The HUSH complex contributes to repression and DNA methylation of an SVA retrotransposon reporter. (A, left) ZNF91 binds the SVA VNTR sequence and represses the reporter. ZNF93 or an empty reporter were used as controls. (enh.) enhancer; (prom.) promoter; (SV40) simian virus 40. (Right) Either luciferase reporter was cotransfected with a Renilla luciferase-encoding control, and relative luciferase light units were measured 48 h later. (B) NTERA-2 cells were verified to express POU5F1 (left) and subject to reporter assays shown in A (middle), here normalized to the empty control, and ZNF expression was measured by qRT-PCR (right). (C) Reporter assays in TRIM28 WT and KO 293T cells, including cotransfection of stated exogenous ZNFs. Data are normalized to the bar on the left (TRIM28 WT, ZNF93). Western blot for TRIM28 using PCNA as a loading control (insets from the same blot). (D) 293T cells were transduced with shRNA vectors against epigenetic factors and puromycin-selected before reporter assays. (E) Following the assay in D, data for ZNF91 were normalized to ZNF93 (left). Unpaired t-tests were used to assess differences between the shControl (transduced with the same vector lacking a hairpin that was puromycin-selected in parallel) and the target shRNA. Experiments were repeated at least three times for each candidate, and representative data are shown. Two-tailed unpaired t-tests were done: (*) P = <0.05; (**) P = <0.01; (***) P = <0.001. qRT-PCR was used to assess the knockdown efficiency of each mRNA (right). (F) DNA methylation analysis of the SV40 promoter 48 h post reporter transfection. Plasmids were produced in dam− bacteria. Methylated and unmethylated CpGs are shown as filled and open circles, respectively. (G) Summary results of levels of de novo DNA methylation of the reporter in control cells versus HUSH-depleted cells. See also Supplemental Figure S1G. Two-tailed, unpaired t-tests were used to compare four controls to four HUSH-depleted samples (from two independent experiments): P = 0.0062.
