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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 2. — ZFP809 is required for transcriptional repression of VL30 elements. (A) RT-qPCR analysis of Zfp809 mRNA levels in Zfp809^GT ESCs (normalized to ActB). Graphs show the mean fold expression (n = 3) relative to wild-type ESCs. Error bars represent SD. (B) Western blot with whole-cell lysates of wild-type and Zfp809^GT ESCs and MEFs using antibodies against ZFP809 (5763) or GAPDH (significantly longer exposure times were used for MEFs to observe signal). (C) ESCs and MEFs were transduced with retroviral vectors containing PBS-pro or the repression escape mutant PBS-B2 upstream of a puro resistance gene and treated with puromycin for 10 d to determine viral titers (colony-forming units per milliliter). Graphs show the mean ratios of PBS-B2/PBS-pro transduction efficiencies (indicating PBS-pro repression activity) as determined in three replicates. Error bars represent SD. (D) Western blot with whole-cell lysates of wild-type ESCs and MEFs using antibodies against ZFP809 or Actin. (E) RNA-seq analysis of Zfp809^GT MEFs (left) and spleens (right). Mapped RNA-seq reads overlapping with annotated internal RepeatMasker LTR repeats were counted and plotted as reads per kilobase of transcript per million mapped reads (RPKM). The three largest ZFP809-targeted ERV groups (repeats located within 10 kb of strong Flag-ZFP809 peaks) are highlighted. Dotted lines mark 10-fold up-regulation and down-regulation. (F) RT-qPCR analysis of VL30-pro expression in whole blood of adult female mice. The genotypes of the animals’ parents are indicated. Values represent mean expression (normalized to Hprt) in arbitrary units (AU) (n = 3 technical replicates). Error bars represent SD. (G) VL30-pro expression in somatic tissues and organs of male adult mice (9–12 wk old) measured by RT-qPCR (normalized to Hprt). Two animals of each genotype were analyzed. Values represent mean fold expression (n = 2 technical replicates) relative to wild-type mouse tissues. Error bars represent SD.