M344 induces NF-kB Nuclear Translocation and Direct RelA DNA Binding at the nuc-1 Region of HIV-1 LTR
To assess whether M344 stimulation provided sufficient stimulus for RelA nuclear translocation and DNA binding, we studied the effect of M344 on the subcellular distribution of p65. We monitored the localization of the endogenous p65 protein during stimulation with M344, TSA, TNF-a by confocal microscopy (Fig. 8). In unstimulated J-Lat clones A7 cells, p65 was localized predominantly in the cytoplasmic compartment. Treatment with M344 for 30 minutes did not alter this subcellular distribution. A 2-hour treatment with M344 caused a translocation of p65 into the nucleus. Treatment with TSA for 30 minutes or 2 hours did not alter this subcellular distribution. Treatment with TNF-a led after 30 minutes to the migration of p65 to the nucleus. Following 2 hours of treatment with TNF-a, we observed the return of the nuclear p65 to the cytoplasm. These results indicate that M344 can induce NF-kB nuclear translocation. To investigate whether RelA is directly recruited to the HIV LTR in vivo following M344 stimulation, chromatin immunoprecipitation assays were performed. J-Lat clones A7 were treated with M344 or TNF-a, respectively, for 4 hours. Next, the DNA from the cross-linked cells was fragmented via digestion with micrococcal nuclease and sonication. Figure 4. Summary of cell viability assays using M344 and TSA. 293- Human Embryonic Kidney (A), J-Lat clones A7 cells (B) and Jurkat T cells (C) were treated with M344 or TSA at the indicated concentrations for 48 hours, and measured by the MTT method. Results are presented as a percentage of the O.D. (P = 550) of untreated controls subtracted for background. Data represent the means6standard deviations of three independent experiments. than one nucleosome and therefore allowed us to examine changes in histone acetylation over a larger region of the LTR. In these experiments, the IGFBP4 promoter was used as a positive-control region to verify the ability of HDAC6 antibodies to work in ChIP assays [58]. As shown in Figure 6B, normal rabbit serum generated no PCR products after immunoprecipitation. AntiHDAC6 antibodies were able to immunoprecipitate the IGFBP4 promoter region but not non-target DNA (Fig. 6B).
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. 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 Figure 6. Subcellular localization and ChIP analysis of HADC6 in the J-Lat clones A7 cells model of latency. (A) HDAC6 is present in the nucleus and cytoplasm of J-Lat clones A7 cells.