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Dr. Giallourakis's research focuses on genetic factors of diseases.
Genetic Analysis of VDJ Recombination
The generation of the B cell receptor gene repertoire is accomplished in a lineage and developmentally regulated manner through the ordered assembly of variable (V), diversity (D) and joining (J) gene segments which are dispersed over the 2.8Mb IgH locus leading to mono-allelic expression of antigen-receptor. The lymphocyte-specific recombination activating gene (RAG) 1 and 2 proteins catalyze the initiation of the VDJ recombination by recognizing and cutting short, conserved recombination signal sequences that flank germline V, D, and J segments followed by the processing and re-ligation of the DMA ends by repair factors of the non-homologous end-joining machinery. Multiple lines of evidence support an accessibility framework whereby the chromosomal structure and chromosomal positioning of immunoglobulin loci undergo lineage and developmental alterations, to allow recombination and enforce allelic exclusion. Yet, the sheer number of VH genes and their chromosomal span has hampered genetic evaluation of certain aspects of accessibility. This proposal aims to evaluate a the role of cis-acting elements in controlling VDJ recombination via the characterization of mutant IgH loci and the creation of simplified endogenous loci towards a long-term effort to dissect the minimal set of cis-acting elements that regulate recombination. Specifically, is there a role for the intergenic region between V and D gene segments in recombination? This will be addressed by analysis of the effect of germ-line deletion of this sequence. Second, can simplified endogenous loci recapitulate critical aspects recombination? This question is being addressed by creation and characterization of mice with only limited numbers of V, D and J segments and monitoring efficiency of recombination, ordered rearrangement, and allelic exclusion. Third, what is the function of germline-sense and anti-sense transcription in controlling accessibility? This will be addressed by using minimal loci to delete sense promoters and further characterize the nature of antisense including mapping of transcriptional start sites. Understanding the cis-regulatory elements in VDJ recombination will enhance our understanding of lymphoid development and the control of gene expression in general.
Integrative Genomic Analysis of Transcriptional Regulation of B cell Development
In collaboration with the laboratory of Ramnik Xavier, MD, I am seeking to elucidate novel regulators of B cell development with regard to NF-kb signal transduction. The NF-kb signal transduction system is critical for B cell development and function. By combining genome-wide analysis of millions of Expressed Sequences Tags (EST) with microarray expression profiling of the repertoire of approximately 1600 potential genes encoding transcriptional regulators, we have identified novel immune specific transcriptional regulators. Furthermore, several of these transcriptional regulators, which are expressed specifically in immune cells and stem cells, are able to activate NF-kb in reporter assays, suggesting that such proteins may function as co-activators in B cell development or responses. In this regard, knockouts these transcriptional factors will help elucidate the role of these factors in vivo.
Integrative Genomic Analysis of PDZ Complexes in Normal GI Development and Malignancy
PDZ domain-containing proteins and their interaction partners are mutated in numerous human diseases and function in complexes regulating epithelial polarity, ion channels, cochlear hair cell development, vesicular sorting, and neuronal synaptic communication. Among several properties of a collection of documented PDZ domain-ligand interactions, we discovered embedded in a large-scale expression data set the existence of a significant level of co-regulation between PDZ domain-encoding genes and these ligands. From this observation, we show how integration of expression data, a comparative genomics catalog of 899 mammalian genes with conserved PDZ-binding motifs, phylogenetic analysis, and literature mining can be utilized to infer PDZ complexes. Using molecular studies we have mapped novel interaction partners for the PDZ proteins DLG1 and CARD11. These results provide insight into the diverse roles of PDZ-ligand complexes in cellular signaling and provide a computational framework for the genome-wide evaluation of PDZ complexes. We are currently extending these initial observations in a variety of directions using genomic approaches. In collaboration with Ramnik Xavier, MD and Randy Hall, PhD, we are using PDZ protein arrays to create a PDZ domain interaction map, identifying and confirming the specific binding partners for numerous potential novel PDZ ligands. The functional consequences of these novel PDZ-ligand interactions is being evaluated in several systems including the establishment of polarity in normal colonic epithelium and in the colorectal CA. Furthermore, genome-wide analysis of PDZ complexes will be evaluated using siRNA approaches to assay for defects in polarity and cell migration. Lastly, further genomic analysis suggests that PDZ complexes may provide a platform for discovery of markers for stem cell populations in the GI tract.
Selected Peer-reviewed Publications
1. Giallourakis C , Cao Z, Green T, Wachtel H, Xie X, Lopez-Illasaca M, Daly M,Rioux J, Xavier R. A molecular-properties-based approach to understanding PDZ domain proteins and PDZ ligands. Genome Research . 2006 Aug;16(8):1056-72. 2. Ishiguro K, Green T, Rapley J, Wachtel H, Giallourakis C , Landry A, Cao Z,Lu N, Takafumi A, Goto H, Daly MJ, Xavier RJ. Ca2+/calmodulin-dependent protein kinase II is a modulator of CARMA1-mediated NF-kappaB activation. Mol Cell Biol. 2006 Jul;26(14):5497-508. 3. Tello-Ruiz MK, Curley C, DelMonte T, Giallourakis C , Kirby A, Miller K, Wild G, Cohen A, Langelier D, Latiano A, Wedemeyer N, Lander E, Schreiber S, Annese V, Daly MJ, Rioux JD. Haplotype-based association analysis of 56 functional candidate genes in the IBD6 locus on chromosome 19. Eur J Hum Genet . 2006 Jun;14(6):780-90. 4. Raby B, Hwang E, Van-Steen K, Tansisara K, Litonjua A, Lazarus R, Giallourakis C , Rioux J, Silverman E, Glimcher L, Weiss S. Tbet polymorphisms are associated with asthma and airway hyperresponsiveness. American J Respir Crit Care Med. 2005 Sep 22 5. Giallourakis C , Stoll M, Miller K, Hampe J, Lander ES, Daly MJ, Schreiber S, Rioux JD. IBD5 is a general risk factor for inflammatory bowel disease: replication of association with Crohn disease and identification of a novel association with ulcerative colitis. Am J Hum Genet . 2003 Jul;73(1):205-11. 6. Giallourakis C , Kashiwada M, Pan PY, Danial N, Jiang H, Cambier J, Coggeshall KM, Rothman P Positive regulation of interleukin-4-mediated proliferation by the SH2-containing inositol-5'-phosphatase. J Biol Chem . 2000 Sep 22;275(38):29275-82.
7. Kashiwada M, Giallourakis CC, Pan PY, Rothman PB. Immunoreceptor tyrosine-based inhibitory motif of the IL-4 receptor associates with SH2-containing phosphatases and regulates IL-4-induced proliferation. J Immunol . 2001 Dec 1;167(11):6382-7.
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