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Identification of Tat-SF1 cellular targets by exon array analysis reveals dual roles in transcription and splicing.

ABSTRACT: Tat specific factor 1 (Tat-SF1) interacts with components of both the transcription and splicing machineries and has been classified as a transcription-splicing factor. Although its function as an HIV-1 dependency factor has been investigated, relatively little is known about the cellular functions of Tat-SF1. To identify target genes of Tat-SF1, we utilized a combination of RNAi and exon-specific microarrays. These arrays, which survey genome-wide changes in transcript and individual exon levels, revealed 450 genes with transcript level changes upon Tat-SF1 depletion. Strikingly, 98% of these target genes were down-regulated upon depletion, indicating that Tat-SF1 generally activates gene expression. We also identified 89 genes that showed differential exon level changes after Tat-SF1 depletion. The 89 genes showed evidence of many different types of alternative exon use consistent with the regulation of transcription initiation sites and RNA processing. Minimal overlap between genes with transcript-level and exon-level changes suggests that Tat-SF1 does not functionally couple transcription and splicing. Biological processes significantly enriched with transcript- and exon-level targets include the cell cycle and nucleic acid metabolism; the insulin signaling pathway was enriched with Tat-SF1 transcript-level targets but not exon-level targets. Additionally, a hexamer, ATGCCG, was over-represented in the promoter region of genes showing changes in transcription initiation upon Tat-SF1 depletion. This may represent a novel motif that Tat-SF1 recognizes during transcription. Together, these findings suggest that Tat-SF1 functions independently in transcription and splicing of cellular genes.

SUBMITTER: Miller HB

PROVIDER: S-EPMC3062177 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Identification of Tat-SF1 cellular targets by exon array analysis reveals dual roles in transcription and splicing.

Miller Heather B HB Robinson Timothy J TJ Gordân Raluca R Hartemink Alexander J AJ Garcia-Blanco Mariano A MA

RNA (New York, N.Y.) 20110131 4

Tat specific factor 1 (Tat-SF1) interacts with components of both the transcription and splicing machineries and has been classified as a transcription-splicing factor. Although its function as an HIV-1 dependency factor has been investigated, relatively little is known about the cellular functions of Tat-SF1. To identify target genes of Tat-SF1, we utilized a combination of RNAi and exon-specific microarrays. These arrays, which survey genome-wide changes in transcript and individual exon level ...[more]

PMID: 21282347

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Project description:BackgroundThe balance between endothelial cell survival and apoptosis during stress is an important cellular process for vessel integrity and vascular homeostasis, and it is also pivotal in angiogenesis during the development of many vascular diseases. However, the underlying molecular mechanisms remain largely unknown. Although both transcription and alternative splicing are important in regulating gene expression in endothelial cells under stress, the regulatory mechanisms underlying this state and their interactions have not yet been studied on a genome-wide basis.ResultsHuman umbilical vein endothelial cells (HUVECs) were treated with cobalt chloride (CoCl2) both to mimic hypoxia and to induce cell apoptosis and alternative splicing responses. Cell apoptosis rate analysis indicated that HUVECs exposed to 300 microM CoCl2 for 24 hrs were initially counterbalancing apoptosis with cell survival. We therefore used the Affymetrix exon array system to determine genome-wide transcript- and exon-level differential expression. Other than 1583 differentially expressed transcripts, 342 alternatively spliced exons were detected and classified by different splicing types. Sixteen alternatively spliced exons were validated by RT-PCR. Furthermore, direct evidence for the ongoing balance between HUVEC survival and apoptosis was provided by Gene Ontology (GO) and protein function, as well as protein domain and pathway enrichment analyses of the differentially expressed transcripts. Importantly, a novel molecular module, in which the heat shock protein (HSP) families play a significant role, was found to be activated under mimicked hypoxia conditions. In addition, 46% of the transcripts containing stress-modulated exons were differentially expressed, indicating the possibility of combinatorial regulation of transcription and splicing.ConclusionThe exon array system effectively profiles gene expression and splicing on the genome-wide scale. Based on this approach, our data suggest that transcription and splicing not only regulate gene expression, but also carry out combinational regulation of the balance between survival and apoptosis of HUVECs under mimicked hypoxia conditions. Since cell survival following the apoptotic challenge is pivotal in angiogenesis during the development of many vascular diseases, our results may advance the knowledge of multilevel gene regulation in endothelial cells under physiological and pathological conditions.

Project description:BACKGROUND: Conventional anti-HIV drug regimens targeting viral enzymes are plagued by the emergence of drug resistance. There is interest in targeting HIV-dependency factors (HDFs), host proteins that the virus requires for replication, as drugs targeting their function may prove protective. Reporter cell lines provide a rapid and convenient method of identifying putative HDFs, but this approach may lead to misleading results and a failure to detect subtle detrimental effects on cells that result from HDF suppression. Thus, alternative methods for HDF validation are required. Cellular Tat-SF1 has long been ascribed a cofactor role in Tat-dependent transactivation of viral transcription elongation. Here we employ sustained RNAi-mediated suppression of Tat-SF1 to validate its requirement for HIV-1 replication in a CD4+ T cell-derived line and its potential as a therapeutic target. RESULTS: shRNA-mediated suppression of Tat-SF1 reduced HIV-1 replication and infectious particle production from TZM-bl reporter cells. This effect was not a result of increased apoptosis, loss of cell viability or an immune response. To validate its requirement for HIV-1 replication in a more relevant cell line, CD4+ SupT1 cell populations were generated that stably expressed shRNAs. HIV-1 replication was significantly reduced for two weeks (~65%) in cells with depleted Tat-SF1, although the inhibition of viral replication was moderate when compared to SupT1 cells expressing a shRNA targeting the integration cofactor LEDGF/p75. Tat-SF1 suppression was attenuated over time, resulting from decreased shRNA guide strand expression, suggesting that there is a selective pressure to restore Tat-SF1 levels. CONCLUSIONS: This study validates Tat-SF1 as an HDF in CD4+ T cell-derived SupT1 cells. However, our findings also suggest that Tat-SF1 is not a critical cofactor required for virus replication and its suppression may affect cell growth. Therefore, this study demonstrates the importance of examining HIV-1 replication kinetics and cytotoxicity in cells with sustained HDF suppression to validate their therapeutic potential as targets.

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Identification of Tat-SF1 cellular targets by exon array analysis reveals dual roles in transcription and splicing.

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Identification of Tat-SF1 cellular targets by exon array analysis reveals dual roles in transcription and splicing.

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