The EcoDam-LEDGF/p75 fusion protein behaves like endogenous LEDGF/p75. (A) Schematic representation of the pLgw-EcoDam-V5-LEDGF/p75 transfer plasmid expressing the EcoDam-V5-LEDGF/p75 fusion protein, and the pLgw-EcoDam-V5 transfer plasmid expressing the EcoDam-V5 control protein. CMV, cytomegalovirus promoter; LTR, long terminal repeat; p75, LEDGF/p75. (B) 293T cells were transiently transfected with the respective transfer plasmids. Total cell lysate was prepared and analyzed by western blotting using an anti-V5 antibody. (C) HeLa CCR5 A3 cells depleted for LEDGF/p75 (34) were transfected with a LEDGF/p75 (upper panels, green) or an EcoDam-V5-LEDGF/p75 expression plasmid (lower panels, green) together with an mRFP-integrase expression plasmid (red). DAPI was used to stain the DNA. An overlay of the green and red panels is shown. (D) HIV-1 replication is rescued by the EcoDam-LEDGF/p75 fusion protein. HeLa CCR5 A3 cells were transfected with the indicated constructs. The 24-h post-transfection cells were infected with two dilutions of an HIV-1 clone encoding firefly luciferase (NL4.3 fLuc) (dark and light gray). Luciferase expression was measured 48 h post-infection. Fluc counts upon transfection of a control plasmid encoding an EcoDam fusion to CBX1 (51) were subtracted. LEDGF/p75 D366A, HIV integrase interaction deficient LEDGF/p75 (40). The experiment was done in triplicate and repeated four times. A representative experiment is shown. Error bars indicate the SD of the triplicate values.

LEDGF/p75 binds TUs downstream of the TSS. (A) Distribution of LEDGF/p75 islands around TUs. The percentage of LEDGF/p75 islands in TUs and overlapping with TSS or transcription ends is shown for RefSeq and Ensembl TUs and for non-overlapping RefSeq TUs. As a control, a random data set of LEDGF/p75 islands was generated 10 times the size of the original data set (MRC). *P < 0.05, **P < 10⁻²⁷ as compared to random as measured by a χ²-test. (B) LEDGF/p75 binding in TUs was plotted relative to the TSS (LEDGF/p75). All genes were divided in 20 equal bins and the MAT score of each LEDGF/p75 positive base was assigned to the corresponding bin. For each LEDGF/p75 island, 10 new islands were picked at a random position within the ENCODE region having the same size and score as the parental island (MRC). As for LEDGF/p75 islands, the random islands were plotted relative to the TSS. Values were divided by 10 for comparison with the LEDGF/p75 islands. HIV-1 integration sites in TUs (28) were assigned to the corresponding bin relative to the TSS (HIV integration). If LEDGF/p75 or integration sites could be assigned to more than one TU, the weight was divided over all TUs. (C) Distribution of LEDGF/p75, MRC and HIV-1 integration sites around the TSS. Scores for LEDGF/p75 and random islands were accumulated for each base in a 20 kb window around the TSS. All TUs were analyzed in 5′–3′ orientation. Data values of the random curve were divided by 10 to be comparable with LEDGF/p75 islands. For HIV-1 integration sites, the 20 kb window was divided into 1-kb bins. Each integration site was assigned to the corresponding bin. Since LEDGF/p75 islands almost never overlap with the TSS, a positive base found in the TU was fully assigned to this TU and not divided over potential neighboring units. (D) Distribution of LEDGF/p75, random islands (MRC) and HIV-1 integration sites around the transcriptional end. Analysis was performed as described in C.

LEDGF/p75 chromatin binding correlates with transcriptional activity. Genes in the ENCODE region were divided into bins according to their expression level. The percentage of LEDGF/p75 positive genes in each bin is shown in black. The 95% confidence interval is shown (gray shading). As a control, 10 MRC sets of equal size as the original data were computed and divided over the bins (gray line). The mean ± SD is shown.

HIV-1 integration is targeted to LEDGF/p75 chromatin binding islands. (A) Schematic representation of LEDGF/p75 islands and HIV-1 integration sites in ENCODE region ENm014 (http://genome.ucsc.edu/). This ENCODE region contains significantly less integration sites than predicted (28). All integration events took place around the same TU that also contains LEDGF/p75 islands. GCC1, GRIP and coiled-coil domain containing 1; ARP5, actin-related protein 5 homolog; FSCN3, fascin 3. (B) The region surrounding the middle of LEDGF/p75 islands was divided in 0.5 kb bins. Integration sites were assigned to the closest LEDGF/p75 island and placed into the corresponding bin. The percentage of integration sites in each bin is shown (bold line). Ten data sets of matched random integration sites (HIV MRC) of equal size as the HIV-1 integration data set were generated and assigned to the corresponding bin (light line). The SD is shown.

Cross-correlation of LEDGF/p75 islands and HIV-1 integration with ENCODE tracks. Scatter plot of the correlation of each of the 215 ENCODE tracks with LEDGF/p75 islands and HIV-1 integration sites. Significance was tested by calculating the 2.5 through 97.5 percentile interval of Pearson’s correlation coefficients for 600 HIV-1 and LEDGF/p75 matched random controls with each ENCODE track of interest as described in the ‘Materials and Methods’ section. Most ENCODE tracks with a positive correlation with HIV-1 integration, positively correlate with LEDGF/p75 binding (in green). Black, no correlation with HIV-1 or LEDGF/p75 track; orange, positive or negative correlation with HIV-1 integration, no correlation with LEDGF/p75 binding; blue, positive or negative correlation with LEDGF/p75 binding, no correlation with HIV-1 integration; red, correlation of the HIV-1 and LEDGF/p75 track with the ENCODE track have the opposite sign.

Shifted cross-correlation of LEDGF/p75 islands and HIV-1 integration with ENCODE tracks. The Pearson’s correlation coefficient between LEDGF/p75 (black) or HIV-1 integration sites (gray) and the indicated ENCODE track was calculated for each lag. Lags indicate the distance the LEDGF/p75 or HIV-1 integration track was shifted as compared to the ENCODE track of interest to calculate the cross-correlation coefficient. Both for LEDGF/p75 islands and HIV-1 integration sites, 600 random tracks of the same size were generated. The correlation coefficient at lag 0 of the depicted ENCODE track with each random track was calculated. The 2.5 through 97.5 percentile interval fell between the dashed lines (black, LEDGF/p75; gray, HIV-1 integration sites). Histones 3 and 4 (H3 and H4) acetylation and Histone 3 Lysine 4 (H3K4) monomethylation are epigenetic markers of active transcription. Histone 3 Lysine 27 (H3K27) trimethylation is a marker of heterochromatin.

Divergent cross-correlation of LEDGF/p75 islands and HIV-1 integration with specific ENCODE tracks. For an explanation of the cross-correlation curves, see legend to Figure 6. The ENCODE tracks shown have a strong positive correlation with LEDGF/p75, but do not correlate with HIV-1 integration. Stat1, signal transducer and activator of transcription; Hnf3b, hepatocyte nuclear factor 3-β; Hnf4a, hepatocyte nuclear factor 4-α; Usf1, upstream transcription factor 1.