Fig. 4

TRIM22 prevents Sp1 binding to the HIV-1 promoter. a 293T cells were transduced with either pAIP/TRIM22 (TRIM22-KI) or pAIP/empty (CTRL-KI) lentiviral vectors [4] and selected in culture by the addition of puromycin (0.5 µM). WCE from 50 × 10⁶ mock-transduced, CTRL-KI and TRIM22-KI cells was incubated overnight at 4 °C with 40 pmol of 5′-biotinilated double-stranded DNA probes containing either the three Sp1 binding sites of the HIV-1 LTR (as shown in Fig. 1a) or the consensus site of Oct-1 (5’-TGTCGAATGCAAATCACTAGAA-3’). Each sample was pulled-down using streptavidin-conjugated beads (Life Technologies) and analyzed by western blot in parallel with the input fraction (1 % of the pull-down) using anti-TRIM22 (Sigma-Aldrich), anti-Sp1 and anti-Actin (Santa Cruz Biotechnologies) Abs. One representative of three independent experiments is shown. b The Sp1 band intensity of the input and LTR-pull-down fractions from either CTRL-KI or TRIM22-KI cells was quantified by ImageJ. The mean ± SD of three independent experiments is reported. p value was calculated using a paired T test. c Modified pREP10 episomal vector containing the Luc reporter driven by the HIV-1 LTR [15] was co-transfected in 1.2 × 10⁸ 293T cells with Flag-TRIM22-expressing plasmid (provided by Dr. Nadir Mechti, Montpellier, France [8]) or empty plasmid. 48 h post-transfection, proteins were crosslinked to the DNA by formaldehyde incubation. Cell extracts were sonicated to shear chromatin obtaining an average fragment size ranging from 50 to 400 base pairs. IP was performed by using anti-Flag (Sigma-Aldrich), anti-Sp1 and anti-IgG (Santa Cruz Biotechnologies) Abs. Both input (1/1000) and immunoprecipitated DNAs were reverse-crosslinked, purified from proteins and quantified by two real-time SYBR-Green qPCR (LTR promoter and leader region). LTR promoter primer sequences: for 5′-GACTTTCCGCTGGGGACTTTC-3′, rev 5′-CTAACCAGAGAGACCCAGTAC-3′. Leader region primer sequences: for 5′-TGGAAAATCTCTAGCAGTGGC-3′, rev 5′-GAGTCCTGCGTCGAGAGATCT-3′. The amount of immunoprecipitated DNA was normalized to the input DNA and expressed as percentage on input fraction. The mean ± SEM of 3 independent experiments is shown. The p value was determined by the two-way ANOVA