Immunology. Antibodies get a break.
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Biomedical subjects
Jayanta Chaudhuri
Publications and source records attributed to Jayanta Chaudhuri.
The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation.
Antibodies, which are produced by B-lineage cells, consist of immunoglobulin heavy (IgH) and light (IgL) chains that have amino-terminal variable regions and carboxy-terminal constant regions. In response to antigens, B cells undergo two types of genomic alterations to increase antibody diversity. Affinity for antigen can be increased by introduction of point mutations into IgH and IgL variable regions by somatic hypermutation. In addition, antibody effector functions can be altered by changing the expressed IgH constant region exons through IgH class switch recombination (CSR). Somatic hypermutation and CSR both require the B-cell-specific activation-induced cytidine deaminase protein (AID), which initiates these reactions through its single-stranded (ss)DNA-specific cytidine deaminase activity. In biochemical assays, replication protein A (RPA), a ssDNA-binding protein, associates with phosphorylated AID from activated B cells and enhances AID activity on transcribed double-stranded (ds)DNA containing somatic hypermutation or CSR target sequences. This AID-RPA association, which requires phosphorylation, may provide a mechanism for allowing AID to access dsDNA targets in activated B cells. Here we show that AID from B cells is phosphorylated on a consensus protein kinase A (PKA) site and that PKA is the physiological AID kinase. Thus, AID from non-lymphoid cells can be functionally phosphorylated by recombinant PKA to allow interaction with RPA and promote deamination of transcribed dsDNA substrates. Moreover, mutation of the major PKA phosphorylation site of AID preserves ssDNA deamination activity, but markedly reduces RPA-dependent dsDNA deamination activity and severely impairs the ability of AID to effect CSR in vivo. We conclude that PKA has a critical role in post-translational regulation of AID activity in B cells.
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Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.
V(D)J recombination is the process by which the variable region exons encoding the antigen recognition sites of receptors expressed on B and T lymphocytes are generated during early development via somatic assembly of component gene segments. In response to antigen, somatic hypermutation (SHM) and class switch recombination (CSR) induce further modifications of immunoglobulin genes in B cells. CSR changes the IgH constant region for an alternate set that confers distinct antibody effector functions. SHM introduces mutations, at a high rate, into variable region exons, ultimately allowing affinity maturation. All of these genomic alteration processes require tight regulatory control mechanisms, both to ensure development of a normal immune system and to prevent potentially oncogenic processes, such as translocations, caused by errors in the recombination/mutation processes. In this regard, transcription of substrate sequences plays a significant role in target specificity, and transcription is mechanistically coupled to CSR and SHM. However, there are many mechanistic differences in these reactions. V(D)J recombination proceeds via precise DNA cleavage initiated by the RAG proteins at short conserved signal sequences, whereas CSR and SHM are initiated over large target regions via activation-induced cytidine deaminase (AID)-mediated DNA deamination of transcribed target DNA. Yet, new evidence suggests that AID cofactors may help provide an additional layer of specificity for both SHM and CSR. Whereas repair of RAG-induced double-strand breaks (DSBs) involves the general nonhomologous end-joining DNA repair pathway, and CSR also depends on at least some of these factors, CSR requires induction of certain general DSB response factors, whereas V(D)J recombination does not. In this review, we compare and contrast V(D)J recombination and CSR, with particular emphasis on the role of the initiating enzymes and DNA repair proteins in these processes.
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An evolutionarily conserved target motif for immunoglobulin class-switch recombination.
Immunoglobulin H class-switch recombination (CSR) occurs between switch regions and requires transcription and activation-induced cytidine deaminase (AID). Transcription through mammalian switch regions, because of their GC-rich composition, generates stable R-loops, which provide single-stranded DNA substrates for AID. However, we show here that the Xenopus laevis switch region S(mu), which is rich in AT and not prone to form R-loops, can functionally replace a mouse switch region to mediate CSR in vivo. X. laevis S(mu)-mediated CSR occurred mostly in a region of AGCT repeats targeted by the AID-replication protein A complex when transcribed in vitro. We propose that AGCT is a primordial CSR motif that targets AID through a non-R-loop mechanism involving an AID-replication protein A complex.
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Replication protein A interacts with AID to promote deamination of somatic hypermutation targets.
Activation-induced cytidine deaminase (AID) is a single-stranded (ss) DNA deaminase required for somatic hypermutation (SHM) and class switch recombination of immunoglobulin genes. Class switch recombination involves transcription through switch regions, which generates ssDNA within R loops. However, although transcription through immunoglobulin variable region exons is required for SHM, it does not generate stable ssDNA, which leaves the mechanism of AID targeting unresolved. Here we characterize the mechanism of AID targeting to in-vitro-transcribed substrates harbouring SHM motifs. We show that the targeting activity of AID is due to replication protein A (RPA), a ssDNA-binding protein involved in replication, recombination and repair. The 32-kDa subunit of RPA interacts specifically with AID from activated B cells in a manner that seems to be dependent on post-translational AID modification. Thus, our study implicates RPA as a novel factor involved in immunoglobulin diversification. We propose that B-cell-specific AID-RPA complexes preferentially bind to ssDNA of small transcription bubbles at SHM 'hotspots', leading to AID-mediated deamination and RPA-mediated recruitment of DNA repair proteins.
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Class-switch recombination: interplay of transcription, DNA deamination and DNA repair.
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Induction of activation-induced cytidine deaminase gene expression by IL-4 and CD40 ligation is dependent on STAT6 and NFkappaB.
Activation-induced cytidine deaminase (AID) is an inducible gene that plays an important role in class switch recombination, somatic hypermutation and gene conversion in B cells. We examined the regulation of AID gene expression in human and mouse B cells by IL-4 and CD40 ligation. IL-4 by itself and, to a much lesser extent, CD40 ligation induced AID mRNA expression in primary B cells. The two stimuli strongly synergized in inducing AID mRNA and protein expression. IL-4 induced STAT6 binding to a site in the 5' upstream region of the AID gene, while CD40 ligation induced NFkappaB binding to two sites in that region. B cells from STAT6-/- mice failed to up-regulate AID in response to IL-4, while B cells from p50-/- mice were impaired in their ability to up-regulate AID in response to CD40 ligation and IL-4. These results suggest that signals delivered via CD40 that activate NFkappaB synergize with signals delivered via the IL-4 receptor that activate STAT6 to induce optimal AID gene expression.
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The role of the non-homologous end-joining pathway in lymphocyte development.
One of the most toxic insults a cell can incur is a disruption of its linear DNA in the form of a double-strand break (DSB). Left unrepaired, or repaired improperly, these lesions can result in cell death or neoplastic transformation. Despite these dangers, lymphoid cells purposely introduce DSBs into their genome to maximize the diversity and effector functions of their antigen receptor genes. While the generation of breaks requires distinct lymphoid-specific factors, their resolution requires various ubiquitously expressed DNA-repair proteins, known collectively as the non-homologous end-joining pathway. In this review, we discuss the factors that constitute this pathway as well as the evidence of their involvement in two lymphoid-specific DNA recombination events.
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Transcription-targeted DNA deamination by the AID antibody diversification enzyme.
Activation-induced cytidine deaminase (AID), which is specific to B lymphocytes, is required for class switch recombination (CSR)--a process mediating isotype switching of immunoglobulin--and somatic hypermutation--the introduction of many point mutations into the immunoglobulin variable region genes. It has been suggested that AID may function as an RNA-editing enzyme or as a cytidine deaminase on DNA. However, the precise enzymatic activity of AID has not been assessed in previous studies. Similarly, although transcription of the target immunoglobulin locus sequences is required for both CSR and somatic hypermutation, the precise role of transcription has remained speculative. Here we use two different assays to demonstrate that AID can deaminate specifically cytidines on single-stranded (ss)DNA but not double-stranded (ds)DNA substrates in vitro. However, dsDNA can be deaminated by AID in vitro when the reaction is coupled to transcription. Moreover, a synthetic dsDNA sequence, which targets CSR in vivo in a manner dependent on transcriptional orientation, was deaminated by AID in vitro with the same transcriptional-orientation-dependence as observed for endogenous CSR. We conclude that transcription targets the DNA deamination activity of AID to dsDNA by generating secondary structures that provide ssDNA substrates.
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Leaky Scid phenotype associated with defective V(D)J coding end processing in Artemis-deficient mice.
Radiosensitive severe combined immune deficiency in humans results from mutations in Artemis, a protein which, when coupled with DNA-dependent protein kinase catalytic subunit (DNA-PKcs), possesses DNA hairpin-opening activity in vitro. Here, we report that Artemis-deficient mice have an overall phenotype similar to that of DNA-PKcs-deficient mice-including severe combined immunodeficiency associated with defects in opening and joining V(D)J coding hairpin ends and increased cellular ionizing radiation sensitivity. While these findings strongly support the notion that Artemis functions with DNA-PKcs in a subset of NHEJ functions, differences between Artemis- and DNA-PKcs-deficient phenotypes, most notably decreased fidelity of V(D)J signal sequence joining in DNA-PKcs-deficient but not Artemis-deficient fibroblasts, suggest additional functions for DNA-PKcs. Finally, Artemis deficiency leads to chromosomal instability in fibroblasts, demonstrating that Artemis functions as a genomic caretaker.
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