DNA methylation and chromatin marks at ERVs in Cbx5-/- and Cbx1-/-mESCs. (A) An approximately 600-bp fragment of the LTR and downstream region of ETnII/MusD ERVs was analyzed by bisulphite sequencing using primers that detect 105 ETnII/MusD elements, according to in silico PCR analysis (UCSC Genome Browser, http://genome.ucsc.edu/). (B) An approximately 400-bp fragment of the LTR and downstream region of IAP ERVs was analyzed by bisulphite sequencing using primers that detect 1,461 IAP elements. (C) Bisulphite-sequenced molecules were binned into four categories, depending on the number of methylated CpG sites detected, and the data are presented as the percentage of all clones for each cell line in each bin. While the ETnII/MusD family shows no difference in DNA methylation, several IAP molecules in the Cbx5-/- cell line exhibit partial demethylation. (D) Native ChIP (N-ChIP) with antibodies against H3K9me3, H4K20me3 and pan-H3 was followed by qPCR using primers specific for major satellite repeats and IAP, MLV and MusD ERVs, and the data are presented as means ± SD for three technical replicates. The level of H4K20me3 was reduced by more than 50% at major satellite repeats in the Cbx5-/- mESCs but remained at the same level at ERVs.