ABSTRACT
The mouse Igh locus is organized into a developmentally regulated topologically associated domain (TAD) that is divided into subTADs. Here we identify a series of distal VH enhancers (EVHs) that collaborate to configure the locus. EVHs engage in a network of long-range interactions that interconnect the subTADs and the recombination center at the DHJH gene cluster. Deletion of EVH1 reduces V gene rearrangement in its vicinity and alters discrete chromatin loops and higher order locus conformation. Reduction in the rearrangement of the VH11 gene used in anti-PtC responses is a likely cause of the observed reduced splenic B1 B cell compartment. EVH1 appears to block long-range loop extrusion that in turn contributes to locus contraction and determines the proximity of distant VH genes to the recombination center. EVH1 is a critical architectural and regulatory element that coordinates chromatin conformational states that favor V(D)J rearrangement.
Subject(s)
B-Lymphocytes , Immunoglobulin Heavy Chains , Regulatory Sequences, Nucleic Acid , Animals , Mice , Chromatin , Chromosome Aberrations , Receptors, Antigen , Immunoglobulin Heavy Chains/geneticsABSTRACT
Class-switch recombination (CSR) involves replacement of the Cµ constant region with another downstream CH region. CSR is initiated by activation-induced cytidine deaminase (AID)-mediated DNA breaks that are targeted to transcriptionally active switch (S) regions. S region promoters (Prs) direct synapsis by associating with the Eµ and 3'Eα enhancers that jointly anchor a chromatin loop. We report that asymmetric loop extrusion allows 3'Eα to track along the locus and form Pr-Pr-E interactions that mediate CSR between downstream S regions, followed by switching to donor Sµ. This alternative pathway bypasses sequential switching and creates immunoglobulin (Ig)E+ B cells in the absence of IgG1 expression. Based on the analysis of diagnostic CSR products in B cell subsets, we identify a BCR-negative cell intermediate that is pivotal to efficient CSR.
Subject(s)
Immunoglobulin Class Switching/genetics , Immunoglobulin Class Switching/physiology , Animals , B-Lymphocytes/immunology , Chromatin/genetics , Chromosome Pairing/genetics , Cytidine Deaminase/genetics , DNA Breaks , Immunoglobulin Class Switching/immunology , Immunoglobulin E/metabolism , Immunoglobulin Heavy Chains/genetics , Lymphocyte Activation/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Promoter Regions, Genetic/genetics , Recombination, Genetic/genetics , Transcription, Genetic/geneticsABSTRACT
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
BACKGROUND: Aedes aegypti and Dermatophagoides pteronyssinus contain important allergens including cross-reactive tropomyosins. However, the functional and clinical relevance of their cross-reactivity is still debated. OBJECTIVE: To analyse the humoral and cellular cross-reactivity of recombinant Aed a 10.01, Aed a 10.02 and Der p 10. METHODS: Sera from 15 Austrian house dust mite-allergic, Der p 10-sensitized individuals were tested for IgE reactivity to recombinant tropomyosins in ELISA, inhibition ELISA and basophil activation tests. BALB/c mice were immunized with Aed a 10.01 or Aed a 10.02, and their sera were assessed for reactivity to all tropomyosins. Splenocytes were stimulated with all tropomyosins and synthetic peptides representing the amino acid sequence of Aed a 10.01. RESULTS: IgE antibodies of Der p 10-sensitized patients cross-reacted with both tropomyosins from A. aegypti. Aed a 10.01 was a more potent inhibitor of IgE binding to Der p 10 and a stronger activator of basophils sensitized with Der p 10-specific IgE than Aed a 10.02. Murine antibodies raised against Aed a 10.01 and Aed a 10.02 cross-reacted with Der p 10. Aed a 10.01-specific antibody showed stronger cross-reactivity with Der p 10 than Aed a 10.02-specific antibody. Splenocytes from both groups of mice proliferated similarly to all tropomyosins. Five cross-reactive T cell-activating regions were identified. CONCLUSION AND CLINICAL RELEVANCE: Tropomyosins from D. pteronyssinus and A. aegypti show humoral and cellular cross-reactivity, involving 5 potential T cell-activating regions. The more pronounced cross-reactivity of Aed a 10.01 and Der p 10 matched the higher sequence similarity of both proteins.
