Tat predominantly associates with host promoter elements in HIV-1-infected T-cells - regulatory basis of transcriptional repression of c-Rel.

HIV-1 Tat is a multifunctional regulatory protein that, in addition to its primary function of transactivating viral transcription, also tends to modulate cellular gene expression, for which the molecular mechanism remains to be clarified. We have reported earlier nuclear factor kappa B (NFκB) enhancer binding activity of Tat and proposed this DNA binding activity as a possible molecular basis for Tat-mediated regulation of cellular gene expression in infected cells. In the present study, we analyzed the genome-wide occupancy of Tat protein on host cell chromatin in HIV-1-infected T-cells to investigate a potential role of Tat on cellular gene expression. The results obtained identify a spectrum of binding sites of Tat protein on the chromatin and reveal that Tat is also recruited on a number of cellular gene promoters in HIV-1-infected T-cells, indicating its possible involvement in the regulation of gene expression of such cellular genes. Tat was identified as a repressor of one such validated gene, c-Rel, because it downregulates the expression of c-Rel in both Tat expressing and HIV-1-infected T-cells. The results also show that Tat downregulates c-Rel promoter activity by interacting with specific NFκB sites on the c-Rel promoter, thus providing a molecular basis of Tat-mediated regulation of cellular gene expression. Thus, in the present study, we have not only identified recruitment sites of Tat on the chromatin in HIV-1-infected T-cells, but also report for the first time that c-Rel is downregulated in HIV-1-infected cells specifically by interaction of Tat with NFκB binding sites on the promoter.

Identification of genomic targets of transcription factor AEBP1 and its role in survival of glioma cells.

A recent transcriptome analysis of graded patient glioma samples led to identification of AEBP1 as one of the genes upregulated in majority of the primary GBM as against secondary GBM. Aebp1 is a transcriptional repressor that is involved in adipogenesis. It binds to AE-1 element present in the proximal promoter of aP2 gene that codes for fatty acid binding protein (FABP4). A comprehensive study was undertaken to elucidate the role of AEBP1 overexpression in glioblastoma. We employed complementary gene silencing approach to identify the genes that are perturbed in a glioma cell line (U87MG). A total of 734 genes were differentially regulated under these conditions (≥ 1.5-fold, P ≤ 0.05) belonging to different GO categories such as transcription regulation, cell growth, proliferation, differentiation, and apoptosis of which perturbation of 114 genes of these pathways were validated by quantitative real time PCR (qRT-PCR). This approach was subsequently combined with ChIP-chip technique using an Agilent human promoter tiling array to identify genomic binding loci of Aebp1 protein. A subset of these genes identified for Aebp1 occupancy was also validated by ChIP-PCR. Bioinformatics analysis of the promoters identified by ChIP-chip technique revealed a consensus motif GAAAT present in 66% of the identified genes. This consensus motif was experimentally validated by functional promoter assay using luciferase as the reporter gene. Both cellular proliferation and survival were affected in AEBP1-silenced U87MG and U138MG cell lines and a significant percentage of these cells were directed towards apoptosis.

Glioblastoma-specific protein interaction network identifies PP1A and CSK21 as connecting molecules between cell cycle-associated genes.

Glioblastoma (GBM; grade IV astrocytoma) is a very aggressive form of brain cancer with a poor survival and few qualified predictive markers. This study integrates experimentally validated genes that showed specific upregulation in GBM along with their protein-protein interaction information. A system level analysis was used to construct GBM-specific network. Computation of topological parameters of networks showed scale-free pattern and hierarchical organization. From the large network involving 1,447 proteins, we synthesized subnetworks and annotated them with highly enriched biological processes. A careful dissection of the functional modules, important nodes, and their connections identified two novel intermediary molecules CSK21 and protein phosphatase 1 alpha (PP1A) connecting the two subnetworks CDC2-PTEN-TOP2A-CAV1-P53 and CDC2-CAV1-RB-P53-PTEN, respectively. Real-time quantitative reverse transcription-PCR analysis revealed CSK21 to be moderately upregulated and PP1A to be overexpressed by 20-fold in GBM tumor samples. Immunohistochemical staining revealed nuclear expression of PP1A only in GBM samples. Thus, CSK21 and PP1A, whose functions are intimately associated with cell cycle regulation, might play key role in gliomagenesis.

The cell death regulator GRIM-19 is involved in HIV-1 induced T-cell apoptosis.

One of the hallmarks of Human Immunodeficiency Virus-1 (HIV-1) infection is progressive depletion of the infected and bystander CD4+ T-cells by apoptosis. Different mitochondrial proteins have been implicated in this apoptotic process; however, the role of different subunits of mitochondrial oxidative phosphorylation (OXPHOS) complexes in apoptosis is not clearly understood. Some of the OXPHOS complex subunits seem to perform other functions in addition to their primary role in energy generating process. GRIM-19 (gene associated with retinoid-interferon-induced-mortality-19), a subunit of mitochondrial complex-I was previously implicated in Interferon-ß and retionoic acid induced apoptosis in many tumor cells. In this study we report, using differential gene expression analysis, that GRIM-19 is up-regulated in HIV-1 infected apoptotic T-cells. A temporal up regulation of this subunit was observed in different HIV-1 infected T-cell lines and human PBMC and the extent of increase correlated to increasing apoptosis and virus production. Moreover, silencing GRIM-19 in HIV-1 infected cells reduced apoptosis, indicating its involvement in HIV-1 induced T-cell death.

Human immunodeficiency virus-1 Nef protein interacts with Tat and enhances HIV-1 gene expression.

The human immunodeficiency virus (HIV-1) Nef protein is now regarded as a regulatory protein responsible not only for establishment of infection and increased pathogenesis but also for enhancement of viral replication. However, the mechanism of Nef-induced activation of viral replication remains to be clearly understood. Using transient transfection assay, co-immunoprecipitation and pull-down analysis, we demonstrate in this report that the HIV-1 Nef protein physically interacts with Tat, the principal transactivating protein of HIV-1. Our observations with single cycle replication experiments further indicate that this interaction results not only in enhancement of Tat-induced HIV-1 long terminal repeat-mediated gene expression but also in virus production.