparing the average MPP8 (h) and FLAG (i) ChIP signal, respectively, over high-confidence MPP8 peaks (n = 55) in indicated Mpp8^mAID; OsTIR1 cell lines + 500 µM IAA (16 h). Input signal (h) and empty parental cells (i), respectively, served as control. Source data are provided as a Source Data file.