Identification and characterization of a positive regulatory region for activation induced cytidine deaminase mediated gene conversion in chicken B cells

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Kim, Yonghwan, 1975-

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B cells have unique machinery to make up a large pool of antibody repertoire. After V(D)J recombination in early B cell development, the rearranged immunoglobulin genes are further diversified by somatic hypermutation (SHM), gene conversion (GC) and class switch recombination (CSR). Acitvation induced cytidine deaminase (AID) is a key initiating factor for SHM, GC and CSR. A majority of research data supports the model that AID modifies Ig genes at the DNA level by deaminating cytosines to uracils. The mutagenic activity of AID is largely restricted to Ig genes to avoid genomic instability in general. The specificity cannot be attributed to the primary sequence of the Ig genes since unrelated DNA is mutated by AID in the context of Ig genes. A clue to this problem is that AID function is dependent on transcription. Since not all transcribed genes are mutated by AID, there must be something special about the transcription of Ig genes, and the reasoning has prompted extensive analysis of Ig promoters and enhancers.
We addressed this question in chicken B cell line DT40. We identified a 2.4-kilobase regulatory region which is important for AID function both within and outside of Ig locus. This regulatory region contains binding sites for multiple transcription factors. Mutation of these binding sites impairs AID mediated gene conversion. In addition, ablation of NF-κB family member, c-Rel and p50, reduces the AID targeting function of this regulatory region. Since the implicated transcription factors have been reported to associate with histone acetylases, the regulatory region may function by facilitating the access of AID to target DNA. To test this hypothesis, we used the I-SceI endonuclease and dam methylase as probes for chromatin structure. We found that the regulatory region does not increase chromatin accessibility to these probes. In fact, the regulatory region appears to interfere with the cleavage of target DNA by I-SceI. Another possible role of the regulatory region could be direct recruitment of AID to Ig genes. To test this hypothesis, we utilized Dam identification method. Surprisingly, we found that the regulatory region facilitates AID targeting to the Igλ locus.




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