The process of somatic hypermutation of immunoglobulin (Ig) genes is one of the key aspects for creating a vast repertoire of antibodies. High-affinity antibodies generated in this process are required to protect us against pathogenic organisms. Recently, the mutator essential for the somatic hypermutation (SHM) was identified as Activation-Induced Cytidine Deaminase (AID), which converts deoxycytidine to deoxyuridine. Previous studies in the lab indicated that somatic hypermutation is dependent on transcription initiation, and the studies on AID also support this idea. While mistargeting of AID to non-Ig genes is thought to be associated with malignant transformation of B cells, the regulatory mechanism and the cis-elements responsible for AID induced somatic hypermutation in Ig genes is largely unknown. In this thesis study, a cis-element, CAGGTG, in the context of Ig enhancers was shown to be sufficient to target somatic hypermutation to a nearby transcribed gene. The effect is independent of enhanced transcription, chromatin acetylation, or overall target gene activity. The other cis-elements of Ig enhancers alone cannot attract the SHM machinery. The results show that the CAGGTG cis-element is likely required to attract AID to Ig targets. Taken together with other recent findings we postulate that AID targets all genes expressed in mutating B cells that are associated with CAGGTG motifs in the appropriate context. Ig genes are the most highly mutated genes presumably because of multiple CAGGTG motifs within the Ig genes and the presence of other co-operating elements in Ig enhancers.;Another regulatory mechanism governing the specific targeting of SHM is the distribution of mutations within Ig genes. In Ig genes, mutations occur about 100 nucleotides downstream of the transcription start and extend to 1-2kb, thereby targeting regions containing rearranged V(D)J gene segments and sparing constant regions. To understand whether uracils resulting from AID activity are faithfully repaired in the very 5' unmutated region, or whether AID does not access or does not act in the 5' region, the distribution of mutations was compared between uracil DNA glycosylase (Ung)-deficient and wild-type mice in endogenous Ig genes and in a hypermutable Ig transgene. If AID gains access to the 5' region which is unmutated in wild-type mice, one would expect an "AID footprint," namely transition mutations from C and G in Ung-deficient mice in the region normally devoid of SHM. The results indicate that the distribution of total mutations and transitions from C and G is indistinguishable in wild-type and Ung-deficient mice. To confirm the finding, Ig genes from mice deficient in both Ung and MSH6 were analyzed and the mutation distributions were also indistinguishable from the wild-type. Thus, AID does not gain access to or cannot act at the 5' region of Ig genes.
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