Figure 4.

UHRF1 is a novel epigenetic repressor of HIV-1 latency. (a) DNA methylation mapping performed by sodium bisulfite sequencing is presented for the promoter CGIs or the intragenic ETR CGI in J-Lat 8.4 cells mock-transduced, shNT-transduced or shUHRF1-transduced, as indicated. Unmethylated and methylated CpG dinucleotides are respectively represented with open and closed circles, where each line corresponds to individual sequenced molecules. The global methylation level presented correspond to mean percentages of methylated CpGs for the twelve clones of each condition, either for the promoter, CGIs considered together (5’LTR + NCR CGIs) or for the ETR CGI. Panel (a) to (d) originates from the same representative experiment out of three. (b) Chromatin was prepared from J-Lat 8.4 cells, either mock-transduced or shUHRF1-transduced. Immunoprecipitations were performed using anti-DNMT1, anti-G9a or purified rabbit IgG, as a negative control. qPCRs were performed with primers hybridizing specifically to the 5’LTR, in the Nuc-1 region. Folds relative to IgG are presented, where fold enrichments for each immunoprecipitated DNA were calculated by the relative standard curve on input DNA. Values represent the means of duplicate samples ± SD. (c) HEK293T cells were transfected either with 600 ng of the pshNT vector or with 600 ng of the pshUHRF1#4. Twenty-four hours after this initial transfection, 400 ng of the pLTR-Fluc, the pLTRme-Fluc or the pLTR(CpG5me)-Fluc reporter constructs together with or without 200 ng of the plasmid overexpressing UHRF1 (pUHRF1) were co-transfected. Luciferase activities were measured in the cell lysates 24 h post-transfection. Results are presented as histograms of “relative luciferase units” (R.L.U.), corresponding to the Fluc activity normalized to the total levels of proteins. Means and standard errors of triplicate samples are represented. An experiment representative of three independent experiments is shown. Statistical significance was assessed by an [unpaired T-test]. (d) UHRF1 and β-actin, serving as a loading control, protein levels were assessed by immunoblot in cell lysates of the corresponding transfection points.