Fig. 6

An active ATPase domain is required for SMARCAD1 function at ERVs. a Schematic of mSMARCAD1 showing the double CUE (dCUE), the ATPase/helicase domains and the K523R mutation. Location of the FLAG and V5 tags are indicated. b Workflow: E14 cells carrying an inducible Smarcad1 shRNA construct were stably transfected with FLAG-SMARCAD1-V5 constructs. Endogenous SMARCAD1 was depleted before RT-qPCR and ChIP was performed. c Characterization of E14 ESCs expressing tagged SMARCAD1 constructs by western blot. Inducible SMARCAD1 knockdown E14 ESCs (lane 1) show depletion of SMARCAD1 after 2 day doxycycline treatment (dox; lane 2). Cells expressing WT (lane 3) and mutant SMARCAD1 (mt1 and mt2, lanes 4–5, K523R) were monitored using an anti-SMARCAD1 antibody, detecting both endogenous and tagged protein, and an anti-V5 antibody, for tagged SMARCAD1. Lamin B1 serves as a loading control. Dotted line indicates discontinuous lanes from the same gel. d RT-qPCR and ChIP-qPCR analysis in SMARCAD1 knockdown cells (S-KD) rescued with SMARCAD1 transgenes; wild-type (WT), K563R (ATPase mt) and F168K, L195K (CUE1 mt). Left panel; RNA was collected 5 days after depletion of endogenous SMARCAD1 to investigate expression of IAPs and nearby genes. Data confirming KD and similar levels of FLAG SMARCAD1 proteins between cell lines are in Supplementary Figure 10c. Relative expression is mean ± S.E. of two biological replicates normalized to three housekeeping genes. Right panel; H3 and H3K9me3 ChIP-qPCR on double-cross-linked chromatin before and 4 days after SMARCAD1 depletion. qPCR was carried out in triplicates and fold binding of H3K9me3 over H3 (±S.E.) is presented. e SMARCAD1 WT and ATPase mutant binding at IAP elements. FLAG ChIP-seq following depletion of endogenous SMARCAD1 in ESCs expressing FLAG tagged SMARCAD1 wild-type (WT) or ATPase mutant (mt) or in ESCs lacking FLAG proteins (Ctrl). For each repeat element the log2 fold ratio over input was plotted as in Fig. 2a, Box lines show the median, 25th and 75th percentiles; whiskers end at the smallest (largest) datum not further than 1.5 times the interquartile range. LINE elements show no enrichment. f ChIP of double-cross-linked chromatin from the cells described in b, c with a FLAG antibody, which detects tagged SMARCAD1 WT and ATPase mutant. qPCR analysis over representative ERV elements of class I (VL30) and class II (IAPs and MMERVK10C) is shown. Analysis of additional IAP elements and SMARCAD1 ChIP is shown in Supplementary Figure 10d, e. g The ATPase mutation does not disrupt the association of SMARCAD1 with KAP1. A FLAG antibody co-immunoprecipitates KAP1 from ESCs expressing FLAG tagged SMARCAD1 protein, both WT (lane 3) or the ATPase mutant K523R (lane 6) in the presence of ethidium bromide and benzonase. Lanes 1 and 4, 3% input. PRMT5 served as a negative control. Endogenous SMARCAD1 was depleted by 2 day doxycycline treatment. Dotted line indicates discontinuous lanes from the same gel. h SMARCAD ATPase function facilitates stable KAP1 occupancy at ERVs. Binding of KAP1 was analysed by ChIP-qPCR over the same sites and in the same cells as in f and Fig. 5b (CUE1 mutant). Additional sites are in Supplementary Figure 10h. qPCR data in f and h are shown as mean ± S.E. of three-independent experiments (except ATPase mt 2 (n = 2) which precludes it from statistical analysis). P-values were calculated using paired two-tailed Student’s t-test: *p < 0.05, **p < 0.01