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. 2015 Mar 1;29(5):538–554. doi: 10.1101/gad.252767.114

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Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 5. — ZFP809 is required for the initiation, but not the maintenance, of ERV silencing. (A) RT-qPCR analysis of VL30-pro expression in ESCs during differentiation (normalized to Hprt). Values represent mean fold expression (n = 3 technical replicates) relative to wild-type ESCs. Results from two independent ESC lines are shown for each genotype. Error bars represent SD. (B) VL30-pro expression in MEFs and iPSCs derived from these MEF lines (normalized to Gapdh). Two iPSC clones were analyzed by RT-qPCR for each MEF line. Values represent mean fold expression (n = 3 technical replicates) over Zfp809^GT/+ MEFs. Error bars represent SD. (C) Dual-luciferase reporter assay in transiently transfected Zfp809^GT/GT cells. LTR activity was normalized to CMV promoter activity on cotransfected renilla luciferase plasmids and is displayed in arbitrary units (AU). Assay was performed in triplicates; error bars represent SD. (D) ERV expression analysis of MEFs treated with Aza-dC and/or TSA by RT-qPCR (normalized to Hprt). Graphs show mean fold expression (n = 3 technical replicates) over Zfp809^GT/+ untreated control. Error bars represent SD. (E) ERV expression in Setdb1^−/+ and Setdb1^−/− MEFs was measured by RT-qPCR (normalized to Hprt) and is shown as mean fold expression (n = 3 technical replicates) relative to Setdb1^-/+ MEFs. Error bars represent SD. (F) RT-qPCR analysis of Zfp809 and VL30-pro expression in inducible Zfp809 knockout (Zfp809^FL/FL; RosaCreERT2) and Zfp809^−/− MEFs. Values represent mean fold expression (n = 3 technical replicates) relative to untreated Zfp809^FL/FL MEFs (normalized to Hprt). Error bars represent SD. (G) Zfp809^GT/+ and Zfp809^GT/GT MEFs stably transfected with a transposon-based Flag-ZFP809 expression vector or an empty control vector were grown under puromycin selection for 2 wk before RT-qPCR analysis. Values represent mean fold expression (n = 3 technical replicates) relative to Zfp809^GT/+ MEFs transfected with control vector (normalized to Hprt). Error bars represent SD.