Fig. 3

SMARCAD1 is required for the maintenance of KAP1, SETDB1, and H3K9me3 at specific ERVs. a ESCs depleted for SMARCAD1 and Ctrl knockdown cells were analysed by ChIP with antibodies specific for SMARCAD1, KAP1, H3K9me3, and unmodified H3. Quantitative real-time PCR was conducted using primers specific for indicated retrotransposon classes, confirming their specific enrichment at the same ERV classes I and II. MuERVL elements, as shown previously, were not enriched in KAP1 or H3K9me3¹⁵. An intergenic region was used as a negative control. Mean enrichment values are presented as percentage of input immunoprecipitated. IAP elements are boxed: the consensus IAP primers shown is for the 5’UTR, the specific IAP primers were designed for the SMARCAD1 bound IAP at the Mier3 locus (Supplementary Figure 4c). b SMARCAD1 is enriched at the 5’UTR of IAPs. Top, schematic of intact IAP structure: LTR, long terminal repeat, gag, and pol genes. Primer amplicons are indicated as black bars. Below, ChIP-qPCR analysis for SMARCAD1, KAP1 and H3K9me3 over IAP elements in control and Smarcad1 knockdown cells. SMARCAD1 depleted cells in b and c show a reduction in KAP1 and H3K9me3 at IAPs. Data are mean ± S.E. from n = 3 (n = 4 ERVK10C) independent experiments in PGK12.1 cells stably depleted for SMARCAD1. c H3K9me3 levels depend on SMARCAD1. ChIP-qPCR data in E14 ESCs showing that SMARCAD1 loss for 4 days results in reduced H3K9me3 levels on ERVs, which are rescued by the expression of exogenous SMARCAD1. Representative target sites and an intergenic control are shown. Real-time qPCR was carried out in technical triplicates and enrichment (±SEM) is the fold change of H3K9me3 over percent input of H3. Western data corresponding to this figure are shown in Supplementary Figure 4f. d SMARCAD1 depletion leads to reduced SETDB1 binding to IAPs. SETDB1 ChIP in single-cross-linked chromatin from PGK12.1 cells depleted for SMARCAD1 compared to a control knockdown. qPCR analysis is representative of two biological replicates and presented as mean ± S.E. of technical triplicates. Asterisks denote statistical significance obtained by paired two-tailed Student’s t-test: *p < 0.05, **p < 0.01, ***p < 0.001