Short-chain fatty acid valerate reduces voluntary alcohol intake in male mice.

BACKGROUND: Despite serious health and social consequences, effective intervention strategies for habitual alcohol binge drinking are lacking. The development of novel therapeutic and preventative approaches is highly desirable. Accumulating evidence in the past several years has established associations between the gut microbiome and microbial metabolites with drinking behavior, but druggable targets and their underlying mechanism of action are understudied. RESULTS: Here, using a drink-in-the-dark mouse model, we identified a microbiome metabolite-based novel treatment (sodium valerate) that can reduce excessive alcohol drinking. Sodium valerate is a sodium salt of valeric acid short-chain fatty acid with a similar structure as γ-aminobutyric acid (GABA). Ten days of oral sodium valerate supplementation attenuates excessive alcohol drinking by 40%, reduces blood ethanol concentration by 53%, and improves anxiety-like or approach-avoidance behavior in male mice, without affecting overall food and water intake. Mechanistically, sodium valerate supplementation increases GABA levels across stool, blood, and amygdala. It also significantly increases H4 acetylation in the amygdala of mice. Transcriptomics analysis of the amygdala revealed that sodium valerate supplementation led to changes in gene expression associated with functional pathways including potassium voltage-gated channels, inflammation, glutamate degradation, L-DOPA degradation, and psychological behaviors. 16S microbiome profiling showed that sodium valerate supplementation shifts the gut microbiome composition and decreases microbiome-derived neuroactive compounds through GABA degradation in the gut microbiome. CONCLUSION: Our findings suggest that sodium valerate holds promise as an innovative therapeutic avenue for the reduction of habitual binge drinking, potentially through multifaceted mechanisms. Video Abstract.

Longitudinal profiling of the microbiome at four body sites reveals core stability and individualized dynamics during health and disease.

To understand the dynamic interplay between the human microbiome and host during health and disease, we analyzed the microbial composition, temporal dynamics, and associations with host multi-omics, immune, and clinical markers of microbiomes from four body sites in 86 participants over 6 years. We found that microbiome stability and individuality are body-site specific and heavily influenced by the host. The stool and oral microbiome are more stable than the skin and nasal microbiomes, possibly due to their interaction with the host and environment. We identify individual-specific and commonly shared bacterial taxa, with individualized taxa showing greater stability. Interestingly, microbiome dynamics correlate across body sites, suggesting systemic dynamics influenced by host-microbial-environment interactions. Notably, insulin-resistant individuals show altered microbial stability and associations among microbiome, molecular markers, and clinical features, suggesting their disrupted interaction in metabolic disease. Our study offers comprehensive views of multi-site microbial dynamics and their relationship with host health and disease.

Longitudinal profiling of the microbiome at four body sites reveals core stability and individualized dynamics during health and disease.

To understand dynamic interplay between the human microbiome and host during health and disease, we analyzed the microbial composition, temporal dynamics, and associations with host multi-omics, immune and clinical markers of microbiomes from four body sites in 86 participants over six years. We found that microbiome stability and individuality are body-site-specific and heavily influenced by the host. The stool and oral microbiome were more stable than the skin and nasal microbiomes, possibly due to their interaction with the host and environment. Also, we identified individual-specific and commonly shared bacterial taxa, with individualized taxa showing greater stability. Interestingly, microbiome dynamics correlated across body sites, suggesting systemic coordination influenced by host-microbial-environment interactions. Notably, insulin-resistant individuals showed altered microbial stability and associations between microbiome, molecular markers, and clinical features, suggesting their disrupted interaction in metabolic disease. Our study offers comprehensive views of multi-site microbial dynamics and their relationship with host health and disease. Study Highlights: The stability of the human microbiome varies among individuals and body sites.Highly individualized microbial genera are more stable over time.At each of the four body sites, systematic interactions between the environment, the host and bacteria can be detected.Individuals with insulin resistance have lower microbiome stability, a more diversified skin microbiome, and significantly altered host-microbiome interactions.

Short-chain-fatty acid valerate reduces voluntary alcohol intake in male mice.

Background: Despite serious health and social consequences, effective intervention strategies for habitual alcohol binge drinking are lacking. Development of novel therapeutic and preventative approaches is highly desirable. Accumulating evidence in the past several years has established associations between the gut microbiome and microbial metabolites with drinking behavior, but druggable targets and their underlying mechanism of action are understudied. Results: Here, using a drink-in-the-dark mouse model, we identified a microbiome metabolite-based novel treatment (sodium valerate) that can reduce excessive alcohol drinking. Sodium valerate is a sodium salt of valeric acidshort-chain-fatty-acid with similar structure as γ-aminobutyric acid (GABA). Ten days of oral sodium valerate supplementation attenuates excessive alcohol drinking by 40%, reduces blood ethanol concentration by 53%, and improves anxiety-like or approach-avoidance behavior in male mice, without affecting overall food and water intake. Mechanistically, sodium valerate supplementation increases GABA levels across stool, blood, and amygdala. It also significantly increases H4 acetylation in the amygdala of mice. Transcriptomics analysis of the amygdala revealed that sodium valerate supplementation led to changes in gene expression associated with functional pathways including potassium voltage-gated channels, inflammation, glutamate degradation, L-DOPA degradation, and psychological behaviors. 16S microbiome profiling showed that sodium valerate supplementation shifts the gut microbiome composition and decreases microbiome-derived neuroactive compounds through GABA degradation in the gut microbiome. Conclusion: Our findings suggest that the sodium valerate holds promise as an innovative therapeutic avenue for the reduction of habitual binge drinking, potentially through multifaceted mechanisms.

Baseline Gut Microbiome Signatures Correlate with Immunogenicity of SARS-CoV-2 mRNA Vaccines.

The powerful immune responses elicited by the mRNA vaccines targeting the SARS-CoV-2 Spike protein contribute to their high efficacy. Yet, their efficacy can vary greatly between individuals. For vaccines not based on mRNA, cumulative evidence suggests that differences in the composition of the gut microbiome, which impact vaccine immunogenicity, are some of the factors that contribute to variations in efficacy. However, it is unclear if the microbiome impacts the novel mode of immunogenicity of the SARS-CoV-2 mRNA vaccines. We conducted a prospective longitudinal cohort study of individuals receiving SARS-CoV-2 mRNA vaccines where we measured levels of anti-Spike IgG and characterized microbiome composition, at pre-vaccination (baseline), and one week following the first and second immunizations. While we found that microbial diversity at all timepoints correlated with final IgG levels, only at baseline did microbial composition and predicted function correlate with vaccine immunogenicity. Specifically, the phylum Desulfobacterota and genus Bilophila, producers of immunostimulatory LPS, positively correlated with IgG, while Bacteroides was negatively correlated. KEGG predicted pathways relating to SCFA metabolism and sulfur metabolism, as well as structural components such as flagellin and capsular polysaccharides, also positively correlated with IgG levels. Consistent with these findings, depleting the microbiome with antibiotics reduced the immunogenicity of the BNT162b2 vaccine in mice. These findings suggest that gut microbiome composition impacts the immunogenicity of the SARS-CoV-2 mRNA vaccines.

Exploratory studies of oral and fecal microbiome in healthy human aging.

Growing evidence has linked an altered host fecal microbiome composition with health status, common chronic diseases, and institutionalization in vulnerable older adults. However, fewer studies have described microbiome changes in healthy older adults without major confounding diseases or conditions, and the impact of aging on the microbiome across different body sites remains unknown. Using 16S ribosomal RNA gene sequencing, we reconstructed the composition of oral and fecal microbiomes in young (23-32; mean = 25 years old) and older (69-94; mean = 77 years old) healthy community-dwelling research subjects. In both body sites, we identified changes in minor bacterial operational taxonomic units (OTUs) between young and older subjects. However, the composition of the predominant bacterial species of the healthy older group in both microbiomes was not significantly different from that of the young cohort, which suggests that dominant bacterial species are relatively stable with healthy aging. In addition, the relative abundance of potentially pathogenic genera, such as Rothia and Mycoplasma, was enriched in the oral microbiome of the healthy older group relative to the young cohort. We also identified several OTUs with a prevalence above 40% and some were more common in young and others in healthy older adults. Differences with aging varied for oral and fecal samples, which suggests that members of the microbiome may be differentially affected by aging in a tissue-specific fashion. This is the first study to investigate both oral and fecal microbiomes in the context of human aging, and provides new insights into interactions between aging and the microbiome within two different clinically relevant sites.

Subgenomic RNA profiling suggests novel mechanism in coronavirus gene regulation and host adaption.

Fundamental to viral biology is identification and annotation of viral genes and their function. Determining the level of coronavirus gene expression is inherently difficult due to the positive stranded RNA genome and the identification of subgenomic RNAs (sgRNAs) that are required for expression of most viral genes. We developed a bioinformatic pipeline to analyze metatranscriptomic data from 20 independent studies encompassing 588 individual samples and 10 coronavirus species. This comparative analysis defined a core sgRNA repertoire for SARS-CoV-2 and found novel sgRNAs that could encode functional short peptides. Relevant to coronavirus infectivity and transmission, we also observed that the ratio of Spike sgRNA to Nucleocapsid one is highest in SARS-CoV-2, among the ß-coronaviruses examined. Furthermore, the adjustment of this ratio can be made by modifications to the viral RNA replication machinery, representing a form of viral gene regulation that may be involved in host adaption.

Alterations of host-gut microbiome interactions in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) has a complex genetic, immune and metabolic pathophysiology. Recent studies implicated the gut microbiome in MS pathogenesis. However, interactions between the microbiome and host immune system, metabolism and diet have not been studied over time in this disorder. METHODS: We performed a six-month longitudinal multi-omics study of 49 participants (24 untreated relapse remitting MS patients and 25 age, sex, race matched healthy control individuals. Gut microbiome composition and function were characterized using 16S and metagenomic shotgun sequencing. Flow cytometry was used to characterize blood immune cell populations and cytokine profiles. Circulating metabolites were profiled by untargeted UPLC-MS. A four-day food diary was recorded to capture the habitual dietary pattern of study participants. FINDINGS: Together with changes in blood immune cells, metagenomic analysis identified a number of gut microbiota decreased in MS patients compared to healthy controls, and microbiota positively or negatively correlated with degree of disability in MS patients. MS patients demonstrated perturbations of their blood metabolome, such as linoleate metabolic pathway, fatty acid biosynthesis, chalcone, dihydrochalcone, 4-nitrocatechol and methionine. Global correlations between multi-omics demonstrated a disrupted immune-microbiome relationship and a positive blood metabolome-microbiome correlation in MS. Specific feature association analysis identified a potential correlation network linking meat servings with decreased gut microbe B. thetaiotaomicron, increased Th17 cell and greater abundance of meat-associated blood metabolites. The microbiome and metabolome profiles remained stable over six months in MS and control individuals. INTERPRETATION: Our study identified multi-system alterations in gut microbiota, immune and blood metabolome of MS patients at global and individual feature level. Multi-OMICS data integration deciphered a potential important biological network that links meat intakes with increased meat-associated blood metabolite, decreased polysaccharides digesting bacteria, and increased circulating proinflammatory marker. FUNDING: This work was supported by the Washington University in St. Louis Institute of Clinical and Translational Sciences, funded, in part, by Grant Number # UL1 TR000448 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award (Zhou Y, Piccio, L, Lovett-Racke A and Tarr PI); R01 NS10263304 (Zhou Y, Piccio L); the Leon and Harriet Felman Fund for Human MS Research (Piccio L and Cross AH). Cantoni C. was supported by the National MS Society Career Transition Fellowship (TA-180531003) and by donations from Whitelaw Terry, Jr. / Valerie Terry Fund. Ghezzi L. was supported by the Italian Multiple Sclerosis Society research fellowship (FISM 2018/B/1) and the National Multiple Sclerosis Society Post-Doctoral Fellowship (FG-190734474). Anne Cross was supported by The Manny & Rosalyn Rosenthal-Dr. John L. Trotter MS Center Chair in Neuroimmunology of the Barnes-Jewish Hospital Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Effects of dietary restriction on gut microbiota and CNS autoimmunity.

Multiple sclerosis (MS) is the most common central nervous system (CNS) autoimmune disease. It is due to the interplay of genetic and environmental factors. Current opinion is that diet could play a pathogenic role in disease onset and development. Dietary restriction (DR) without malnutrition markedly improves health and increases lifespan in multiple model organisms. DR regimens that utilize continuous or intermittent food restriction can induce anti-inflammatory, immuno-modulatory and neuroendocrine adaptations promoting health. These adaptations exert neuroprotective effects in the main MS animal model, experimental autoimmune encephalomyelitis (EAE). This review summarizes the current knowledge on DR-induced changes in gut microbial composition and metabolite production and its impact on underlying functional mechanisms. Studies demonstrating the protective effects of DR regimens on EAE and people with MS are also presented. This is a rapidly developing research field with important clinical implications for personalized dietary interventions in MS prevention and treatment.

Procedures for Fecal Microbiota Transplantation in Murine Microbiome Studies.

Fecal microbiota transplantation (FMT) has been widely recognized as an approach to determine the microbiome's causal role in gut dysbiosis-related disease models and as a novel disease-modifying therapy. Despite potential beneficial FMT results in various disease models, there is a variation and complexity in procedural agreement among research groups for performing FMT. The viability of the microbiome in feces and its successful transfer depends on various aspects of donors, recipients, and lab settings. This review focuses on the technical practices of FMT in animal studies. We first document crucial factors required for collecting, handling, and processing donor fecal microbiota for FMT. Then, we detail the description of gut microbiota depletion methods, FMT dosages, and routes of FMT administrations in recipients. In the end, we describe assessments of success rates of FMT with sustainability. It is critical to work under the anaerobic condition to preserve as much of the viability of bacteria. Utilization of germ- free mice or depletion of recipient gut microbiota by antibiotics or polyethylene glycol are two common recipient preparation approaches to achieve better engraftment. Oral-gastric gavage preferred by most researchers for fast and effective administration of FMT in mice. Overall, this review highlights various methods that may lead to developing the standard and reproducible protocol for FMT.

Alterations of the gut mycobiome in patients with MS.

BACKGROUND: The mycobiome is the fungal component of the gut microbiome and is implicated in several autoimmune diseases. However, its role in MS has not been studied. METHODS: In this case-control observational study, we performed ITS sequencing and characterised the gut mycobiome in people with MS (pwMS) and healthy controls at baseline and after six months. FINDINGS: The mycobiome had significantly higher alpha diversity and inter-subject variation in pwMS than controls. Saccharomyces and Aspergillus were over-represented in pwMS. Saccharomyces was positively correlated with circulating basophils and negatively correlated with regulatory B cells, while Aspergillus was positively correlated with activated CD16+ dendritic cells in pwMS. Different mycobiome profiles, defined as mycotypes, were associated with different bacterial microbiome and immune cell subsets in the blood. Initial treatment with dimethyl fumarate, a common immunomodulatory therapy which also has fungicidal activity, did not cause uniform gut mycobiome changes across all pwMS. INTERPRETATION: There is an alteration of the gut mycobiome in pwMS, compared to healthy controls. Further study is required to assess any causal association of the mycobiome with MS and its direct or indirect interactions with bacteria and autoimmunity. FUNDING: This work was supported by the Washington University in St. Louis Institute of Clinical and Translational Sciences, funded, in part, by Grant Number # UL1 TR000448 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award (Zhou Y, Piccio, L, Lovett-Racke A and Tarr PI); R01 NS102633-04 (Zhou Y, Piccio L); the Leon and Harriet Felman Fund for Human MS Research (Piccio L and Cross AH). Cantoni C. was supported by the National MS Society Career Transition Fellowship (TA-1805-31003) and by donations from Whitelaw Terry, Jr. / Valerie Terry Fund. Ghezzi L. was supported by the Italian Multiple Sclerosis Society research fellowship (FISM 2018/B/1) and the National Multiple Sclerosis Society Post-Doctoral Fellowship (FG- 1907-34474). Anne Cross was supported by The Manny & Rosalyn Rosenthal-Dr. John L. Trotter MS Center Chair in Neuroimmunology of the Barnes-Jewish Hospital Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Intermittent Fasting Confers Protection in CNS Autoimmunity by Altering the Gut Microbiota.

Multiple sclerosis (MS) is more common in western countries with diet being a potential contributing factor. Here we show that intermittent fasting (IF) ameliorated clinical course and pathology of the MS model, experimental autoimmune encephalomyelitis (EAE). IF led to increased gut bacteria richness, enrichment of the Lactobacillaceae, Bacteroidaceae, and Prevotellaceae families and enhanced antioxidative microbial metabolic pathways. IF altered T cells in the gut with a reduction of IL-17 producing T cells and an increase in regulatory T cells. Fecal microbiome transplantation from mice on IF ameliorated EAE in immunized recipient mice on a normal diet, suggesting that IF effects are at least partially mediated by the gut flora. In a pilot clinical trial in MS patients, intermittent energy restriction altered blood adipokines and the gut flora resembling protective changes observed in mice. In conclusion, IF has potent immunomodulatory effects that are at least partially mediated by the gut microbiome.

HCoDES reveals chromosomal DNA end structures with single-nucleotide resolution.

The structure of broken DNA ends is a critical determinant of the pathway used for DNA double-strand break (DSB) repair. Here, we develop an approach involving the hairpin capture of DNA end structures (HCoDES), which elucidates chromosomal DNA end structures at single-nucleotide resolution. HCoDES defines structures of physiologic DSBs generated by the RAG endonuclease, as well as those generated by nucleases widely used for genome editing. Analysis of G1 phase cells deficient in H2AX or 53BP1 reveals DNA ends that are frequently resected to form long single-stranded overhangs that can be repaired by mutagenic pathways. In addition to 3' overhangs, many of these DNA ends unexpectedly form long 5' single-stranded overhangs. The divergence in DNA end structures resolved by HCoDES suggests that H2AX and 53BP1 may have distinct activities in end protection. Thus, the high-resolution end structures obtained by HCoDES identify features of DNA end processing during DSB repair.

KAP-1 promotes resection of broken DNA ends not protected by γ-H2AX and 53BP1 in G1-phase lymphocytes.

The resection of broken DNA ends is required for DNA double-strand break (DSB) repair by homologous recombination (HR) but can inhibit normal repair by nonhomologous end joining (NHEJ), the main DSB repair pathway in G1-phase cells. Antigen receptor gene assembly proceeds through DNA DSB intermediates generated in G1-phase lymphocytes by the RAG endonuclease. These DSBs activate ATM, which phosphorylates H2AX, forming γ-H2AX in flanking chromatin. γ-H2AX prevents CtIP from initiating resection of RAG DSBs. Whether there are additional proteins required to promote resection of these DNA ends is not known. KRAB-associated protein 1 (KAP-1) (TRIM28) is a transcriptional repressor that modulates chromatin structure and has been implicated in the repair of DNA DSBs in heterochromatin. Here, we show that in murine G1-phase lymphocytes, KAP-1 promotes resection of DSBs that are not protected by H2AX and its downstream effector 53BP1. In these murine cells, KAP-1 activity in DNA end resection is attenuated by a single-amino-acid change that reflects a KAP-1 polymorphism between primates and other mammalian species. These findings establish KAP-1 as a component of the machinery that can resect DNA ends in G1-phase cells and suggest that there may be species-specific features to this activity.

Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination.

V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA-PKcs deficiency leads to a nearly complete block in coding join formation, as DNA-PKcs is required to activate Artemis, the endonuclease that opens hairpin-sealed coding ends. In contrast to loss of DNA-PKcs protein, here we show that inhibition of DNA-PKcs kinase activity has no effect on coding join formation when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA-PKcs. Mutation of these threonine residues to alanine (DNA-PKcs(3A)) renders DNA-PKcs dependent on its intrinsic kinase activity during coding end joining, at a step downstream of opening hairpin-sealed coding ends. Thus, DNA-PKcs has critical functions in coding end joining beyond promoting Artemis endonuclease activity, and these functions can be regulated redundantly by the kinase activity of either ATM or DNA-PKcs.

Ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases have overlapping activities during chromosomal signal joint formation.

Lymphocyte antigen receptor gene assembly occurs through the process of V(D)J recombination, which is initiated when the RAG endonuclease introduces DNA DSBs at two recombining gene segments to form broken DNA coding end pairs and signal end pairs. These paired DNA ends are joined by proteins of the nonhomologous end-joining (NHEJ) pathway of DSB repair to form a coding joint and signal joint, respectively. RAG DSBs are generated in G1-phase developing lymphocytes, where they activate the ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases to orchestrate diverse cellular DNA damage responses including DSB repair. Paradoxically, although Atm and DNA-PKcs both function during coding joint formation, Atm appears to be dispensible for signal joint formation; and although some studies have revealed an activity for DNA-PKcs during signal joint formation, others have not. Here we show that Atm and DNA-PKcs have overlapping catalytic activities that are required for chromosomal signal joint formation and for preventing the aberrant resolution of signal ends as potentially oncogenic chromosomal translocations.

H2AX prevents CtIP-mediated DNA end resection and aberrant repair in G1-phase lymphocytes.

DNA double-strand breaks (DSBs) are generated by the recombination activating gene (RAG) endonuclease in all developing lymphocytes as they assemble antigen receptor genes. DNA cleavage by RAG occurs only at the G1 phase of the cell cycle and generates two hairpin-sealed DNA (coding) ends that require nucleolytic opening before their repair by classical non-homologous end-joining (NHEJ). Although there are several cellular nucleases that could perform this function, only the Artemis nuclease is able to do so efficiently. Here, in vivo, we show that in murine cells the histone protein H2AX prevents nucleases other than Artemis from processing hairpin-sealed coding ends; in the absence of H2AX, CtIP can efficiently promote the hairpin opening and resection of DNA ends generated by RAG cleavage. This CtIP-mediated resection is inhibited by γ-H2AX and by MDC-1 (mediator of DNA damage checkpoint 1), which binds to γ-H2AX in chromatin flanking DNA DSBs. Moreover, the ataxia telangiectasia mutated (ATM) kinase activates antagonistic pathways that modulate this resection. CtIP DNA end resection activity is normally limited to cells at post-replicative stages of the cell cycle, in which it is essential for homology-mediated repair. In G1-phase lymphocytes, DNA ends that are processed by CtIP are not efficiently joined by classical NHEJ and the joints that do form frequently use micro-homologies and show significant chromosomal deletions. Thus, H2AX preserves the structural integrity of broken DNA ends in G1-phase lymphocytes, thereby preventing these DNA ends from accessing repair pathways that promote genomic instability.

Role of the translocation partner in protection against AID-dependent chromosomal translocations.

Chromosome translocations between Ig (Ig) and non-Ig genes are frequently associated with B-cell lymphomas in humans and mice. The best characterized of these is c-myc/IgH translocation, which is associated with Burkitt's lymphoma. These translocations are caused by activation-induced cytidine deaminase (AID), which produces double-strand DNA breaks in both genes. c-myc/IgH translocations are rare events, in part because ATM, p53, and p19 actively suppress them. To further define the mechanism of protection against the accumulation of cells that bear c-myc/IgH translocation, we assayed B cells from mice that carry mutations in cell-cycle and apoptosis regulator proteins that act downstream of p53. We find that PUMA, Bim, and PKCdelta are required for protection against c-myc/IgH translocation, whereas Bcl-XL and BAFF enhance c-myc/IgH translocation. Whether these effects are general or specific to c-myc/IgH translocation and whether AID produces dsDNA breaks in genes other than c-myc and Ig is not known. To examine these questions, we developed an assay for translocation between IgH and Igbeta, both of which are somatically mutated by AID. Igbeta/IgH, like c-myc/IgH translocations, are AID-dependent, and AID is responsible for lesions on IgH and the non-IgH translocation partners. However, ATM, p53, and p19 do not protect against Igbeta/IgH translocations. Instead, B cells are protected against Igbeta/IgH translocations by a BAFF- and PKCdelta-dependent pathway. We conclude that AID-induced double-strand breaks in non-Ig genes other than c-myc lead to their translocation, and that at least two nonoverlapping pathways protect against translocations in primary B cells.

Haploinsufficiency of activation-induced deaminase for antibody diversification and chromosome translocations both in vitro and in vivo.

The humoral immune response critically relies on the secondary diversification of antibodies. This diversification takes places through somatic remodelling of the antibody genes by two molecular mechanisms, Class Switch Recombination (CSR) and Somatic Hypermutation (SHM). The enzyme Activation Induced Cytidine Deaminase (AID) initiates both SHM and CSR by deaminating cytosine residues on the DNA of immunoglobulin genes. While crucial for immunity, AID-catalysed deamination is also the triggering event for the generation of lymphomagenic chromosome translocations. To address whether restricting the levels of AID expression in vivo contributes to the regulation of its function, we analysed mice harbouring a single copy of the AID gene (AID(+/-)). AID(+/-) mice express roughly 50% of normal AID levels, and display a mild hyperplasia, reminiscent of AID deficient mice and humans. Moreover, we found that AID(+/-) cells have an impaired competence for CSR and SHM, which indicates that AID gene dose is limiting for its physiologic function. We next evaluated the impact of AID reduction in AID(+/-) mice on the generation of chromosome translocations. Our results show that the frequency of AID-promoted c-myc/IgH translocations is reduced in AID(+/-) mice, both in vivo and in vitro. Therefore, AID is haploinsufficient for antibody diversification and chromosome translocations. These findings suggest that limiting the physiologic levels of AID expression can be a regulatory mechanism that ensures an optimal balance between immune proficiency and genome integrity.

AID is required for the chromosomal breaks in c-myc that lead to c-myc/IgH translocations.

Chromosomal translocation requires formation of paired double-strand DNA breaks (DSBs) on heterologous chromosomes. One of the most well characterized oncogenic translocations juxtaposes c-myc and the immunoglobulin heavy-chain locus (IgH) and is found in Burkitt's lymphomas in humans and plasmacytomas in mice. DNA breaks in IgH leading to c-myc/IgH translocations are created by activation-induced cytidine deaminase (AID) during antibody class switch recombination or somatic hypermutation. However, the source of DNA breaks at c-myc is not known. Here, we provide evidence for the c-myc promoter region being required in targeting AID-mediated DNA damage to produce DSBs in c-myc that lead to c-myc/IgH translocations in primary B lymphocytes. Thus, in addition to producing somatic mutations and DNA breaks in antibody genes, AID is also responsible for the DNA lesions in oncogenes that are required for their translocation.