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The Sauer Lab

Research

1. Using Functional Genomics to Identify Novel Genes Involved in Lymphocyte Development and Function

In collaborations with the Genomics Institute of the Novartis Research Foundation (GNF) and with academic colleagues, we are combining various functional genomics approaches with traditional methods to identify and then functionally investigate novel genes with critical roles in lymphocyte development or function (Fig. 1)1.

functional genomics overview

Fig. 1. Approaches used in our lab to identify and then analyze novel genes involved in lymphocyte development and function. We take advantage of large scale functional genomics approaches to identify novel genes with important roles in the adaptive immune system, or novel functions for known genes in lymphocyte signaling. An advantage of this approach is that a functional validation of the genes in tissue culture cells or whole animals occurs at the beginning of the project, ensuring that the gene studied is important for the process of interest and allowing to formulate hypotheses about its in vivo function that can then be tested experimentally. The following thorough, hypothesis-driven functional analysis of selected genes relies on traditional approaches such as co-immunoprecipitations, immunoblots, in vitro biochemistry, microscopy or detailed analyses in immunological model systems to gain novel biological insights.

In one approach, we use gene expression profiling and proteomics analyses to identify genes whose expression indicates potential involvement in lymphocyte development or function. This approach is relatively simple and provides near 100% genome coverage in a short time frame. However, a disadvantage of this approach is that an interesting expression pattern does not necessarily indicate an important function of the gene.

Therefore, we complement the gene expression/proteomics approach with cell-based screens of arrayed cDNA or siRNA libraries. The advantage of this method is that genes are identified based on their functional involvement in the process of interest.

However, functional involvement in tissue culture cells, which are often transformed, does not always indicate functional importance in whole organisms. Therefore, we use reverse genetics (the generation of knockout, transgenic or knockin mice) to investigate the in vivo functions of selected candidate genes in detail. While time consuming, this approach is required to demonstrate that a gene identified in the broad genomic approaches is really important in vivo.

To directly identify in vivo modulators of lymphocyte development and function, we are participating in forward genetic mutagenesis screens for mice harboring chemically induced gene mutations which impair lymphocyte development or function. This approach has the advantage that the phenotype, for example a lack of T cells, immediately indicates that the mutated gene is important for lymphocyte development or function. No other presumptions are made about the gene. A major advantage of forward genetics is that it can lead to the identification of novel genes that have not been studied before, opening a new area of research. In other cases, the mutation, usually a point mutation, demonstrates an entirely novel function for an already known gene, or unveils an important novel insight about how the gene functions mechanistically. Our ENU induced mouse mutant Ms. T-less is an interesting example, as it unveiled novel functions for the enzyme Inositol (1,4,5)-trisphosphate 3-kinase B (ItpkB) and its soluble small molecule product IP4 in T and B cell development2-4.

Once we have identified high priority genes of interest, we combine focused genetic, biochemical, molecular biological, cell biological, pharmacological and immunological approaches to determine the in vivo functions of these genes and their precise mechanisms of action in detail (Fig. 1). This allows us to formulate novel hypotheses which then provide a basis for further, hypothesis-driven focused research to gain novel biological insights. One example for this approach is again Ms. T-less. Originally identified as a mouse lacking peripheral T cells2, detailed functional analyses lead us to identify a novel function for the ItpkB product IP4, a soluble inositolpolyphosphate, as a positive regulator of PH domain function and protein recruitment to cellular membranes3. This function is essential for thymocyte positive selection3.

Further Reading

  1. Huang, Y.H., Barouch-Bentov, R., Herman, A., Walker, J. & Sauer, K. Integrating traditional and postgenomic approaches to investigate lymphocyte development and function. Adv Exp Med Biol 584, 245-276 (2006).
  2. Wen, B.G. et al. Inositol (1,4,5) trisphosphate 3 kinase B controls positive selection of T cells and modulates Erk activity. Proc Natl Acad Sci U S A 101, 5604-5609 (2004).
  3. Huang, Y.H. et al. Positive Regulation of Itk PH Domain Function by Soluble IP4. Science 316, 886-889 (2007).
  4. Miller, A.T. et al. Production of Ins(1,3,4,5)P(4) mediated by the kinase Itpkb inhibits store-operated calcium channels and regulates B cell selection and activation. Nat Immunol 8, 514-521 (2007).