Expression of heterochromatin protein 1 genes and ERVs in the Cbx5-/- and Cbx1-/- mESCs. (A) qRT-PCR with primers specific for Cbx5 (encoding HP1α) Cbx1 (encoding HP1β), Cbx3 (encoding HP1γ) and the pluripotency factor Nanog in the Cbx5-/- and Cbx1-/- mESC lines confirms the KOs and reveals compensatory upregulation of the genes encoding the remaining HP1 proteins in the Cbx5-/- line. Expression levels were normalized to β-actin relative to wt, and the data are presented as means ± SD for three technical replicates. (B) Western blot analysis of whole-cell lysates confirms the lack of expression of HP1α and HP1β in the Cbx5-/- and Cbx1-/- mESC lines, respectively. (C) Expression of representative ERV families in the HM1 (wt), Cbx5-/- and Cbx1-/- mESCs was determined by qRT-PCR. Expression levels were normalized to β-actin relative to wt. Data are presented as means ± SD of four independent experiments, each of which was performed in triplicate. (D) Northern blot analysis of RNA isolated from the parental HM1,Cbx5-/- and Cbx1-/- mESC lines using probes specific for ETnII, MusD and IAP ERVs are shown. RNA from Dnmt1-/- mESCs, in which IAP elements are upregulated approximately fourfold [20, 86] and MusD elements are upregulated approximately 1.5-fold , was used as a control.