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A method for isolating prosurvival targets of NF-kappaB/Rel transcription factors
Christian Kuntzen1, Francesca Zazzeroni, Can G Pham
1The Ben May Institute for Cancer Research, The University of Chicago, IL, USA.
Abstract:
NF-KappaB/Rel transcription factors are critical regulators of immunity, inflammation, development, and cell survival. Activation of NF-KB inhibits programmed cell death (PCD) triggered by tumor necrosis factor alpha (TNFalpha) and several other stimuli. The prosurvival activity of NF-KB is also crucial to lymphopoiesis, neuroprotection, tumorigenesis, and cancer chemoresistance. The characterization of the downstream targets that mediate the prosurvival activity of NF-KB is therefore a topic of intense investigation. Early screens aimed at identifying these genes were mainly based on expression criteria and so were poised to only isolate genes already known to have protective effects. Here, we describe a new method for the identification of these genes, whereby expression libraries are screened for their ability to halt PCD in NF-KB-deficient cells. This complementation approach provides substantial advantages over other approaches, as it enables functional assessment of isolated genes without any preconceived notion about their sequence or presumed role. Expression libraries are generated from cells that are resistant to TNFalpha-induced cytotoxicity and are then enriched in prosurvival genes upon selection with TNFa in NF-kappaB/RelA-null cells, which are highly susceptible instead to this cytotoxicity. Upon enrichment, libraries are screened through a randomized two-step approach, whereby cDNAs are first tested for cytoprotective function and then for differential expression in NF-kappaB-proficient and NF-KappaB-deficient cells.
Insights
Researchers developed a novel method to identify genes that promote cell survival by screening expression libraries in cells lacking NF-KappaB/Rel. This approach functionally assesses genes without prior assumptions, aiding cancer and immunity research.
Area of Science:
- Molecular Biology
- Cell Biology
- Immunology
Background:
- Nuclear factor kappa B (NF-κB)/Rel transcription factors are vital for immunity, inflammation, and cell survival.
- NF-κB activation inhibits programmed cell death (PCD) induced by tumor necrosis factor alpha (TNFα) and other factors.
- Understanding NF-κB's prosurvival targets is crucial for cancer chemoresistance and lymphopoiesis.
Purpose of the Study:
- To develop a new functional screening method for identifying NF-κB target genes.
- To overcome limitations of previous expression-based screens that favored known protective genes.
- To functionally assess cytoprotective genes without preconceived notions of their sequence or role.
Main Methods:
- A complementation approach using expression libraries from TNFα-resistant cells.
- Enrichment of prosurvival genes by selection with TNFα in NF-κB/RelA-null cells.
- A two-step screening process: first for cytoprotective function, then for differential expression.
Main Results:
- The new method successfully identified genes mediating prosurvival activity.
- This functional screening approach identified novel protective genes beyond those previously known.
- The method enables unbiased assessment of gene function in cell survival pathways.
Conclusions:
- The described complementation screening method is effective for identifying novel NF-κB-regulated prosurvival genes.
- This approach offers significant advantages over traditional expression-based screens.
- The findings contribute to understanding cell survival mechanisms in immunity and cancer.
