ABSTRACT
Immunoglobulin (Ig) diversification occurs via somatic hypermutation (SHM) and class switch recombination (CSR), and is initiated by activation-induced deaminase (AID), which converts cytosine to uracil. Variable (V) region genes undergo SHM to create amino acid substitutions that produce antibodies with higher affinity for antigen. The conversion of cytosine to uracil in DNA promotes mutagenesis. Two distinct DNA repair mechanisms regulate uracil processing in Ig genes. The first involves base removal by the uracil DNA glycosylase (UNG), and the second detects uracil via the mismatch repair (MMR) complex. Methyl binding domain protein 4 (MBD4) is a uracil glycosylase and an intriguing candidate for involvement in somatic hypermutation because of its interaction with the MMR MutL homolog 1 (MLH1). We found that the DNA uracil glycosylase domain of MBD4 is highly conserved among mammals, birds, shark, and insects. Conservation of the human and chicken MBD4 uracil glycosylase domain structure is striking. Here we examined the function of MBD4 in chicken DT40 B cells which undergo constitutive SHM. We constructed structural variants of MBD4 DT40 cells using CRISPR/Cas9 genome editing. Disruption of the MBD4 uracil glycosylase catalytic region increased SHM frequency in IgM loss assays. We propose that MBD4 plays a role in SHM.
Subject(s)
Avian Proteins/immunology , Chickens/immunology , Endodeoxyribonucleases/immunology , Immunoglobulins/genetics , Somatic Hypermutation, Immunoglobulin , Uracil-DNA Glycosidase/immunology , Animals , Aphids/genetics , Aphids/immunology , B-Lymphocytes/immunology , Cell Line , Chickens/genetics , Fishes/genetics , Fishes/immunology , Humans , Immunoglobulin Class Switching , Immunoglobulins/immunology , Mice , Platypus/genetics , Platypus/immunology , Protein Domains , Sharks/genetics , Sharks/immunologyABSTRACT
IL-1 is a key cytokine known to drive chronic inflammation and to regulate many physiological, immunological, and neuroimmunological responses via actions on diverse cell types of the body. To determine the mechanisms of IL-1 actions as part of the inflammatory response in vivo, we generated a conditional IL-1 receptor 1 (IL-1R1) mouse mutant using the Cre/LoxP system (IL-1R1(fl/fl) ). In the mutant generated, exon 5, which encodes part of the extracellular-binding region of the receptor, is flanked by LoxP sites, thereby inactivating the two previously described functional IL-1R1 gene transcripts after Cre-mediated recombination. Using keratin 14-Cre driver mice, new IL-1R1 deficient (-/-) mice were subsequently generated, in which all signaling IL-1 receptor isoforms are deleted ubiquitously. Furthermore, using vav-iCre driver mice, we deleted IL-1 receptor isoforms in the hematopoietic system. In these mice, we show that both the IL-17 and IL-22 cytokine response is reduced, when mice are challenged by the helminth Trichuris muris. We are currently crossing IL-1R1(fl/fl) mice with different Cre-expressing mice in order to study mechanisms of acute and chronic inflammatory diseases.
