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Patients with common variable immunodeficiency associated with autoimmune cytopenia (CVID+AIC) generate few isotypeswitched B cells with severely decreased frequencies of somatic hypermutations (SHMs), but their underlying molecular defects remain poorly characterized. We identified a CVID+AIC patient who displays a rare homozygous missense M466V mutation in [beta]-catenin-like protein 1 (CTNNBL1). Because CTNNBL1 binds activation-induced cytidine deaminase (AID) that catalyzes SHM, we tested AID interactions with the CTNNBL1 M466V variant. We found that the M466V mutation interfered with the association of CTNNBL1 with AID, resulting in decreased AID in the nuclei of patient EBV-transformed B cell lines and of CTNNBL1 [466.sup.V/V] Ramos B cells engineered to express only CTNNBL1 M466V using CRISPR/Cas9 technology. As a consequence, the scarce IgG+ memory B cells from the CTNNBL1 [466.sup.V/V] patient showed a low SHM frequency that averaged 6.7 mutations compared with about 18 mutations per clone in healthy-donor counterparts. In addition, CTNNBL1 [466.sup.V/V] Ramos B cells displayed a decreased incidence of SHM that was reduced by half compared with parental WT Ramos B cells, demonstrating that the CTNNBL1 M466V mutation is responsible for defective SHM induction. We conclude that CTNNBL1 plays an important role in regulating AID-dependent antibody diversification in humans., Introduction Isotype-switched high-affinity antibodies are essential for protection against a vast range of pathogens. Activation-induced cytidine deaminase (AID) is the enzyme that mediates somatic hypermutation (SHM) and class-switch recombination (CSR), [...]

Transposons have had a pivotal role in genome evolution.sup.1 and are believed to be the evolutionary progenitors of the RAG1-RAG2 recombinase.sup.2, an essential component of the adaptive immune system in jawed vertebrates.sup.3. Here we report one crystal structure and five cryo-electron microscopy structures of Transib.sup.4,5, a RAG1-like transposase from Helicoverpa zea, that capture the entire transposition process from the apo enzyme to the terminal strand transfer complex with transposon ends covalently joined to target DNA, at resolutions of 3.0-4.6 Å. These structures reveal a butterfly-shaped complex that undergoes two cycles of marked conformational changes in which the 'wings' of the transposase unfurl to bind substrate DNA, close to execute cleavage, open to release the flanking DNA and close again to capture and attack target DNA. Transib possesses unique structural elements that compensate for the absence of a RAG2 partner, including a loop that interacts with the transposition target site and an accordion-like C-terminal tail that elongates and contracts to help to control the opening and closing of the enzyme and assembly of the active site. Our findings reveal the detailed reaction pathway of a eukaryotic cut-and-paste transposase and illuminate some of the earliest steps in the evolution of the RAG recombinase. Analysis of multiple structures of the Helicoverpa zea DNA transposase Transib, determined by X-ray crystallography and cryo-electron microscopy, reveals the detailed pathway of the transposition reaction and sheds light on the evolution of the RAG recombinase., Author(s): Chang Liu [sup.1] , Yang Yang [sup.2] [sup.3] , David G. Schatz [sup.1] Author Affiliations: (1) Department of Immunobiology, Yale School of Medicine, New Haven, USA (2) Department of [...]

Cohesin enables post-replicative DNA repair and chromosome segregation by holding sister chromatids together from the time of DNA replication in S phase until mitosis (1). There is growing evidence that cohesin also forms long-range chromosomal cis-interactions (2-4) and may regulate gene expression (2-10) in association with CTCF (8,9), mediator (4) or tissue-specific transcription factors (10). Human cohesin opathies such as Cornelia de Lange syndrome are thought to result from impaired non-canonical cohesin functions (7), but a clear distinction between the cell-division-related and cell-division-independent functions of cohesion--as exemplified in Drosophila (11-13)--has not been demonstrated in vertebrate systems. To address this, here we deleted the cohesin locus Rad21 in mouse thymocytes at a time in development when these cells stop cycling and rearrange their T-cell receptor (TCR) a locus (Tcra). Rad21-deficient thymocytes had a normal lifespan and retained the ability to differentiate, albeit with reduced efficiency. Loss of Rad21 led to defective chromatin architecture at the Tcra locus, where cohesion-binding sites flank the TEA promoter and the Eα enhancer, and demarcate Tcra from interspersed Tcrd elements and neighbouring housekeeping genes. Cohesin was required for long-range promoter-enhancer interactions, Tcra transcription, H3K4me3 histone modifications that recruit the recombination machinery (14,15) and Tcra rearrangement. Provision of pre-rearranged TCR transgenes largely rescued thymocyte differentiation, demonstrating that among thousands of potential target genes across the genome (4,8-10), defective Tcra rearrangement was limiting for the differentiation of cohesin-deficient thymocytes. These findings firmly establish a cell-division-independent role for cohesin in Tcra locus rearrangement and provide a comprehensive account of the mechanisms by which cohesin enables cellular differentiation in a well-characterized mammalian system., The somatic rearrangement of lymphocyte receptor loci is central to adaptive immunity16. Gene segments distributed over millions of base pairs of genomic DNA are transcribed, brought into proximity with each [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2010.03.010 Byline: Yanhong Ji (1), Wolfgang Resch (2), Elizabeth Corbett (1)(4), Arito Yamane (2), Rafael Casellas (2)(3), David G. Schatz (1)(4) Keywords: MOLIMMUNO; DNA; PROTEINS Abstract: The critical initial step in V(D)J recombination, binding of RAG1 and RAG2 to recombination signal sequences flanking antigen receptor V, D, and J gene segments, has not previously been characterized in vivo. Here, we demonstrate that RAG protein binding occurs in a highly focal manner to a small region of active chromatin encompassing Ig[kappa] and Tcr[alpha] J gene segments and Igh and Tcr[beta] J and J-proximal D gene segments. Formation of these small RAG-bound regions, which we refer to as recombination centers, occurs in a developmental stage- and lineage-specific manner. Each RAG protein is independently capable of specific binding within recombination centers. While RAG1 binding was detected only at regions containing recombination signal sequences, RAG2 binds at thousands of sites in the genome containing histone 3 trimethylated at lysine 4. We propose that recombination centers coordinate V(D)J recombination by providing discrete sites within which gene segments are captured for recombination. Author Affiliation: (1) Department of Immunobiology, Yale University School of Medicine, 300 Cedar Street, Box 208011, New Haven, CT 06520-8011, USA (2) Genomics and Immunity, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA (3) Center of Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA (4) Howard Hughes Medical Institute Article History: Received 15 June 2009; Revised 12 February 2010; Accepted 3 March 2010 Article Note: (miscellaneous) Published online: April 15, 2010