title: A critical context-dependent role for E boxes in the targeting of somatic hypermutation.
abstract: Secondary B cell repertoire diversification occurs by somatic hypermutation (SHM) in germinal centers following Ag stimulation. In SHM, activation-induced cytidine deaminase mutates the V region of the Ig genes to increase the affinity of Abs. Although SHM acts primarily at Ig loci, low levels of off-target mutation can result in oncogenic DNA damage, illustrating the importance of understanding SHM targeting mechanisms. A candidate targeting motif is the E box, a short DNA sequence (CANNTG) found abundantly in the genome and in many SHM target genes. Using a reporter assay in chicken DT40 B cells, we previously identified a 1928-bp portion of the chicken IgL locus capable of supporting robust SHM. In this article, we demonstrate that mutation of all 20 E boxes in this fragment reduces SHM targeting activity by 90%, and that mutation of subsets of E boxes reveals a functional hierarchy in which E boxes within "core" targeting regions are of greatest importance. Strikingly, when the sequence and spacing of the 20 E boxes are preserved but surrounding sequences are altered, SHM targeting activity is eliminated. Hence, although E boxes are vital SHM targeting elements, their function is completely dependent on their surrounding sequence context. These results suggest an intimate cooperation between E boxes and other sequence motifs in SHM targeting to Ig loci and perhaps also in restricting mistargeting to certain non-Ig loci.
authors.affiliation: Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Jessica J
authors.initials: JJ
authors.lastname: McDonald
authors.forename: Jukka
authors.initials: J
authors.lastname: Alinikula
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Buerstedde
authors.forename: David G
authors.initials: DG
authors.lastname: Schatz
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D001665:Binding Sites; D002478:Cells, Cultured; D002645:Chickens; D003564:Cytidine Deaminase / Q000502:physiology; D004274:DNA, Recombinant / Q000235:genetics; D024721:E-Box Elements / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D050439:Genes, Immunoglobulin Light Chain / Q000235:genetics; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D009154:Mutation; D011485:Protein Binding; D027041:Somatic Hypermutation, Immunoglobulin / Q000235:genetics*; D058127:Transcription Factor 3 / Q000378:metabolism; D014162:Transfection; D019076:Transgenes; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D004274:DNA, Recombinant; D007135:Immunoglobulin Variable Region; D058127:Transcription Factor 3; D049452:Green Fluorescent Proteins; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase