- Open Access
Signaling advances from immunogenetics to immunogenomics
© BioMed Central Ltd 2003
- Published: 19 November 2003
Recent studies describe new genome-wide mutagenesis strategies, coupled with phenotypic screening, and demonstrate the power of such approaches to provide new insights into the genetics of the immune response.
- Green Fluorescent Protein
- Mutant Mouse Strain
- Mutate Embryonic Stem Cell
- Carma1 Expression
A spate of recent articles in Nature, Journal of Biology, and Immunity [1–4] herald the ripened fruit of mutagenic and genomic approaches to dissecting the molecular mechanisms of immunity. Genetics has long been of central importance in elucidating the nature of immune responses, its roots extending at least as deeply as the establishment of systems for understanding histocompatibility and immune regulation. The 'pregenomic' era produced insights into autoimmune-disease susceptibility using the non-obese diabetic (NOD) strain of mice . Somatic-cell mutants unresponsive to interferons were generated and characterized, thereby leading understanding of Jak-STAT signaling pathways [6–8]. The TLR4 gene was identified as the basis for lipopolysaccharide (LPS) unresponsiveness in C3H/HeJ mice , and a mutated NF-κB-inducing kinase gene was found to be the cause of the alymphoid (aly) mutant mouse strain . Moving into the post-genomic era, these exciting precedents have inspired systematic attacks on the generation and analysis of mutant mouse strains to achieve a greater saturation of the gene pool and screening of these strains for immune system phenotypes. Papers exemplifying each of several mutational approaches establish that global mutagenic strategies are already paying dividends in new understanding and promise even greater yields in the future.
At least four systematic approaches are now in use, employing oncoretroviruses or chemical mutagenesis, in cell lines or mice. Because of the greater ease and lower costs of screening and selection strategies with mammalian cell lines rather than whole animals, cellular approaches have provided some of the earliest targets and success stories of genome-wide approaches [6, 7, 11, 12]. Evidence that Carma1/CARD11, a member of the MAGUK (signaling-associated scaffold) family of proteins, is essential for induction of NF-κB transcription factors by the T-cell receptor (TCR) and CD28, is one recent finding that exemplifies the success of such strategies for studying immune regulation . In this study, a powerful screening strategy was implemented in a widely used human T-cell line, Jurkat. A synthetic NF-κB-dependent promoter was used to govern the expression of green fluorescent protein (GFP) and chemically mutagenized cells were identified that were deficient in the induction of GFP in response to activation of the TCR but normal in terms of the response to stimulation by tumor necrosis factor α (TNFα). From a panel of such cells with a selective block to signaling, one variant was shown to be deficient in Carma1, a defect reversed by restoration of Carma1 expression. This mutation caused a loss of NF-κB induction as a result of defective relays between the cell surface and the transcription factor, and also diminished TCR-induced activation of the JNK but not the ERK subtype of mitogen activated protein (MAP) kinase .
Another triumph of functional screening at a genome-wide level has emerged from a separate program, resulting from similar ENU mutagenesis and recessive screening in the same C57BL/6 background [3, 4]. This large-scale program focussed on the identification of mutations leading to immune dysregulation . Founder mice were identified by a quantitative screen that detected differences in the profile of surface immunoglobulin M (sIgM) on circulating B lymphocytes that also carried IgD. In addition to a two-fold increase in sIgM, closer examination also revealed a modest increase in another molecule linked to signaling, CD21. Gene mapping localized the point mutation to the Carma1/CARD11 gene. Rather than a loss of expression, as found in the prior work in Jurkat cells (see above, ) and concomitant papers using gene targeting [24, 25], this ENU-induced point mutation led to normal levels of a full-length protein with altered function. Comparison of the consequences of this mutation to the outcome of gene-targeting experiments which eliminated Carma1 expression  is illuminating in terms of what each strategy can offer. The ENU mutant, which affects a coiled-coil region of the protein, led to a phenotype far more prominent in B lymphocytes than their T-cell counterparts, while the lack of Carma1 in the loss-of-expression studies led to a profound impairment of TCR/CD28-induced production of interleukin-2 (IL-2) and proliferation, along with B-cell signaling defects [13, 22, 23]. Conversely, the point-mutant Carma1 led to a partially penetrant hyper-IgE syndrome with other atopic manifestations in the mice, whereas this abnormality was not noted in the conventional knockouts. Thus, the random generation of a point mutant uncovered new features of the protein's function in vivo and some selectivity in the impact of these functional traits on particular lymphoid-cell types.
So, what are the global lessons from these advances, and what future refinements incorporating genome-wide approaches may be possible? The first lesson is that these approaches establish the ability of large, well-funded, and well-organized operations to generate new insights into immune regulation using functional-genomic screens. In addition, some of the findings will differ from those obtained using conventional knockouts. Subsequent to these initial 'test-of-principle' papers, the pace of discovery using this type of approach will accelerate. The second lesson is that although there undoubtedly is some selection bias, a strikingly high frequency of 'old friends' turn up in these screens. Thus far, most of the listed gene products have been previously identified and implicated in the relevant signaling pathways by conventional approaches. This suggests that intensive gene discovery by pregenomic techniques has been quite successful. Nonetheless, because a substantial fraction of predicted genes do not fall into known functional groups it is to be expected that the gene products uncovered will extend more into groups not previously linked to the functions being screened. Finally, the collected findings indicate that it is already timely to be generating systems-based strategies to exploit these advances. Approaches of this type will gain power as they are combined and linked to other forms of genomics [26–28], proteomics and systems analysis. Thus, for example, the tetracycline-regulated dominant retrovector system can be combined with transductions of hematopoietic cells from mutant mice and to microarrray analyses of gene-expression profiles. Such approaches may allow the identification of selective roles of protein modules and quantitation of the effects on function of a wide range of sequence variants generated in vitro. Similarly, when coupled to screens of protein-protein interactions in cell lines, genome-scale approaches will rapidly increase our insights into structure-function relationships in immunity and disease.
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