Induction of OGG1 Gene Expression by HIV-1 Tat

Abstract

To identify the cellular gene target for Tat, we performed gene expression profile analysis and found that Tat up-regulates the expression of the OGG1 (8-oxoguanine-DNA glycosylase-1) gene, which encodes an enzyme responsible for repairing the oxidatively damaged guanosine, 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG). We observed that Tat induced OGG1 gene expression by enhancing its promoter activity without changing its mRNA stability. We found that the upstream AP-4 site within the OGG1 promoter is responsible and that Tat interacted with AP-4 and removed AP-4 from the OGG1 promoter by in vivo chromatin immunoprecipitation assay. Thus, Tat appears to activate OGG1 expression by sequestrating AP-4. Interestingly, although Tat induces oxidative stress known to generate 8-oxo-dG, which causes the G:C to T:A transversion, we observed that the amount of 8-oxo-dG was reduced by Tat. When OGG1 was knocked down by small interfering RNA, Tat increased the amount of 8-oxo-dG, thus confirming the role of OGG1 in preventing the formation of 8-oxo-dG. These findings collectively indicate the possibility that Tat may play a role in maintenance of the genetic integrity of the proviral and host cellular genomes by upregulating OGG1 as a feed-forward mechanism.

Transcriptional Repression of Human Immunodeficiency Virus Type 1 by AP-4

“…Although AP-4 was initially identified as a cellular protein that binds to the simian virus 40 (SV40) enhancer and activates the viral late gene transcription (21), transcriptional repression by AP-4 was reported in a number of other genes including angiotensinogen (21) and E7 oncoprotein of human papillomavirus type 16 (23). In addition, we recently found that AP-4 negatively regulates transcription of 8-oxo guanine DNA glycosylase 1 (OGG1) gene (24). However, because AP-4 sites are not located adjacent to the TATA box in these promoters, the molecular mechanism of its repressive action is yet to be clarified.…”

“…Immunoprecipitation and Immunoblot Assays-The experimental procedures for immunoprecipitation and immunoblotting were performed as described (24,27). Briefly, cells were harvested with lysis buffer (25 mM HEPES-NaOH, pH 7.9, 150 mM NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.3% Nonidet P-40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride).…”

“…Plasmids-Construction of mammalian expression plasmids pMyc-AP-4, containing the full-length AP-4 cDNA, pCMV-Tat, and pNL4-3 were described previously (24,25). pCMV-TBP was a generous gift from T. Tamura (Chiba University).…”

“…Electrophoretic Mobility Shift Assay (EMSA)-The experimental procedure was carried out as described previously (24). Purified recombinant TBP and GST-TBP proteins were purchased from Promega and Santa Cruz Biotechnology, respectively.…”

Proteome analyses of the growth inhibitory effects of NCH-51, a novel histone deacetylase inhibitor, on lymphoid malignant cells

“…27 After treatment with HDACi, the cells were incubated with an oxidation-sensitive fluorescent probe 2 0 , 7 0 -dichlorofluorescein diacetate (H 2 -DCFDA) (Molecular Probes Inc., Eugene, OR, USA) at a final concentration of 5 mM for 30 min. The cells were washed and resuspended in PBS, and then ROS amount was measured by flowcytometry.…”