Fig. 4

Depletion of FUS expression activates transcription of HIV. a Depletion of FUS activates HIV gene activation. Luciferase readings were determined in J-LTR-Luc FUS KO and J-LTR-Tat-FUS KO cells, where FUS expression was depleted. To obtain these cells, Jurkat (J)-LTR-Luc were transduced with lentivirus driving the expression of Cas9/sgRNA (FUS specific) and following puromycin drug selection, mono-clonal cells where FUS expression was knockout (KO) were obtained (J-LTR-Luc-FUS KO; gray bar). Control cells were similarly generated, where their CRISPR/sgRNA vector harbored a scrambled sgRNA (black bar). To generate J-LTR-Tat-FUS KO, a selected J-LTR Luc FUS KO clone was further transduced with lentivirus expressing HA-Tat-BFP, and cells were sorted based on their BFP expression (black bar). Luciferase activity was monitored 48 h post transduction according to the manufacturer protocols. Data are presented relatively to luciferase readings in control cells J-LTR-Luc—set to 1, and are representative of three independent experiments. The error bars represent mean ± SD from three independent reactions. Asterisks indicate levels of statistical significance as calculated by two-tailed student T test (**p ≤ 0.01). When asterisks are not shown, no statistically significant difference was observed. b Western Blot analysis of J-LTR-Luc FUS KO cells using FUS antibody, confirming depletion of FUS expression (lane 2). Endogenous expression of FUS in control J-LTR-Luc cells is also presented (ct; lane 1). c Western Blot analysis of J-LTR-Tat-Luc FUS KO cells confirming HA-Tat expression. J-LTR-Luc-FUS KO cells were transduced with lentivirus that drive the expression of HA-Tat. Cells were sorted based on their BFP expression (linked to Tat via IRES). Sorted cells were then harvested and subjected to luciferase assay and western blotting using an HA IgG. Tat expression in control J-LTR-Tat Luc cells was also monitored (lane 1). d Characterization of J-LTR-Luc-FUS knockout clones. Genotyping of genomic DNA isolated from the two J-LTR-Luc-FUS KO clones, where the gene encoding for FUS was disrupted following the introduction of Cas9/sgRNA. Presented is the nucleotide and amino acid residues of FUS around the region where the sgRNA oligos was located. Two independent clones are presented (a and b), where deletions were generated around the sgRNA sequence targets. a—in Clone #11 two sgRNA were used (sgRNA 1 and 2) and generated a 122 bp gap; b—in Clone #1, a single sgRNA was used (sgRNA 1) formed a short deletion as well. e Overexpression of FUS restores HIV gene silencing in FUS KO cells. J-LTR-Luc-FUS KO cells were transduced with increasing MOI of lentivirus that drive the expression of Flag-FUS. Cells were sorted based on their GFP (linked to FUS via IRES) intensities to obtain different levels of FUS expression in cells. Sorted cells were further grown, harvested and subjected to luciferase assay according to the manufacturer protocol. Results are presented relatively to luciferase readings in J-LTR-Luc control cells that express scrambled sgRNA—set to 1. Error bars show mean ± SD from three independent reactions. Asterisks indicate levels of statistical significance as calculated by two-tailed student T test (p < 0.01). Also presented is a western blot verifying increasing amounts of FUS expression. Control J-LTR-Luc express endogenous FUS (lane 1), while J-LTR-Luc-FUS KO cells do not (lane 2). J-LTR-FUS-KO that express increasing concentrations of Flag FUS are also presented (lanes 3–5)