Fig. 1: dCas9-targeted HIV-1 LTR-interactome analysis identified PRMT3 as a LTR-binding factor. | Nature Communications

Fig. 1: dCas9-targeted HIV-1 LTR-interactome analysis identified PRMT3 as a LTR-binding factor.

From: PRMT3 reverses HIV-1 latency by increasing chromatin accessibility to form a TEAD4-P-TEFb-containing transcriptional hub

Fig. 1

a Schematic of dCas9-targeted proteome analysis workflow. (1) sgRNAs targeting the LTR were complex formation with dCas9-3 × Flag; (2) Crosslinking of proteins bound to the LTR; (3) dCas9-3 × Flag-sgRNA complex was incubated with sheared fragments from NH1 cells that stably expressed LTR-luciferase reporter; (4) Immunoprecipitation was used to capture LTR bound proteins; (5) LTR-interacted proteins were identified by mass spectrometry. b Schematic of the position of 7 sgRNAs. c Luciferase activity was measured in NH1 cells transfected with a plasmid encoding each sgRNA and control sgRNA (sgGal4), which expresses the Cas9 in the presence of Tat (p = 0.0016, p = 0.0024, p = 0.0042). d ChIP-qPCR analyses were conducted to assess the occupancy of 3 × Flag-dCas9 at the Promoter (Left) or Nascent region (Right) of LTR when cells were transfected with control plasmid, a mixture of plasmids containing sgLTRs-5, 6, 7 or the sgGal4 plasmid (Promoter: p = 0.0009, p = 0.0011, Nascent: p = 0.000004, p = 0.000003). e Proteins showing over 1.5-fold enrichment relative to the control sample identified from 3 × Flag-dCas9 immunoprecipitation following by mass spectrometry analysis were listed. f ChIP-qPCR analyses were conducted to assess the occupancy of PRMT3 at LTR-luciferase region (p = 0.0001, p = 0.0012, p = 0.0005, p = 0.0008). The schematic display of the four primers was shown on the top of the panel. g ChIP-qPCR analyses were conducted to assess the occupancy of PRMT3 at LTR-luciferase regions in the absence or presence of Tat (p = 0.0022, p = 0.0012, p = 0.0045, p = 0.0017). h CUT&Tag sequencing was performed in primary CD4+ T cells isolated from virologically suppressed HIV-1-infected patient cells that were treated with PMA, by using IgG or PRMT3 antibody. The alignment results of sequencing with the HIV-1 genome in three patients were displayed. Error bars = mean +/− SD of three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t test. Source data are provided as a Source Data file.

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