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Results for “Class switch recombination”

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6 recordsLinked to original sources
PubMed2026

Isolation of region-specific factors driving antibody class-switch recombination from the immunoglobulin heavy chain locus.

Activation-Induced Cytidine Deaminase (AID) induces DNA double-strand breaks (DSBs) at the switch (S) regions of the Immunoglobulin heavy chain (IgH) locus, which are essential for class switch recombination (CSR) and somatic hypermutation (SHM), key processes for effective antibody production. While AID activity is critical, its off-target effects, such as DSBs at the Myc locus, can cause chromosomal translocations like IgH-Myc fusions, contributing to B-cell lymphomas. The factors assembled on the IgH locus that help restrict AID-induced DSBs and subsequently CSR, remain unknown. To address this, we developed a method to isolate CSR-specific factors by inserting a 5×-GAL4-UAS sequence at the switch-mu (Sμ) region in CH12 cells. This engineered site enables recruitment of a 3-FLAG-GAL4 DNA-binding protein (3F-GAL4-DBD), allowing specific pulldown of proteins enriched at the Sμ region. Successful recovery of the known CSR regulator BRD2 from the Sμ region, along with enrichment of the DNA repair factors 53BP1 and gH2AX, validated this approach. Identification and characterization of IgH-enriched factors establish a validated methodological framework to facilitate future proteomic discovery of CSR regulators and highlight mechanisms that balance antibody diversification with genomic integrity in B cells.

Immunoglobulin Class Switching↗
PubMed2026

Chronic psychological stress potentiates IgE class switch recombination via glucocorticoid receptor-mediated epigenetic reprogramming of B cells.

BACKGROUND: Chronic psychological stress is a well-recognized factor in the exacerbation of allergic diseases, with IgE playing a central role in their pathophysiology. However, the exact molecular mechanisms by which stress hormones directly influence IgE production and contribute to allergic responses remain largely uncharacterized. OBJECTIVE: This study aimed to elucidate the direct mechanisms through which chronic psychological stress, via elevated cortisol, regulates IgE class switch recombination (CSR) in B cells and contributes to stress-aggravated allergic inflammation in vivo. METHODS: We employed a chronic restraint stress (CRS) mouse model to investigate the impact of psychological stress on humoral immunity. In vitro experiments utilized primary murine B cells treated with physiological cortisol concentrations (250 nM), incorporating molecular techniques such as CRISPR-Cas9-mediated gene knockdown, chromatin immunoprecipitation (ChIP), whole-genome bisulfite sequencing, and pharmacological inhibitors of epigenetic enzymes. Primary human B cells and the U266 human myeloma cell line were used for translational validation. In vivo validation was performed using an ovalbumin (OVA)-induced allergic airway inflammation model with B cell-specific glucocorticoid receptor (GR) knockout mice. RESULTS: Chronic psychological stress significantly elevated plasma corticosterone and serum IgE levels in mice, with no changes in IgG1 or IgM. In purified in vitro B-cell cultures, cortisol promotes epigenetic remodeling at the Iε promoter region and enhances Iε germline transcript expression in an isotype-specific manner, and this effect was recapitulated in human B cells. GR bound to the Iε promoter's Amp_1 region (-154 to -62 bp), and CRISPR-Cas9-mediated GR knockdown abolished cortisol-induced IgE production. Mechanistically, cortisol increases enrichment of activating histone marks (H3K27ac, H3K4me3) and reduces H3K27me3 at the Iε promoter region, and induces site-specific DNA hypomethylation; inhibition of histone acetyltransferases (HATs) or DNA demethylation attenuated this effect. In vivo, B cell-specific GR knockout completely abrogated stress-induced exacerbation of allergic airway inflammation, including elevated serum IgE, eosinophilic inflammation, and airway hyperresponsiveness (AHR). CONCLUSION: Our findings support a mechanistic model in which chronic psychological stress, through elevated glucocorticoids, acts via GR to promote epigenetic remodeling at the Iε promoter region in B cells to enhance IgE synthesis and exacerbate allergic responses. This study provides a critical molecular link between the neuroendocrine system and adaptive immunity, offering promising therapeutic targets for stress-aggravated IgE-mediated diseases.

Animals↗
PubMed2025

Biochemical assays for AID/APOBECs and the identification of AID/APOBEC inhibitors.

Activation-induced cytidine deaminase (AID) and apolipoprotein B-mRNA editing catalytic polypeptide 3 (APOBEC3 or A3) proteins belong to the AID/APOBEC family of cytidine deaminases. While AID mediates somatic hypermutation and class-switch recombination in adaptive immunity, A3s restrict viruses and retroelements by hypermutation. Mis-regulated expression and off-target activity of AID/A3 can cause genome-wide mutations promoting oncogenesis, immune evasion, and therapeutic resistance due to tumor and viral evolution. In these contexts, inhibition of AID/A3 represents a promising therapeutic approach. Competitive inhibition could be achieved with different strategies: one class would be small molecules that bind in the catalytic pocket (active site) and block access for the substrate cytidine. Another type of larger molecule inhibitor would bind the enzymes' surface more broadly and compete with the binding of the polynucleotide substrates prior to deamination catalysis. Several biochemical assays developed to assess AID/A3 activity can be employed to screen for potential inhibitors. These include in cellulo and in vitro activity-based as well as binding-based assays. In this chapter, we discuss the key considerations for designing robust enzyme assays and provide an overview of assays that we and others have established or modified for specific applications in AID/A3 enzymology, including measurement of inhibition. We provide detailed protocols for the two most widely used in vitro enzyme assays that directly measure the activities of purified AID/A3s on DNA and/or RNA substrates, namely, the gel-based alkaline cleavage assay and multiple variations of PCR/sequencing-based assays.

Cytidine Deaminase↗
PubMed2026

X-linked hyper-IgM syndrome presenting as severe Pneumocystis pneumonia in a 6-month-old infant: a case report.

BACKGROUND: X-linked hyper-immunoglobulin M (XHIGM) syndrome is a rare primary immunodeficiency caused by mutations in the CD40 ligand gene (CD40LG), characterized by defective T-cell-dependent B-cell class-switch recombination. Patients typically present with recurrent infections in early childhood, but diagnosis is often delayed due to heterogeneous clinical manifestations and the fact that serum IgM levels may remain within the normal range in a significant proportion of patients. Here we report a case of XHIGM in an infant whose diagnostic journey began with recurrent lymphadenitis and culminated in life-threatening Pneumocystis jirovecii pneumonia (PJP). CASE PRESENTATION: A 6-month-old male infant was admitted with severe respiratory distress and hypoxemia. He had recurrent axillary lymphadenitis at 1 and 2 months of age and significant failure to thrive. Chest CT showed bilateral consolidative and interstitial opacities. Immunological evaluation revealed markedly decreased IgA, normal IgM and IgG, and profound T-cell lymphopenia. Sputum metagenomic sequencing identified Pneumocystis jirovecii. Whole-genome sequencing identified a hemizygous likely pathogenic CD40LG mutation (NM_000074.3:c.520C>T, p.Q174*); his mother was a carrier. He was treated with mechanical ventilation, trimethoprim-sulfamethoxazole (TMP-SMX), micafungin, corticosteroids, and intravenous immunoglobulin (IVIG), and was discharged after 36 days. CONCLUSIONS: In infants with recurrent or opportunistic infections, persistently low IgA and declining T-cell counts-even when initial screening appears normal-should raise suspicion for underlying immunodeficiency and prompt genetic evaluation. Early aggressive management and evaluation for hematopoietic stem cell transplantation are essential to improve outcomes.Serial immunological evaluation is essential in infants with recurrent or opportunistic infections, as persistently low IgA and declining T-cell counts-even when initial screening appears normal-should prompt genetic evaluation for underlying immunodeficiency. Early aggressive management and evaluation for hematopoietic stem cell transplantation are essential to improve outcomes.

Humans↗
PubMed2025

DIS3 licenses B cells for plasma cell differentiation in humans.

DIS3 is the main catalytic subunit of the nuclear RNA exosome, a complex playing a crucial role in RNA processing and the degradation of various noncoding RNA substrates. In mice, DIS3 is essential for genomic rearrangements during B cell development, but its role in terminal plasma cell (PC) differentiation has not been explored. Although DIS3 gene alterations are frequent in multiple myeloma (MM), a PC malignancy, their molecular impact remains poorly understood. In this study, we developed an antisense oligonucleotide strategy to knock down DIS3 expression in a well-characterized model of human PC differentiation. Reducing DIS3 expression systematically led to decreased B cell proliferation and impaired PC differentiation with lower levels of switched immunoglobulin secretion. Transcriptome analyses confirmed alterations in the proliferation and differentiation programs, alongside an accumulation of noncoding RNAs. Notably, centromere-associated noncoding RNAs were highly sensitive to DIS3 activity, and their accumulation in DIS3-deficient cells, either as transcripts or DNA-associated RNAs, correlated with the mislocalization of the centromere-specific histone variant CENP-A. We finally observed reduced physiological DNA recombination and somatic hypermutation but increased genomic instability in DIS3-deficient cells, in agreement with the higher levels of IGH translocations observed in our large cohort of DIS3-mutant MM patients. Together, these results underscore the essential role of DIS3 in regulating B cell proliferation, DNA recombination, and physiological or malignant PC differentiation in humans.

Humans↗
PubMed2026

R-loops and D-loops: a delicate balance in genomic stability and instability.

R-loops and D-loops are three-stranded nucleic acid structures that have emerged as central regulators of genome stability, gene expression, and DNA metabolism. R-loops form co-transcriptionally or post-transcriptionally when nascent RNA re-anneals with the template DNA strand, generating an RNA: DNA hybrid that displaces the non-template strand into a single-stranded state. These structures are enriched at CpG island promoters, transcription termination sites, and immunoglobulin class-switch regions, where they coordinate transcription regulation, chromatin remodeling, and DNA damage signaling. D-loops are formed when a single-stranded DNA segment pairs with one strand of a duplex and displaces the other, arising through context-dependent mechanisms that include RAD51- or DMC1-mediated strand invasion in homologous recombination, shelterin-assisted invasion at telomeres, and replication-coupled strand displacement at the mitochondrial DNA origin. They serve as indispensable intermediates in double-strand break repair, telomere maintenance, and mitochondrial DNA replication. Recent cryo-electron microscopy studies have resolved the stepwise RAD51-mediated strand exchange mechanism at near-atomic resolution, substantially advancing structural understanding of D-loop biogenesis. Despite their differences in molecular composition, both structures remodel Watson-Crick base pairing and, when dysregulated, are associated with replication fork stalling, transcription-replication conflicts, and aberrant recombination. This review systematically compares the structural features, formation mechanisms, regulatory networks, and biological functions of R-loops and D-loops, with emphasis on their convergent roles in safeguarding genome integrity. We further discuss rapidly evolving detection technologies and emerging therapeutic strategies targeting these structures in cancer and neurodegeneration, identifying key unresolved questions for future investigation.

Genomic Instability↗