Publications (74)1096.86 Total impact
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Article: Multiple Transcription Factor Binding Sites Predict AID Targeting in Non-Ig Genes.
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ABSTRACT: Aberrant targeting of the enzyme activation-induced cytidine deaminase (AID) results in the accumulation of somatic mutations in ∼25% of expressed genes in germinal center B cells. Observations in Ung(-/-) Msh2(-/-) mice suggest that many other genes efficiently repair AID-induced lesions, so that up to 45% of genes may actually be targeted by AID. It is important to understand the mechanisms that recruit AID to certain genes, because this mistargeting represents an important risk for genome instability. We hypothesize that several mechanisms combine to target AID to each locus. To resolve which mechanisms affect AID targeting, we analyzed 7.3 Mb of sequence data, along with the regulatory context, from 83 genes in Ung(-/-) Msh2(-/-) mice to identify common properties of AID targets. This analysis identifies three transcription factor binding sites (E-box motifs, along with YY1 and C/EBP-β binding sites) that may work together to recruit AID. Based on previous knowledge and these newly discovered features, a classification tree model was built to predict genome-wide AID targeting. Using this predictive model, we were able to identify a set of 101 high-interest genes that are likely targets of AID.The Journal of Immunology 03/2013; · 5.79 Impact Factor -
Article: Higher-Order Looping and Nuclear Organization of Tcra Facilitate Targeted RAG Cleavage and Regulated Rearrangement in Recombination Centers.
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ABSTRACT: V(D)J recombination is essential for generating a diverse array of B and T cell receptors that can recognize and combat foreign antigens. As with any recombination event, tight control is essential to prevent the occurrence of genetic anomalies that drive cellular transformation. One important aspect of regulation is directed targeting of the RAG recombinase. Indeed, RAG accumulates at the 3' end of individual antigen receptor loci poised for rearrangement; however, it is not known whether focal binding is involved in regulating cleavage, and what mechanisms lead to enrichment of RAG in this region. Here, we show that monoallelic looping out of the 3' end of the T cell receptor α (Tcra) locus, coupled with transcription and increased chromatin/nuclear accessibility, is linked to focal RAG binding and ATM-mediated regulation of monoallelic cleavage on looped-out 3' regions. Our data identify higher-order loop formation as a key determinant of directed RAG targeting and the maintenance of genome stability.Cell reports. 02/2013; 3(2):359-70. -
Article: The Ataxia Telangiectasia mutated kinase controls Igκ allelic exclusion by inhibiting secondary Vκ-to-Jκ rearrangements.
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ABSTRACT: Allelic exclusion is enforced through the ability of antigen receptor chains expressed from one allele to signal feedback inhibition of V-to-(D)J recombination on the other allele. To achieve allelic exclusion by such means, only one allele can initiate V-to-(D)J recombination within the time required to signal feedback inhibition. DNA double-strand breaks (DSBs) induced by the RAG endonuclease during V(D)J recombination activate the Ataxia Telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) kinases. We demonstrate that ATM enforces Igκ allelic exclusion, and that RAG DSBs induced during Igκ recombination in primary pre-B cells signal through ATM, but not DNA-PK, to suppress initiation of additional Igκ rearrangements. ATM promotes high-density histone H2AX phosphorylation to create binding sites for MDC1, which functions with H2AX to amplify a subset of ATM-dependent signals. However, neither H2AX nor MDC1 is required for ATM to enforce Igκ allelic exclusion and suppress Igκ rearrangements. Upon activation in response to RAG Igκ cleavage, ATM signals down-regulation of Gadd45α with concomitant repression of the Gadd45α targets Rag1 and Rag2. Our data indicate that ATM kinases activated by RAG DSBs during Igκ recombination transduce transient H2AX/MDC1-independent signals that suppress initiation of further Igκ rearrangements to control Igκ allelic exclusion.Journal of Experimental Medicine 02/2013; · 13.85 Impact Factor -
Article: Cooperative recruitment of HMGB1 during V(D)J recombination through interactions with RAG1 and DNA.
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ABSTRACT: During V(D)J recombination, recombination activating gene (RAG)1 and RAG2 bind and cleave recombination signal sequences (RSSs), aided by the ubiquitous DNA-binding/-bending proteins high-mobility group box protein (HMGB)1 or HMGB2. HMGB1/2 play a critical, although poorly understood, role in vitro in the assembly of functional RAG-RSS complexes, into which HMGB1/2 stably incorporate. The mechanism of HMGB1/2 recruitment is unknown, although an interaction with RAG1 has been suggested. Here, we report data demonstrating only a weak HMGB1-RAG1 interaction in the absence of DNA in several assays, including fluorescence anisotropy experiments using a novel Alexa488-labeled HMGB1 protein. Addition of DNA to RAG1 and HMGB1 in fluorescence anisotropy experiments, however, results in a substantial increase in complex formation, indicating a synergistic binding effect. Pulldown experiments confirmed these results, as HMGB1 was recruited to a RAG1-DNA complex in a RAG1 concentration-dependent manner and, interestingly, without strict RSS sequence specificity. Our finding that HMGB1 binds more tightly to a RAG1-DNA complex over RAG1 or DNA alone provides an explanation for the stable integration of this typically transient architectural protein in the V(D)J recombinase complex throughout recombination. These findings also have implications for the order of events during RAG-DNA complex assembly and for the stabilization of sequence-specific and non-specific RAG1-DNA interactions.Nucleic Acids Research 01/2013; · 8.03 Impact Factor -
Article: Localized epigenetic changes induced by D(H) recombination restricts recombinase to DJ(H) junctions.
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ABSTRACT: Genes encoding immunoglobulin heavy chains (Igh) are assembled by rearrangement of variable (V(H)), diversity (D(H)) and joining (J(H)) gene segments. Three critical constraints govern V(H) recombination. These include timing (V(H) recombination follows D(H) recombination), precision (V(H) gene segments recombine only to DJ(H) junctions) and allele specificity (V(H) recombination is restricted to DJ(H)-recombined alleles). Here we provide a model for these universal features of V(H) recombination. Analyses of DJ(H)-recombined alleles showed that DJ(H) junctions were selectively epigenetically marked, became nuclease sensitive and bound RAG recombinase proteins, which thereby permitted D(H)-associated recombination signal sequences to initiate the second step of Igh gene assembly. We propose that V(H) recombination is precise, because these changes did not extend to germline D(H) segments located 5' of the DJ(H) junction.Nature Immunology 10/2012; · 26.01 Impact Factor -
Article: Identification of Core DNA Elements That Target Somatic Hypermutation.
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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.The Journal of Immunology 10/2012; · 5.79 Impact Factor -
Article: AID-targeting and hypermutation of non-immunoglobulin genes does not correlate with proximity to immunoglobulin genes in germinal center B cells.
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ABSTRACT: Upon activation, B cells divide, form a germinal center, and express the activation induced deaminase (AID), an enzyme that triggers somatic hypermutation of the variable regions of immunoglobulin (Ig) loci. Recent evidence indicates that at least 25% of expressed genes in germinal center B cells are mutated or deaminated by AID. One of the most deaminated genes, c-Myc, frequently appears as a translocation partner with the Ig heavy chain gene (Igh) in mouse plasmacytomas and human Burkitt's lymphomas. This indicates that the two genes or their double-strand break ends come into close proximity at a biologically relevant frequency. However, the proximity of c-Myc and Igh has never been measured in germinal center B cells, where many such translocations are thought to occur. We hypothesized that in germinal center B cells, not only is c-Myc near Igh, but other mutating non-Ig genes are deaminated by AID because they are near Ig genes, the primary targets of AID. We tested this "collateral damage" model using 3D-fluorescence in situ hybridization (3D-FISH) to measure the distance from non-Ig genes to Ig genes in germinal center B cells. We also made mice transgenic for human MYC and measured expression and mutation of the transgenes. We found that there is no correlation between proximity to Ig genes and levels of AID targeting or gene mutation, and that c-Myc was not closer to Igh than were other non-Ig genes. In addition, the human MYC transgenes did not accumulate mutations and were not deaminated by AID. We conclude that proximity to Ig loci is unlikely to be a major determinant of AID targeting or mutation of non-Ig genes, and that the MYC transgenes are either missing important regulatory elements that allow mutation or are unable to mutate because their new nuclear position is not conducive to AID deamination.PLoS ONE 01/2012; 7(6):e39601. · 4.09 Impact Factor -
Article: Recombination centres and the orchestration of V(D)J recombination.
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ABSTRACT: The initiation of V(D)J recombination by the recombination activating gene 1 (RAG1) and RAG2 proteins is carefully orchestrated to ensure that antigen receptor gene assembly occurs in the appropriate cell lineage and in the proper developmental order. Here we review recent advances in our understanding of how DNA binding and cleavage by the RAG proteins are regulated by the chromatin structure and architecture of antigen receptor genes. These advances suggest novel mechanisms for both the targeting and the mistargeting of V(D)J recombination, and have implications for how these events contribute to genome instability and lymphoid malignancy.Nature Reviews Immunology 03/2011; 11(4):251-63. · 32.25 Impact Factor -
Article: Uracil residues dependent on the deaminase AID in immunoglobulin gene variable and switch regions.
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ABSTRACT: Activation-induced deaminase (AID) initiates diversity of immunoglobulin genes through deamination of cytosine to uracil. Two opposing models have been proposed for the deamination of DNA or RNA by AID. Although most data support DNA deamination, there is no physical evidence of uracil residues in immunoglobulin genes. Here we demonstrate their presence by determining the sensitivity of DNA to digestion with uracil DNA glycosylase (UNG) and abasic endonuclease. Using several methods of detection, we identified uracil residues in the variable and switch regions. Uracil residues were generated within 24 h of B cell stimulation, were present on both DNA strands and were found to replace mainly cytosine bases. Our data provide direct evidence for the model that AID functions by deaminating cytosine residues in DNA.Nature Immunology 01/2011; 12(1):70-6. · 26.01 Impact Factor -
Article: A role for cohesin in T-cell-receptor rearrangement and thymocyte differentiation.
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ABSTRACT: 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. There is growing evidence that cohesin also forms long-range chromosomal cis-interactions and may regulate gene expression in association with CTCF, mediator or tissue-specific transcription factors. Human cohesinopathies such as Cornelia de Lange syndrome are thought to result from impaired non-canonical cohesin functions, but a clear distinction between the cell-division-related and cell-division-independent functions of cohesion--as exemplified in Drosophila--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) α 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 and Tcra rearrangement. Provision of pre-rearranged TCR transgenes largely rescued thymocyte differentiation, demonstrating that among thousands of potential target genes across the genome, 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.Nature 01/2011; 476(7361):467-71. · 36.28 Impact Factor -
Article: Uracil residues dependent on the deaminase AID in immunoglobulin gene variable and switch regions
Nature Immunology 12/2010; 12(1):70-76. · 26.01 Impact Factor -
Article: Promoters, enhancers, and transcription target RAG1 binding during V(D)J recombination.
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ABSTRACT: V(D)J recombination assembles antigen receptor genes in a well-defined order during lymphocyte development. This sequential process has long been understood in the context of the accessibility model, which states that V(D)J recombination is regulated by controlling the ability of the recombination machinery to gain access to its chromosomal substrates. Indeed, many features of "open" chromatin correlate with V(D)J recombination, and promoters and enhancers have been strongly implicated in creating a recombinase-accessible configuration in neighboring chromatin. An important prediction of the accessibility model is that cis-elements and transcription control binding of the recombination-activating gene 1 (RAG1) and RAG2 proteins to their DNA targets. However, this prediction has not been tested directly. In this study, we use mutant Tcra and Tcrb alleles to demonstrate that enhancers control RAG1 binding globally at Jα or Dβ/Jβ gene segments, that promoters and transcription direct RAG1 binding locally, and that RAG1 binding can be targeted in the absence of RAG2. These findings reveal important features of the genetic mechanisms that regulate RAG binding and provide a direct confirmation of the accessibility model.Journal of Experimental Medicine 12/2010; 207(13):2809-16. · 13.85 Impact Factor -
Article: V(D)J recombination: mechanisms of initiation.
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ABSTRACT: V(D)J recombination assembles immunoglobulin and T cell receptor genes during lymphocyte development through a series of carefully orchestrated DNA breakage and rejoining events. DNA cleavage requires a series of protein-DNA complexes containing the RAG1 and RAG2 proteins and recombination signals that flank the recombining gene segments. In this review, we discuss recent advances in our understanding of the function and domain organization of the RAG proteins, the composition and structure of RAG-DNA complexes, and the pathways that lead to the formation of these complexes. We also consider the functional significance of RAG-mediated histone recognition and ubiquitin ligase activities, and the role played by RAG in ensuring proper repair of DNA breaks made during V(D)J recombination. Finally, we propose a model for the formation of RAG-DNA complexes that involves anchoring of RAG1 at the recombination signal nonamer and RAG2-dependent surveillance of adjoining DNA for suitable spacer and heptamer sequences.Annual Review of Genetics 11/2010; 45:167-202. · 22.23 Impact Factor -
Article: Sin1-mTORC2 suppresses rag and il7r gene expression through Akt2 in B cells.
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ABSTRACT: Mammalian target of rapamycin (mTOR) is an important mediator of phosphoinositol-3-kinase (PI3K) signaling. PI3K signaling regulates B cell development, homeostasis, and immune responses. However, the function and molecular mechanism of mTOR-mediated PI3K signaling in B cells has not been fully elucidated. Here we show that Sin1, an essential component of mTOR complex 2 (mTORC2), regulates B cell development. Sin1 deficiency results in increased IL-7 receptor (il7r) and RAG recombinase (rag1 and rag2) gene expression, leading to enhanced pro-B cell survival and augmented V(D)J recombinase activity. We further show that Akt2 specifically mediates the Sin1-mTORC2 dependent suppression of il7r and rag gene expression in B cells by regulating FoxO1 phosphorylation. Finally, we demonstrate that the mTOR inhibitor rapamycin induces rag expression and promotes V(D)J recombination in B cells. Our study reveals that the Sin1/mTORC2-Akt2 signaling axis is a key regulator of FoxO1 transcriptional activity in B cells.Molecular cell 08/2010; 39(3):433-43. · 14.61 Impact Factor -
Article: The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci.
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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.Cell 04/2010; 141(3):419-31. · 32.40 Impact Factor -
Article: RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci.
Nature Immunology 04/2010; 11(4):356. · 26.01 Impact Factor -
Article: Imatinib resistance and progression of CML to blast crisis: somatic hypermutation AIDing the way.
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ABSTRACT: Very little is known about how acquired oncogenic mutations arise. In this issue of Cancer Cell, Klemm and colleagues present evidence supporting a role for the antibody diversification enzyme activation-induced deaminase (AID) in the generation of mutations associated with disease progression and drug resistance in chronic myeloid leukemia.Cancer cell 10/2009; 16(3):174-6. · 25.29 Impact Factor -
Article: Corrigendum: RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci
Nature Immunology 08/2009; 10(9):1034-1034. · 26.01 Impact Factor -
Article: RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci
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ABSTRACT: Coordinated recombination of homologous antigen receptor loci is thought to be important for allelic exclusion. Here we show that homologous immunoglobulin alleles pair in a stage-specific way that mirrors the recombination patterns of these loci. The frequency of homologous immunoglobulin pairing was much lower in the absence of the RAG-1–RAG-2 recombinase and was restored in Rag1−/− developing B cells with a transgene expressing a RAG-1 active-site mutant that supported DNA binding but not cleavage. The introduction of DNA breaks on one immunoglobulin allele induced ATM-dependent repositioning of the other allele to pericentromeric heterochromatin. ATM activated by the cleaved allele acts in trans on the uncleaved allele to prevent biallelic recombination and chromosome breaks or translocations.Nature Immunology 05/2009; 10(6):655-664. · 26.01 Impact Factor -
Article: Balancing AID and DNA repair during somatic hypermutation.
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ABSTRACT: Somatic hypermutation (SHM) of Ig genes in B cells is crucial for antibody affinity maturation. The reaction is initiated by cytosine deamination of Ig loci by activation induced deaminase (AID) and is completed by error-prone DNA repair enzyme processing of AID-generated uracils. The mechanisms that target SHM specifically to Ig loci are poorly understood. Recently, it has been demonstrated that although AID preferentially targets Ig loci, it acts surprisingly widely on non-Ig loci, many of which are protected from mutation accumulation by high-fidelity DNA repair. We propose that breakdown of this high fidelity repair process helps explain oncogene mutations observed in B-cell tumors, and further, that many oncogenes are vulnerable to AID-mediated DNA breaks and translocations in normal activated B cells.Trends in Immunology 05/2009; 30(4):173-81. · 10.40 Impact Factor
Top co-authors
Top Journals
Institutions
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2013
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New York University USA
- Department of Pathology
New York City, NY, USA
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2002–2013
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Howard Hughes Medical Institute
Chevy Chase, MD, USA -
The Rockefeller University
- Laboratory of Lymphocyte Biology
New York City, NY, USA
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2002–2012
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Yale University
- • Department of Immunobiology
- • Department of Molecular Biophysics and Biochemistry
- • Department of Genetics
- • Department of Orthopaedics and Rehabilitation
New Haven, CT, USA
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2009
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University of Michigan
- Department of Internal Medicine
Ann Arbor, MI, USA
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2003–2006
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Yale-New Haven Hospital
New Haven, CT, USA
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