title: Identification of core DNA elements that target somatic hypermutation.
abstract: Somatic hypermutation (SHM) diversifies the V region of Ig genes and underlies the process of affinity maturation, in which B lymphocytes producing high-affinity Abs are generated and selected. SHM is triggered in activated B cells by deamination of deoxycytosine residues mediated by activation-induced deaminase (AID). Whereas mistargeting of SHM and AID results in mutations and DNA damage in many non-Ig genes, they act preferentially at Ig loci. The mechanisms responsible for preferential targeting of SHM and AID activity to Ig loci are poorly understood. Using an assay involving an SHM reporter cassette inserted into the Ig L chain locus (IgL) of chicken DT40 B cells, we have identified a 1.9-kb DIVAC (diversification activator) element derived from chicken IgL that supports high levels of AID-dependent mutation activity. Systematic deletion analysis reveals that targeting activity is spread throughout much of the sequence and identifies two core regions that are particularly critical for function: a 200-bp region within the IgL enhancer, and a 350-bp 3' element. Chromatin immunoprecipitation experiments demonstrate that whereas DIVAC does not alter levels of several epigenetic marks in the mutation cassette, it does increase levels of serine-5 phosphorylated RNA polymerase II in the mutation target region, consistent with an effect on transcriptional elongation/pausing. We propose that multiple, dispersed DNA elements collaborate to recruit and activate the mutational machinery at Ig gene variable regions during SHM.
authors.affiliation: Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Kohler
authors.forename: Jessica J
authors.initials: JJ
authors.lastname: McDonald
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Duke
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Arakawa
authors.forename: Sally
authors.initials: S
authors.lastname: Tan
authors.forename: Steven H
authors.initials: SH
authors.lastname: Kleinstein
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Buerstedde
authors.forename: David G
authors.initials: DG
authors.lastname: Schatz
mesh_terms: D024509:3' Flanking Region; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D002478:Cells, Cultured; D002645:Chickens; D047369:Chromatin Immunoprecipitation; D003564:Cytidine Deaminase / Q000235:genetics / Q000276:immunology; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D004742:Enhancer Elements, Genetic; D005803:Genes, Immunoglobulin / Q000276:immunology; D056426:Genetic Loci; D007118:Immunoassay; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D009154:Mutation; D010766:Phosphorylation; D012319:RNA Polymerase II / Q000235:genetics / Q000276:immunology; D012694:Serine / Q000378:metabolism; D027041:Somatic Hypermutation, Immunoglobulin / Q000235:genetics* / Q000276:immunology; D014158:Transcription, Genetic / Q000276:immunology; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D007135:Immunoglobulin Variable Region; D012694:Serine; D004247:DNA; D012319:RNA Polymerase II; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase