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A functional genome-wide RNAi screen identifies TAF1 as a regulator for apoptosis in response to genotoxic stress.

ABSTRACT: Evasion from apoptotic cell death is a characteristic of cancer; genes that modulate this process may be optimal for therapeutic attack. Identifying key regulators of apoptosis is thus a central goal in cancer therapy. Here, we describe a loss-of-function screen that uses RNA interference libraries to identify genes required for induction of apoptosis. We used a short-hairpin RNA expressing vector with high gene-expression silencing activity that contained fetal brain cDNAs. Survived cells from genotoxic stress were isolated to determine knock-down of molecules that are crucial for induction of apoptosis. We identified TBP-associated factor 1 (TAF1), a gene previously implicated as an essential component of transcription machinery. Depletion of TAF1 was associated with substantial attenuation of apoptosis induced by oxidative as well as genotoxic stress. Microarray analysis further demonstrated that a number of genes were transcriptionally declined in cells silenced for TAF1. Surprisingly, knocking down TAF1 exhibited a marked decrease in p27(Kip1) expression, allowing cells resistant from oxidative stress-induced apoptosis. These results suggest that TAF1 regulates apoptosis by controlling p27(Kip1) expression. Our system provides a novel approach to identifying candidate genes that modulate apoptosis.

SUBMITTER: Kimura J

PROVIDER: S-EPMC2532742 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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A functional genome-wide RNAi screen identifies TAF1 as a regulator for apoptosis in response to genotoxic stress.

Kimura Junko J Nguyen Su Tien ST Liu Hanshao H Taira Naoe N Miki Yoshio Y Yoshida Kiyotsugu K

Nucleic acids research 20080806 16

Evasion from apoptotic cell death is a characteristic of cancer; genes that modulate this process may be optimal for therapeutic attack. Identifying key regulators of apoptosis is thus a central goal in cancer therapy. Here, we describe a loss-of-function screen that uses RNA interference libraries to identify genes required for induction of apoptosis. We used a short-hairpin RNA expressing vector with high gene-expression silencing activity that contained fetal brain cDNAs. Survived cells from ...[more]

PMID: 18684994

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Project description:Glioblastoma multiforme is the most common and most aggressive type of primary brain tumor. The brain-infiltrative character of glioblastoma makes complete surgical removal of the tumor impossible and neither radiation nor current chemotherapy provide cure. Recent evidence shows that glioblastoma multiforme consists of heterogeneous cell populations which differ in tumor-forming potential. Enriched tumor-initiating capacity has been linked to poorly differentiated glioblastoma cells sharing features with neural stem cells. Thus, these cells are important targets for new therapeutic strategies. We aim to identify novel targets controlling maintenance and differentiation in glioblastoma-initiating cells through high throughput screening. To this end, we utilized libraries of small chemical compounds and small interference RNAs in combination with automated imaging and data analysis. Patient-derived glioblastoma cells were expanded and characterized using neural stem cell conditions. In culture, the cells showed low differentiation but expression of neural stem cell markers such as Nestin and Sox2. Upon intracranial injection into SCID mice these cells gave rise to tumors displaying the hallmarks of the human disease. Differentiation of glioblastoma-initiating cells (for example elicited through bone morphogenetic protein, BMP) was associated with strong morphological changes. Hence, cellular morphology, as well as markers specific for differentiation or death were used as screen readout. Lentiviral RNA interference-based screening yielded several gene knockdowns leading to ‘forced’ differentiation of glioblastoma-initiating cells. For example, knockdown of TRRAP (transformation/transcription domain associated protein) led to strongly increased differentiation and loss of proliferative and self-renewing capacity in these cells. TRRAP is an adapter protein implicated in oncogenic transformation through c-MYC transcription activation, also participating in chromatin remodeling and DNA repair. Glioblastoma-initiating cells with reduced TRRAP displayed increased apoptosis upon treatment with the genotoxic agent temozolomide. In vivo, Trapp knockdown cells were not able to give rise to glioblastoma upon transplantation into the brain of SCID mice. Together, these findings support a crucial role for TRRAP in maintenance and tumorigenicity of glioblastoma-initiating cells and might offer future therapeutic options. Two treatments compared to control: two different shRNA sequences for TRRAP were compared to a control shRNA sequence in their effects on global transcription in brain tumor initiating cells

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A functional genome-wide RNAi screen identifies TAF1 as a regulator for apoptosis in response to genotoxic stress.

Publications

A functional genome-wide RNAi screen identifies TAF1 as a regulator for apoptosis in response to genotoxic stress.

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