Copyright (c) 2013 University of Massachusetts Medical School All rights reserved. http://escholarship.umassmed.edu/maps_pubs Recent documents in Microbiology and Physiological Systems Publications and Presentations en-us Fri, 17 May 2013 11:30:54 PDT 3600 http://escholarship.umassmed.edu/maps_pubs/10 http://escholarship.umassmed.edu/maps_pubs/10 Thu, 21 Mar 2013 11:15:20 PDT Jeroen E. J. Guikema et al. Animals B-Lymphocytes Endonucleases Genes, Immunoglobulin Immunoglobulin Class Switching Mice Somatic Hypermutation, Immunoglobulin http://escholarship.umassmed.edu/maps_pubs/9 http://escholarship.umassmed.edu/maps_pubs/9 Thu, 21 Mar 2013 11:15:19 PDT Ig class switch recombination (CSR) occurs in activated mature B cells, and causes an exchange of the IgM isotype for IgG, IgE, or IgA isotypes, which increases the effectiveness of the humoral immune response. DNA ds breaks in recombining switch (S) regions, where CSR occurs, are required for recombination. Activation-induced cytidine deaminase initiates DNA ds break formation by deamination of cytosines in S regions. This reaction requires reactive oxygen species (ROS) intermediates, such as hydroxyl radicals. In this study we show that the ROS scavenger N-acetylcysteine inhibits CSR. We also demonstrate that IFN-gamma treatment, which is used to induce IgG2a switching, increases intracellular ROS levels, and activates p53 in switching B cells, and show that p53 inhibits IgG2a class switching through its antioxidant-regulating function. Finally, we show that p53 inhibits DNA breaks and mutations in S regions in B cells undergoing CSR, suggesting that p53 inhibits the activity of activation-induced cytidine deaminase.

]]> Jeroen E. J. Guikema et al. Animals Antioxidants Blotting, Western Cell Separation Cytidine Deaminase DNA Breaks, Double-Stranded Flow Cytometry Immunoglobulin Class Switching Immunoglobulin G Mice Mice, Inbred C57BL Mice, Knockout Reactive Oxygen Species Reverse Transcriptase Polymerase Chain Reaction Tumor Suppressor Protein p53 http://escholarship.umassmed.edu/maps_pubs/7 http://escholarship.umassmed.edu/maps_pubs/7 Thu, 21 Mar 2013 11:15:18 PDT After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). We asked if, during B cell activation, AID also induces DNA breaks at genes other than IgH genes. Using a nonbiased genome-wide approach, we have identified hundreds of reproducible, AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. Unlike AID-induced DSBs in Ig genes, genome-wide AID-dependent DSBs are not restricted to transcribed regions and frequently occur within repeated sequence elements, including CA repeats, non-CA tandem repeats, and SINEs.

]]> Ori Staszewski et al. Amino Acid Motifs Animals B-Lymphocytes Binding Sites Carrier Proteins Cytidine Deaminase *DNA Breaks, Double-Stranded Genes, myc Immunoglobulin Class Switching Lymphocyte Activation Mice Nuclear Proteins Repetitive Sequences, Nucleic Acid http://escholarship.umassmed.edu/maps_pubs/8 http://escholarship.umassmed.edu/maps_pubs/8 Thu, 21 Mar 2013 11:15:18 PDT Class switch recombination (CSR) is induced upon B cell activation and occurs within special DNA regions, termed switch (S) regions, which consist of tandem repeats of G-rich sequences. CSR occurs by introduction of double-strand breaks (DSBs) into each S region, and recombination by nonhomologous end-joining (NHEJ). The recombination event occurs during the G1 phase of the cell cycle in cells that are rapidly dividing. By examination of patients and mouse knock-out strains lacking various DNA-damage response factors and enzymes involved in DNA repair, much has been learned about which factors are important for CSR, how DSBs are introduced into S regions, and how the donor and acceptor S regions are then recombined. One of the approaches for analyzing the steps involved in CSR is to determine the nucleotide sequence of S-S junctions. Many of the DNA repair deficiencies alter the sequence of the recombination junctions, generally increasing the use of microhomologies, interpreted as a shift from classical (C)-NHEJ to alternative end-joining (A-EJ). However, it is clear that A-EJ, is not simply one pathway; rather, recombination is likely to occur using various subsets of end-joining factors, which will vary depending on the structure of the DSBs provided by the initial phases of CSR. Herein we review the results of analyses of S-S junctions, suggest minimal information required for these analyses, and attempt to integrate these results in order to increase our understanding of the complex process of CSR.

]]> Janet Stavnezer et al. Animals B-Lymphocytes *DNA Repair Humans *Immunoglobulin Class Switching Immunoglobulin Isotypes Mice http://escholarship.umassmed.edu/maps_pubs/6 http://escholarship.umassmed.edu/maps_pubs/6 Thu, 21 Mar 2013 11:15:17 PDT Activation-induced cytidine deaminase (AID) instigates mutations and DNA breaks in Ig genes that undergo somatic hypermutation and class switch recombination during B cell activation in response to immunization and infection. This review discusses how AID expression and activity are regulated, including recent discoveries of AID-interacting proteins that might recruit AID to Ig genes, and allow it to target both DNA strands. Also discussed is the accumulating evidence that AID binds to, mutates, and creates breaks at numerous non-Ig sites in the genome, which initiates cell transformation and malignancies.

]]> Janet Stavnezer Animals B-Lymphocytes Cytidine Deaminase DNA, Superhelical *Gene Expression Regulation, Enzymologic Humans Immunoglobulin Class Switching Lymphocyte Activation Mice RNA Stability Somatic Hypermutation, Immunoglobulin http://escholarship.umassmed.edu/maps_pubs/5 http://escholarship.umassmed.edu/maps_pubs/5 Thu, 21 Mar 2013 11:15:16 PDT Frances Lund et al. Animals B-Lymphocytes Humans *Lymphocyte Activation *Periodicals as Topic T-Lymphocytes http://escholarship.umassmed.edu/maps_pubs/4 http://escholarship.umassmed.edu/maps_pubs/4 Thu, 21 Mar 2013 11:15:15 PDT Activation-induced cytidine deaminase (AID) is induced in B cells during an immune response and is essential for both class-switch recombination (CSR) and somatic hypermutation of Ab genes. The C-terminal 10 aa of AID are required for CSR but not for somatic hypermutation, although their role in CSR is unknown. Using retroviral transduction into mouse splenic B cells, we show that the C terminus is not required for switch (S) region double-strand breaks (DSBs) and therefore functions downstream of DSBs. Using chromatin immunoprecipitation, we show that AID binds cooperatively with UNG and the mismatch repair proteins Msh2-Msh6 to Ig Smu and Sgamma3 regions, and this depends on the C terminus and the deaminase activity of AID. We also show that mismatch repair does not contribute to the efficiency of CSR in the absence of the AID C terminus. Although it has been demonstrated that both UNG and Msh2-Msh6 are important for introduction of S region DSBs, our data suggest that the ability of AID to recruit these proteins is important for DSB resolution, perhaps by directing the S region DSBs toward accurate and efficient CSR via nonhomologous end joining.

]]> Sanjay Ranjit et al. Animals B-Lymphocytes Cell Separation Chromatin Immunoprecipitation Cytidine Deaminase DNA-Binding Proteins Flow Cytometry Immunoglobulin Class Switching Immunoglobulin G Immunoglobulin Switch Region Mice Mice, Inbred C57BL Mice, Knockout MutS Homolog 2 Protein Protein Binding Reverse Transcriptase Polymerase Chain Reaction Uracil-DNA Glycosidase http://escholarship.umassmed.edu/maps_pubs/3 http://escholarship.umassmed.edu/maps_pubs/3 Thu, 21 Mar 2013 11:15:13 PDT During activation of B cells to undergo class switching, B cell metabolism is increased, and levels of reactive oxygen species (ROS) are increased. ROS can oxidize DNA bases resulting in substrates for the DNA glycosylases Ogg1 and Nth1. Ogg1 and Nth1 excise oxidized bases, and nick the resulting abasic sites, forming single-strand DNA breaks (SSBs) as intermediates during the repair process. In this study, we asked whether splenic B cells from mice deficient in these two enzymes would show altered class switching and decreased DNA breaks in comparison with wild-type mice. As the c-myc gene frequently recombines with the IgH S region in B cells induced to undergo class switching, we also analyzed the effect of deletion of these two glycosylases on DSBs in the c-myc gene. We did not detect a reduction in S region or c-myc DSBs or in class switching in splenic B cells from Ogg1- or Nth1-deficient mice or from mice deficient in both enzymes.

]]> Anna J. Ucher et al. Animals B-Lymphocytes Cell Proliferation Cells, Cultured DNA Breaks, Double-Stranded DNA Glycosylases Deoxyribonuclease (Pyrimidine Dimer) Gene Knockout Techniques Genes, myc *Immunoglobulin Class Switching Immunoglobulin Heavy Chains Mice Mice, Inbred C57BL Mice, Knockout Real-Time Polymerase Chain Reaction Recombination, Genetic Spleen Transcription, Genetic http://escholarship.umassmed.edu/maps_pubs/2 http://escholarship.umassmed.edu/maps_pubs/2 Thu, 21 Mar 2013 11:15:11 PDT Activation-induced cytidine deaminase (AID) initiates DNA double-strand breaks (DSBs) in the IgH gene (Igh) to stimulate isotype class switch recombination (CSR), and widespread breaks in non-Igh (off-target) loci throughout the genome. Because the DSBs that initiate class switching occur during the G(1) phase of the cell cycle, and are repaired via end joining, CSR is considered a predominantly G(1) reaction. By contrast, AID-induced non-Igh DSBs are repaired by homologous recombination. Although little is known about the connection between the cell cycle and either induction or resolution of AID-mediated non-Igh DSBs, their repair by homologous recombination implicates post-G(1) phases. Coordination of DNA breakage and repair during the cell cycle is critical to promote normal class switching and prevent genomic instability. To understand how AID-mediated events are regulated through the cell cycle, we have investigated G(1)-to-S control in AID-dependent genome-wide DSBs. We find that AID-mediated off-target DSBs, like those induced in the Igh locus, are generated during G(1). These data suggest that AID-mediated DSBs can evade G(1)/S checkpoint activation and persist beyond G(1), becoming resolved during S phase. Interestingly, DSB resolution during S phase can promote not only non-Igh break repair, but also Ig CSR. Our results reveal novel cell cycle dynamics in response to AID-initiated DSBs, and suggest that the regulation of the repair of these DSBs through the cell cycle may ensure proper class switching while preventing AID-induced genomic instability.

]]> Muneer G. Hasham et al. Animals B-Lymphocytes Cells, Cultured Cytidine Deaminase *DNA Breaks, Double-Stranded DNA Repair G1 Phase Immunoglobulin Class Switching Immunoglobulin Isotypes Mice Mice, Inbred C57BL Mice, Knockout S Phase http://escholarship.umassmed.edu/maps_pubs/1 http://escholarship.umassmed.edu/maps_pubs/1 Wed, 29 Feb 2012 10:55:48 PST Signaling mechanisms used by Haemophilus influenzae to adapt to conditions it encounters during stages of infection and pathogenesis are not well understood. The ArcAB two-component signal transduction system controls gene expression in response to respiratory conditions of growth and contributes to resistance to bactericidal effects of serum and to bloodstream infection by H. influenzae. We show that ArcA of nontypeable H. influenzae (NTHI) activates expression of a glycosyltransferase gene, lic2B. Structural comparison of the lipooligosaccharide (LOS) of a lic2B mutant to that of the wild-type strain NT127 revealed that lic2B is required for addition of a galactose residue to the LOS outer core. The lic2B gene was crucial for optimal survival of NTHI in a mouse model of bacteremia and for evasion of serum complement. The results demonstrate that ArcA, which controls cellular metabolism in response to environmental reduction and oxidation (redox) conditions, also coordinately controls genes that are critical for immune evasion, providing evidence that NTHI integrates redox signals to regulate specific countermeasures against host defense.

]]> Sandy M. S. Wong et al. Animals Bacterial Proteins Blotting, Western Cell Separation Complement System Proteins Flow Cytometry Gene Expression Gene Expression Profiling Gene Expression Regulation, Bacterial Haemophilus Infections Haemophilus influenzae Humans Immune Evasion Lipopolysaccharides Mice Oxidation-Reduction Reverse Transcriptase Polymerase Chain Reaction Signal Transduction