Fig. 5

SMARCAD1 binding to ERVs is not dependent on SETDB1 but on KAP1. a Western blot analysis in E14 mESCs transfected with a Setdb1 shRNA (KD) or a non-targeting control (Ctrl) 96 h post transfection shows efficient knockdown. SMARCAD1 levels were not affected. Lamin B1 serves as loading control. Single-cross-linked chromatin was prepared from these cells and ChIP was carried out with H3, H3K9me3, SMARCAD1, and KAP1-specific antibodies. qPCR over retrotransposons reveals a clear reduction of H3K9me3 but not of SMARCAD1 over class I (VL30) and class II (IAPs, MMERVK10C) ERVs upon Setdb1 knockdown. Data are representative of two immunoprecipitations and the error bars denote the mean ± S.E. of technical triplicates. Corresponding H3 and KAP1 ChIPs are shown in Supplementary Figure 9a. b Stable association of SMARCAD1 with ERV subfamilies depends on an intact CUE1 domain in SMARCAD1. ChIP-qPCR analysis in ESCs depleted for 2 days of endogenous SMARCAD1 protein but expressing 3X FLAG tagged SMARCAD1, either WT or a mutant that affects its interaction with KAP1 (CUE1 mt, F168K, L195K²⁵). Western blot shows that tagged WT and CUE1 mutant SMARCAD1 proteins are expressed at similar levels in the cells utilized for ChIP. ChIP was carried out with a FLAG-specific antibody on double-cross-linked chromatin and analysed by qPCR. Depicted is the mean ± S.E. from three biological replicates (n = 3). P-values are from paired two-tailed Student’s t-test: *p < 0.05, **p < 0.01. Additional analysis with specific IAP primers is shown in Supplementary Figure 9b