Figure 5. Histone acetylation modification at HIV LTR promoter. (A) Western blot detection of acetylated histone H3 levels in latently infected cells treatment with M344. J-Lat clones A7 cells were mock treated or treated with M344 (100 nM, 200 nM, 400 nM), and cell lysates were harvested after 8 hours. Western blot analysis was performed with antibodies acetylated histone H3. The amount of protein was normalized by comparison to levels of b-actin. (B) Diagram shows the positions of nucleosomes bound to the HIV-1 LTR and the location of primer used for PCR amplification in the ChIP assay. (C)Chromatin fragments from J-Lat clones A7 cells cultured for 4 hours with or without M344 (200 nM) or TSA (200 nM) were immunoprecipitated with antibody to acetylated histones H3 (AcH3) and H4 (AcH4) or control normal rabbit serum (IgG). PCR primers for the LTR promoter were used to amplify the DNA isolated from the immunoprecipitated chromatin as described in Materials and Methods. (D) Each ChIP experiment was repeated three times to confirm reproducibility of results and real-time quantitation of the fold change relative to untreated control is shown. doi:10.1371/journal.pone.0048832.g005
pitated with anti-p65 or anti-p50 antibodies or rabbit preimmune IgG. These precipitates were then investigated for fragments of HIV-LTR sequences. In the absence of stimulation, samples immunoprecipitated with anti-p65 amplified low to undetectable levels of HIV LTR kB binding site DNA. Following stimulation M344 or TNF-a, anti-p65 immunoprecipitated samples from both J-Lat clones A7 amplified significant quantities of HIV LTR DNA. In contrast, stimulated and unstimulated samples with M344 or TNF-a immunoprecipitated with NF-kB p50 antibodies were enriched in HIV LTR DNA. Fold increase in immunoprecipitation over mock antibody immunoprecipitation is shown in Figure 9. In addition, these samples did not amplify b-actin negative controls beyond background levels (data not shown), demonstrating specificity of the DNA immunoprecipitation.
Discussion
The latently HIV-1-infected monocytic cell lines U1 and ACH2 have long been used to study cellular models of postintegration latency [62,63]. However, mutations in Tat (U1) or in its RNA target TAR (ACH2) have been demonstrated to be causative of the latent phenotype of the proviruses integrated in these two cell
lines. More recently established J-Lat cells, developed with an HIV-1-based vector containing an intact Tat/TAR axis [64,65], were selected for a lack of GFP expression under basal conditions. However, upon appropriate stimulation, such as with the NF-kB inducer, TNF-a, or the HDAC inhibitor TSA, viral transcription is activated and viral expression can be measured by cytometric detection of GFP epifluorescence [64,65]. When the stimuli are removed, the cells return to their latent state. J-Lat cells represent valuable tools for studying HIV transcriptional silencing mechanism and for screening small molecules that can reactivate latent HIV. For this reason, we chose to employ J-Lat Tat-GFP Clone A7 cells in this experiment. Previous studies have demonstrated the transcriptional activation of the HIV-1 promoter in response to HDAC inhibitors such as TSA, trapoxin (TPX), and valproic acid (VPA) [40,48,66]. However, the fact that these inhibitors are not class-specific, and interrupt many cellular parthways, their toxicity issues have raised a fair amount of concerns in the field [67?9]. Class I HDACs, such as HDAC1-3 and 8, are predominantly nuclear enzymes [70]. Class II HDACs include HDAC4-7, 9, and 10, which transport between the nucleus and the cytoplasm [71,72]. Like in many such cases, the choice of an inhibitor most specific to the
