Signaling advances from immunogenetics to immunogenomics
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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.
KeywordsGreen Fluorescent Protein Ethylnitrosourea Mutant Mouse Strain Mutate Embryonic Stem Cell Carma1 Expression
A spate of recent articles in Nature, Journal of Biology, and Immunity [1, 2, 3, 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, 7, 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 .
Retroviral mutagenesis methods
Such a cell-based, dominant selection system offers a powerful tool for the identification of molecules that impact a signaling pathway, albeit at the cost of several drawbacks. Discovery will be limited to those molecules for which dominant negatives have an effect on the pathway, and there will be inherent pitfalls in the use of any cell line: for example, lipid-kinase signaling in most Jurkat T cells will be abnormal because of pre-existing loss-of-function mutations in the PTEN lipid phosphatase/tumor suppressor genes. Nonetheless, this or analogous retrovector systems can be used for the transduction of primary lymphocytes to perform further validation. Intriguingly, variations on this approach offer potentially great power in structure-function analyses with selected molecules. Single cDNAs could be subjected to high-density mutagenesis and, through the on-off capabilities of the approach, microarray analyses would allow measurement of the changes in global gene-expression profiles that result from graded interference with a given pathway.
The applicability of a finding to immune regulation ultimately depends on its validation in the immune response of animals. In principle, such retroviral technologies could also be applied to the transduction of hematopoietic cells (for example, lymphocytes or dendritic cells) derived from mouse lines in which loss-of-function mutations have been engineered by other approaches. This extension of the approach could allow high-throughput structure-function screening in primary cells independent of the need to achieve dominant-negative effects. In parallel, however, enhanced techniques for random mutagenesis, phenotypic screening, and gene identification have been important goals. To facilitate the process of cloning mutated genes after mutagenesis, another class of mutagen takes advantage of the fact that retrovirus insertion in the genome can be engineered so as to achieve loss-of-function mutations at high efficiencies (; reviewed in ). In particular, vectors based on oncoretroviruses are modified to encode a selectable trait whose expression depends on insertion into an active transcription unit. After bulk infection of pluripotent embryonic stem (ES) cells with high-titre retrovirions, individual clones can be micro-injected and transmitted to the germ line, so that progeny can be mated to homozygosity. As an example of the early success and potential of this strategy of retroviral mutagenesis, a novel link was identified between the mouse FUS gene, chromosomal stability, and B-lymphoid proliferation . This technology promises to provide further successes, which will be facilitated by the creation of banks of mutated ES cells for which a sequence tag identifying the mutated gene is publicly accessible. Two examples of ES-cell banks currently available are from a for-profit company, Lexicon Genetics , and a program funded by the National Heart Lung and Blood Institute and linked to BayGenomics .
Ethylnitrosourea mutagenesis screens
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, 27, 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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