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A cis-Acting Diversification Activator Both Necessary and Sufficient for AID-Mediated Hypermutation

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Author Summary It remains an open question how AID-mediated gene diversification is targeted to the immunoglobulin loci. Here we define a cis-acting sequence, named DIVAC for diversification activator, which is required for hypermutation of the Ig light chain gene and sufficient to activate hypermutation at various non-Ig loci in the DT40 B cell line. DIVAC is composed of multiple interacting sequences and able to work over considerable distances both upstream and downstream of its target gene. This work provides the first conclusive evidence that AID-mediated gene diversification is targeted to the Ig loci by cis-acting sequences. The conservation of AID-mediated Ig gene diversification during vertebrate evolution suggests that DIVACs also play a role in gene conversion, hypermutation, and switch recombination in mammalian B cells. The findings should be of general interest not only for molecular immunology and the pathogenesis of B cell lymphomas but also the whole field of biology as a unique example of how locus-specific gene diversification is controlled. The described experimental system offers unique advantages to further clarify the molecular mechanism of DIVAC.
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... To uncover the targets of TET3-mediated DNA demethylation, we performed bisulfite sequencing at several candidate loci in WT and TET3-KO cells (Figure 4b-e). In this study, we focused on 5 loci including 3 pseudogenes (ψ7, ψ8 and ψ10, which were employed in GCV either of WT or TET3-KO cells as explained in the previous section), the IgVλ segment and the core region of the DIVAC (diversification activator) element, downstream of IgVλ required for the enhancement of SHM and GCV (Blagodatski et al., 2009;Kohler et al., 2012;Kothapalli et al., 2008). ...
... Previous studies demonstrated that cis-acting DIVAC elements are needed for AID targeting to Ig variable regions (Blagodatski et al., 2009;Kohler et al., 2012). The DIVAC elements reside in the region spanning from the transcription start site to 9.8kb downstream of DT40 IgVλ. ...
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... Further, the crucial role of Ig enhancers in AID targeting has been well documented in Ig transgene studies, where deletion analysis of intronic or 3 enhancers revealed substantial decrease or complete elimination of SHM [63]. Mutation analysis studies of endogenous IgV region sequences associates the mutation frequency in SHM with the recurrence of AID hotspots [64], signifying that these specific nucleotide patterns such as WRC/GYW are important for recruiting AID to its individual target. ...
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