The regulation of immunoglobulin gene expression
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Antibody is diversified during B cell development to achieve high affinity to encountered antigens and multiple effecter functions. Somatic hypermutation (SHM), gene conversion (GC) and class switch recombination (CSR) are involved in diversification process. Activation induced cytidine deaminase (AID) is a B cell specific protein that initiates all three distinctive reactions by introducing cytosine to uracil mutation in immunoglobulin (Ig) genes. The mutagenic process is largely confined to specific regions of the Ig loci to prevent genomic instability. The targeting mechanism is unclear. To address this question, we used chicken B cell line DT40 as a working system to characterize the cis-acting elements and trans-activating factors involved in the control machinery. DT40 cells undergo gene conversion at the Ig loci. A major advantage of the system is that homologous recombination is efficient in this cell line. This property permits genetic analyses. Taking advantage of this property, we have made a series of deletions on the endogenous locus of Ig lambda (Igl) light chain gene. Based on the phenotypes of these mutations, we have defined the regions that control the expression of Igl. Moreover, these deletions revealed aberrant Igl transcripts. We are currently analyzing the effects of these mutations on gene conversion activity. In a complementary approach, we are knocking out the genes that may encode trans-acting factors for Igl gene expression and gene conversion. We have generated complete knockouts of interferon regulatory factor 4 (IRF4) and knocked out one allele of PU.1 gene. The targeted cell lines will be analyzed for Igl transcription and gene conversion.