Immunoglobulin heavy chain (IgH) variable region exons are assembled from VH, D and JH gene segments in developing B lymphocytes. Within the 2.7 megabase (Mb) mouse IgH locus (IgH), V(D)J recombination is regulated to ensure specific and diverse antibody repertoires. Herein, we report a key IgH V(D)J recombination regulatory region, termed InterGenic Control Region-1 (IGCR1), that lies between the VH and D clusters. Functionally, IGCR1 employs CTCF looping/insulator factor binding elements and, correspondingly, mediates IgH loops containing distant enhancers. IGCR1 promotes normal B cell development and balances antibody repertoires by inhibiting transcription and rearrangement of DH-proximal VHs and promoting rearrangement of distal VHs. IGCR1 maintains ordered and lineage-specific VH(D)JH recombination, respectively, by suppressing VH joining to Ds not joined to JHs and VH to DJH joins in thymocytes. IGCR1 also is required to allow feedback regulation and allelic exclusion of proximal VH to DJH recombination. Our studies elucidate a long-sought IgH V(D)J recombination control region and implicate a new role for the generally expressed CTCF protein.

Classical non-homologous DNA end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Deficiencies for C-NHEJ factors, such as XRCC4, abrogate lymphocyte development, owing to a strict requirement for C-NHEJ to join V(D)J recombination DSB intermediates1,2. The XRCC4-like factor (XLF) is mutated in certain immunodeficient human patients and has been implicated in C-NHEJ3,4,5,6. Yet, XLF-deficient mice have relatively normal lymphocyte development and their lymphocytes support normal V(D)J recombination5. The Ataxia Telangiectasia-Mutated protein (“ATM”) detects DSBs and activates DSB responses by phosphorylating substrates including histone H2AX7. However, ATM-deficiency causes only modest V(D)J recombination and lymphocyte developmental defects, and H2AX-deficiency does not measurably impact these processes7,8,9. Here, we show that XLF, ATM, and H2AX all have fundamental roles in processing and joining ends during V(D)J recombination; but that these roles have been masked by unanticipated functional redundancies. Thus, combined ATM/XLF-deficiency nearly blocks mouse lymphocyte development due inability to process and join chromosomal V(D)J recombination DSB intermediates. Combined XLF and ATM deficiency also severely impairs C-NHEJ, but not alternative end-joining, during IgH class switch recombination. Redundant ATM and XLF functions in C-NHEJ are mediated via ATM kinase activity and are not required for extra-chromosomal V(D)J recombination, suggesting a role for chromatin-associated ATM substrates. Correspondingly, conditional H2AX inactivation in XLF-deficient pro-B lines leads to V(D)J recombination defects associated with marked degradation of unjoined V(D)J ends, revealing that H2AX indeed has a role in this process.

The classical nonhomologous DNA end-joining (C-NHEJ) doublestrand break (DSB) repair pathway in mammalian cells maintains genome stability and is required for V(D)J recombination and lymphocyte development. Mutations in the XLF C-NHEJ factor or ataxia telangiectasia-mutated (ATM) DSB response protein cause radiosensitivity and immunodeficiency in humans. Although potential roles for XLF in C-NHEJ are unknown, ATM activates a general DSB response by phosphorylating substrates, including histone H2AX and 53BP1, which are assembled into chromatin complexes around DSBs. In mice, C-NHEJ, V(D)J recombination, and lymphocyte development are, at most, modestly impaired in the absence of XLF or ATM, but are severely impaired in the absence of both. Redundant functions of XLF and ATM depend on ATM kinase activity; correspondingly, combined XLF and H2AX deficiency severely impairs V(D)J recombination, even though H2AX deficiency alone has little impact on this process. These and other findings suggest that XLF may provide functions that overlap more broadly with assembled DSB response factors on chromatin. As one test of this notion, we generated mice and cells with a combined deficiency for XLF and 53BP1. In this context, 53BP1 deficiency, although leading to genome instability, has only modest effects on V(D)J recombination or lymphocyte development. Strikingly, we find that combined XLF/53BP1 deficiency in mice severely impairs C-NHEJ, V(D)J recombination, and lymphocyte development while also leading to general genomic instability and growth defects. We conclude that XLF is functionally redundant with multiple members of the ATM-dependent DNA damage response in facilitating C-NHEJ and discuss implications of our findings for potential functions of these factors.ataxia telangiectasia-mutated | double-strand DNA break repair | NHEJ1 | Cernunnos I n mammalian cells, double-strand break (DSBs) can be generated extrinsically by ionizing radiation (IR) or by general physiologic factors such as oxidative stress, DNA replication, or transcription. Programmed DSBs are introduced into antigen receptor loci during V(D)J recombination in developing lymphocytes and during Ig heavy chain (IgH) class switch recombination (CSR) in activated mature B lymphocytes. There are two major DSB repair pathways in mammalian cells (1, 2). Homologous recombination (HR) accurately repairs DSBs in the S and G2 phases of the cell cycle; classical nonhomologous DNA end-joining (C-NHEJ) fuses broken DNA ends that lack or have limited homology throughout the cell cycle but predominantly in G1. There are multiple well-characterized C-NHEJ factors (2). The Ku70/Ku80 (Ku) complex recognizes DSBs, and the XRCC4/DNA Ligase IV (Lig4) complex ligates DSBs. DNA-dependent protein kinase, catalytic subunit (DNAPKcs) is activated by Ku at DSBs and, among other functions, activates Artemis, which processes DSBs. The precise role for the XLF (also called NHEJ1 or Cernunnos) C-NHEJ factor remains unknown; however, XLF recently was found to be functionally redundant ...