Catalytically active SETDB1 is required for endogenous retrovirus silencing. (A) Profiling of trimethylated lysine 9 of histone 3 (H3K9me3) along the length of IAPEz endogenous retroviruses (ERVs) in the TT2 wild type (TT2 wt) and Setdb1 knockout (Setdb1 KO) mouse embryonic stem cells (mESCs) (see Figure S3 in Additional file 1 for profiles of murine leukaemia virus (MLV), MusD, MMERVK10C and GLN ERVs). The profile was generated by aligning chromatin immunoprecipitation assay sequencing (ChIP-seq) reads from TT2 wt and Setdb1 KO mESCs  to the consensus sequence of IAPEz. H3K9me3 enrichment levels are presented as reads per kilobase per million mapped reads values (RPKM). (B) Profiling of H3K9me3 and H4K20me3 in the genomic regions flanking 599 IAPEz elements in TT2 wt and Setdb1 KO mESCs (see Figure S4 in Additional file 1 for MusD and MLV profiles). H3K9me3 ChIP-seq reads from TT2 wt (C57BL/6 ± CBA) and Setdb1 KO mESCs  were used, along with H4K20me3 ChIP-seq from the wt V6.5 mESCs (129SvJae ± C57BL/6) . Reads were aligned to the mouse genome (mm9), and the density of reads mapping to the 7-kb regions flanking intact IAPEz ERV families was plotted for H3K9me3 in TT2 wt and Setdb1 KO mESCs and for H4K20me3 in V6.5 wt mESCs. Vertical lines indicate the 5' and 3' boundaries of the ERV. The average mappability for 50-bp reads was confirmed to be, on average, uniform in the assayed 7 kb region (data not shown), ruling out the possibility of mapping bias. (C) Setdb1 deletion was induced with 4-hydroxytamoxifen (4-OHT) in mESCs containing no transgene (KO), a wt transgene (KO TG+) or a transgene with a mutation rendering SETDB1 catalytically inactive (KO C1243A) . Expression is normalized to β-actin relative to wt. Data are presented as means ± standard deviations (SD) for three technical replicates. (D) To establish the expression levels of Setdb1 in the KO and transgenic lines, quantitative RT-PCR (qRT-PCR) was performed with Setdb1-specific primers, and expression was normalized to β-actin relative to wt. Data are presented as means ± SD for three technical replicates.