title: The mouse immunoglobulin heavy chain V-D intergenic sequence contains insulators that may regulate ordered V(D)J recombination.
abstract: During immunoglobulin heavy chain (Igh) V(D)J recombination, D to J precedes V to DJ recombination in an ordered manner, controlled by differential chromatin accessibility of the V and DJ regions and essential for correct antibody assembly. However, with the exception of the intronic enhancer Emu, which regulates D to J recombination, cis-acting regulatory elements have not been identified. We have assembled the sequence of a strategically located 96-kb V-D intergenic region in the mouse Igh and analyzed its activity during lymphocyte development. We show that Emu-dependent D antisense transcription, proposed to open chromatin before D to J recombination, extends into the V-D region for more than 30 kb in B cells before, during, and after V(D)J recombination and in T cells but terminates 40 kb from the first V gene. Thus, subsequent V antisense transcription before V to DJ recombination is actively prevented and must be independently activated. To find cis-acting elements that regulate this differential chromatin opening, we identified six DNase I-hypersensitive sites (HSs) in the V-D region. One conserved HS upstream of the first D gene locally regulates D genes. Two further conserved HSs near the D region mark a sharp decrease in antisense transcription, and both HSs bind CTCF in vivo. Further, they both possess enhancer-blocking activity in vivo. Thus, we propose that they are enhancer-blocking insulators preventing Emu-dependent chromatin opening extending into the V region. Thus, they are the first elements identified that may control ordered V(D)J recombination and correct assembly of antibody genes.
authors.affiliation: Laboratory of Chromatin and Gene Expression, Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom.
authors.forename: Karen
authors.initials: K
authors.lastname: Featherstone
authors.affiliation: Laboratory of Chromatin and Gene Expression, Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Wood
authors.affiliation: Laboratory of Chromatin and Gene Expression, Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Bowen
authors.affiliation: Laboratory of Chromatin and Gene Expression, Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom. Electronic address: anne.corcoran@bbsrc.ac.uk.
authors.forename: Anne E
authors.initials: AE
authors.lastname: Corcoran
mesh_terms: D000483:Alleles; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002843:Chromatin / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D021901:DNA, Intergenic / Q000235:genetics; D015321:Gene Rearrangement; D050438:Genes, Immunoglobulin Heavy Chain; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D018448:Models, Immunological; D016376:Oligonucleotides, Antisense / Q000235:genetics; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D013601:T-Lymphocytes / Q000378:metabolism
chemical_list: D002843:Chromatin; D021901:DNA, Intergenic; D007143:Immunoglobulin Heavy Chains; D016376:Oligonucleotides, Antisense