BRD2 promotes antibody class switch recombination by facilitating DNA repair in collaboration with NIPBL.

Efficient repair of DNA double-strand breaks in the Ig heavy chain gene locus is crucial for B-cell antibody class switch recombination (CSR). The regulatory dynamics of the repair pathway direct CSR preferentially through nonhomologous end joining (NHEJ) over alternative end joining (AEJ). Here, we demonstrate that the histone acetyl reader BRD2 suppresses AEJ and aberrant recombination as well as random genomic sequence capture at the CSR junctions. BRD2 deficiency impairs switch (S) region synapse, optimal DNA damage response (DDR), and increases DNA break end resection. Unlike BRD4, a similar bromodomain protein involved in NHEJ and CSR, BRD2 loss does not elevate RPA phosphorylation and R-loop formation in the S region. As BRD2 stabilizes the cohesion loader protein NIPBL in the S regions, the loss of BRD2 or NIPBL shows comparable deregulation of S-S synapsis, DDR, and DNA repair pathway choice during CSR. This finding extends beyond CSR, as NIPBL and BRD4 have been linked to Cornelia de Lange syndrome, a developmental disorder exhibiting defective NHEJ and Ig isotype switching. The interplay between these proteins sheds light on the intricate mechanisms governing DNA repair and immune system functionality.

Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition.

Antibody class switch recombination (CSR) is a locus-specific genomic rearrangement mediated by switch (S) region transcription, activation-induced cytidine deaminase (AID)-induced DNA breaks, and their resolution by non-homologous end joining (NHEJ)-mediated DNA repair. Due to the complex nature of the recombination process, numerous cofactors are intimately involved, making it important to identify rate-limiting factors that impact on DNA breaking and/or repair. Using an siRNA-based loss-of-function screen of genes predicted to encode PHD zinc-finger-motif proteins, we identify the splicing factor Phf5a/Sf3b14b as a novel modulator of the DNA repair step of CSR. Loss of Phf5a severely impairs AID-induced recombination, but does not perturb DNA breaks and somatic hypermutation. Phf5a regulates NHEJ-dependent DNA repair by preserving chromatin integrity to elicit optimal DNA damage response and subsequent recruitment of NHEJ factors at the S region. Phf5a stabilizes the p400 histone chaperone complex at the locus, which in turn promotes deposition of H2A variant such as H2AX and H2A.Z that are critical for the early DNA damage response and NHEJ, respectively. Depletion of Phf5a or p400 blocks the repair of both AID- and I-SceI-induced DNA double-strand breaks, supporting an important contribution of this axis to programmed as well as aberrant recombination.

SAMHD1-mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination.

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1), a dNTP triphosphohydrolase, regulates the levels of cellular dNTPs through their hydrolysis. SAMHD1 protects cells from invading viruses that depend on dNTPs to replicate and is frequently mutated in cancers and Aicardi-Goutières syndrome, a hereditary autoimmune encephalopathy. We discovered that SAMHD1 localizes at the immunoglobulin (Ig) switch region, and serves as a novel DNA repair regulator of Ig class switch recombination (CSR). Depletion of SAMHD1 impaired not only CSR but also IgH/c-Myc translocation. Consistently, we could inhibit these two processes by elevating the cellular nucleotide pool. A high frequency of nucleotide insertion at the break-point junctions is a notable feature in SAMHD1 deficiency during activation-induced cytidine deaminase-mediated genomic instability. Interestingly, CSR induced by staggered but not blunt, double-stranded DNA breaks was impaired by SAMHD1 depletion, which was accompanied by enhanced nucleotide insertions at recombination junctions. We propose that SAMHD1-mediated dNTP balance regulates dNTP-sensitive DNA end-processing enzyme and promotes CSR and aberrant genomic rearrangements by suppressing the insertional DNA repair pathway.

RNA-binding motifs of hnRNP K are critical for induction of antibody diversification by activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) is the key enzyme for class switch recombination (CSR) and somatic hypermutation (SHM) to generate antibody memory. Previously, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was shown to be required for AID-dependent DNA breaks. Here, we defined the function of major RNA-binding motifs of hnRNP K, GXXGs and RGGs in the K-homology (KH) and the K-protein-interaction (KI) domains, respectively. Mutation of GXXG, RGG, or both impaired CSR, SHM, and cMyc/IgH translocation equally, showing that these motifs were necessary for AID-dependent DNA breaks. AID-hnRNP K interaction is dependent on RNA; hence, mutation of these RNA-binding motifs abolished the interaction with AID, as expected. Some of the polypyrimidine sequence-carrying prototypical hnRNP K-binding RNAs, which participate in DNA breaks or repair bound to hnRNP K in a GXXG and RGG motif-dependent manner. Mutation of the GXXG and RGG motifs decreased nuclear retention of hnRNP K. Together with the previous finding that nuclear localization of AID is necessary for its function, lower nuclear retention of these mutants may worsen their functional deficiency, which is also caused by their decreased RNA-binding capacity. In summary, hnRNP K contributed to AID-dependent DNA breaks with all of its major RNA-binding motifs.

B cell-specific and stimulation-responsive enhancers derepress Aicda by overcoming the effects of silencers.

Activation-induced cytidine deaminase (AID) is essential for the generation of antibody memory but also targets oncogenes, among other genes. We investigated the transcriptional regulation of Aicda (which encodes AID) in class switch-inducible CH12F3-2 cells and found that Aicda regulation involved derepression by several layers of positive regulatory elements in addition to the 5' promoter region. The 5' upstream region contained functional motifs for the response to signaling by cytokines, the ligand for the costimulatory molecule CD40 or stimuli that activated the transcription factor NF-kappaB. The first intron contained functional binding elements for the ubiquitous silencers c-Myb and E2f and for the B cell-specific activator Pax5 and E-box-binding proteins. Our results show that Aicda is regulated by the balance between B cell-specific and stimulation-responsive elements and ubiquitous silencers.

Chromatin reader Brd4 functions in Ig class switching as a repair complex adaptor of nonhomologous end-joining.

Class switch recombination (CSR) is a B cell-specific genomic alteration induced by activation-induced cytidine deaminase (AID)-dependent DNA break at the immunoglobulin heavy-chain locus, followed by repair. Although chromatin-associated factors in promoting AID-induced DNA break have been widely reported, the involvement of chromatin adaptors at the repair phase of CSR remains unknown. Here, we show that the acetylated histone reader Brd4 is critical for nonhomologous end-joining (NHEJ) repair of AID- and I-SceI-induced DNA breaks. Brd4 was recruited to the DNA break regions, and its depletion from the chromatin caused CSR impairment without affecting the DNA break generation. Inhibition of Brd4 suppressed the accumulation of 53BP1 and uracil DNA glycosylase at the switch regions, perturbed the switch junctional microhomology, and reduced Igh/c-myc translocation. We conclude that Brd4 serves as a chromatin platform required for the recruitment of repair components during CSR and general DNA damage.

Inclusion of Veterinary Services in national emergency management plans.

With human population growth, rapid urbanisation, increasing globalisation, and climate change, the interdependency of human health and animal health is mounting. Therefore, the importance of national emergency management plans (NEMPs) for the mitigation of, and preparedness for, all hazards, including disease epidemics, both zoonotic and zootic, is ever increasing. The authors decided to take a One Health approach by assessing the inclusion of Veterinary Services and animal health in NEMPs, based on geographical region, the date of the NEMP, national income status, and the proportion of the agricultural sector in national gross domestic product (GDP). To carry out the assessment, the authors analysed the publicly available NEMPs of 86 Members of the World Organisation for Animal Health. Of the 86 NEMPs reviewed, only a third expressly mentioned Veterinary Services, almost 60% mentioned zoonotic and/or zootic diseases, and about two-thirds mentioned animals to some extent. The highest correlating factor to the inclusion of animal health in NEMPs was the level of the agricultural sector's contributions to the national GDP. Fisheries and aquaculture were not a major consideration in any of the reviewed NEMPs, especially not in relation to diseases. Based on region, Latin America and the Caribbean exhibited the lowest inclusion rate of animal health in NEMPs. The results demonstrate that the omission of animal health is still a problem. A multi-disciplinary approach that includes veterinary medicine as well as human medicine is vital in the construction and/or revision of NEMPs. Future studies should consider whether or not there is a connection between countries' veterinary capacities and the inclusion of Veterinary Services in their NEMPs and whether or not they have the infrastructure and human resources to put into operation the roles of Veterinary Services as identified in their NEMPs.

La croissance démographique humaine, l'urbanisation accélérée, la mondialisation accrue et le changement climatique sont autant de facteurs qui intensifient l'interdépendance de la santé humaine et de la santé animale. De ce fait, les plans nationaux de gestion des urgences jouent un rôle de plus en plus important pour atténuer les dangers, quels qu'ils soient, et pour se préparer à leur survenue, y compris les dangers liés aux épidémies zoonotiques ou zootiques. Les auteurs ont entrepris d'évaluer le niveau d'intégration des Services vétérinaires et de la santé animale dans les plans nationaux de gestion des urgences dans une perspective Une seule santé, en se basant sur les critères suivants : la région géographique, la date du plan national de gestion des urgences, le niveau de revenu du pays et la part du secteur agricole dans le produit intérieur brut (PIB). Pour les besoins de cette évaluation, les auteurs ont analysé les plans nationaux de gestion des urgences publiés par 86 Membres de l'Organisation mondiale de la santé animale. Parmi ces 86 plans nationaux, un tiers seulement mentionnait expressément les Services vétérinaires, près de 60 % mentionnaient les maladies zoonotiques ou les épizooties et environ deux tiers prenaient en compte les animaux pour une raison ou pour une autre. Le facteur présentant la corrélation la plus élevée avec la prise en compte de la santé animale dans les plans nationaux de gestion des urgences était le niveau de contribution du secteur agricole dans le PIB national. Aucun des plans nationaux de gestion des urgences examinés ne prenait en compte la pêche et l'aquaculture en tant qu'aspect important, en particulier en lien avec des maladies. À l'échelle régionale, c'est en Amérique latine et aux Caraïbes que l'intégration de la santé animale dans les plans nationaux de gestion des urgences était la plus faible. Ces résultats montrent que le problème de l'omission de la santé animale est toujours d'actualité. Il est d'une importance capitale qu'une approche pluridisciplinaire intégrant la médecine vétérinaire et la médecine humaine soit adoptée lors de la conception et/ou de la révision des plans nationaux de gestion des urgences. Il conviendrait que de nouvelles études déterminent à l'avenir s'il existe ou non un lien entre les capacités vétérinaires des pays et la prise en compte des Services vétérinaires dans les plans nationaux de gestion des urgences, et si les pays disposent ou non des infrastructures et des ressources humaines permettant à leurs Services vétérinaires de mener à bien les interventions prévues dans les plans nationaux de gestion des urgences.

El crecimiento demográfico, la rápida urbanización, la creciente mundialización y el cambio climático son otros tantos factores que traen consigo una dependencia recíproca cada vez más acusada entre la salud humana y la sanidad animal. De ahí la creciente importancia que van adquiriendo los planes nacionales de gestión de emergencias destinados a prepararse para todo tipo de peligros, incluidas las enfermedades epidémicas, tanto zoonóticas como epizoóticas, y, llegado el caso, a mitigar sus consecuencias. Los autores, partiendo de las premisas de Una sola salud, decidieron evaluar la integración de los Servicios Veterinarios y la sanidad animal en los planes nacionales de gestión de emergencias, utilizando como criterios de evaluación la región geográfica, la fecha del plan nacional en cuestión, el nivel de renta del país y el porcentaje del producto interno bruto (PIB) que representa el sector agrícola. Para llevar a cabo la evaluación los autores analizaron los planes nacionales de gestión de emergencias que están a disposición pública de 86 Miembros de la Organización Mundial de Sanidad Animal. De esos 86 planes nacionales examinados, solo en un tercio se mencionaban explícitamente los Servicios Veterinarios, en casi un 60% se aludía a enfermedades zoonóticas y/o epizoóticas y en cerca de dos tercios se hablaba en alguna medida de los animales. El factor que mayor correlación presentaba con la integración de la sanidad animal en los planes nacionales de gestión de emergencias era la aportación del sector agrícola al PIB. En ninguno de los planes examinados ocupaban un lugar relevante ni la pesca ni la acuicultura, especialmente en relación con las enfermedades. Por regiones, América Latina y el Caribe presentaba el menor porcentaje de integración de la sanidad animal en los planes nacionales de gestión de emergencias. Los resultados demuestran que la omisión de la sanidad animal sigue suponiendo un problema. A la hora de elaborar o revisar los planes nacionales de gestión de emergencias es crucial hacerlo desde planteamientos multidisciplinares que incluyan tanto la medicina veterinaria como la humana. En estudios ulteriores convendría determinar si existe una correlación entre la capacidad veterinaria de los países y la integración de los Servicios Veterinarios en su plan nacional de gestión de emergencias y si los países disponen de la infraestructura y el personal requeridos para que los Servicios Veterinarios cumplan las funciones que se les asignan en el plan nacional de gestión de emergencias.

Liquiritin elicitation can increase the content of medicinally important glucosinolates and phenolic compounds in Chinese kale plants.

BACKGROUND: Brassica oleracea var. alboglabra (Chinese kale) is an important vegetable grown in southern China. This study was aimed at searching for environmentally friendly and affordable approaches to increase the production of medicinally relevant glucosinolates and phenolic compounds in Chinese kale plants. For this purpose, the foliar application of liquiritin at 0 (control), 250, 500 and 750 ppm was tested starting from the four-leaf stage and repeated every two weeks until plants were two months old. RESULTS: Foliar application of liquiritin in Chinese kale plants significantly increased glucosinolates and total phenolic content, in a dose-dependent manner. Compared with control plants, 2.3- and 1.9-fold increases in yields of glucosinolates and total phenolic content, respectively, were corroborated in Chinese kale plants treated with 750 ppm of liquiritin. Along with rises in the content of eight different glucosinolates, liquiritin elicitation effectively increased the concentration of glycosilated and acylated flavonoids and hydroxycinnamic acids. The expression of genes involved in glucosinolate and phenolic biosynthesis was significantly higher in liquiritin-treated plants as compared to controls. CONCLUSIONS: Liquiritin elicitation is a feasible and environmentally friendly practice for increasing the production of medicinally important glucosinolates and phenolic compounds in Chinese kale, which may improve this plant's value as a nutraceutical food. This study also contributes to understanding the molecular mechanisms underlying liquiritin elicitation. This is the first report documenting the use of liquiritin for an elicitation purpose in plants. © 2019 Society of Chemical Industry.

Functional requirements of AID's higher order structures and their interaction with RNA-binding proteins.

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID's structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.

Depletion of recombination-specific cofactors by the C-terminal mutant of the activation-induced cytidine deaminase causes the dominant negative effect on class switch recombination.

Activation-induced cytidine deaminase (AID) is essential for class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes. Studies on in vitro mutagenized AID as well as its mutations in human patients with hyper-IgM (HIGM)-syndrome type II revealed that C-terminal AID mutations were defective in CSR whereas their DNA cleavage and SHM activities remained intact. The C-terminal mutants of AID were speculated to exert the dominant negative effect on wild-type (WT) AID whereas its mechanism remains unknown. We generated the JP41 (R190X) mutation in one allele and a null mutation on the other allele in a mouse B cell line (CH12F3-2A) using CRISPR/Cas9 genome-editing tools and studied the effect of JP41 expression on the function of exogenously introduced WT AID fused with estrogen receptor (AIDER) in AIDJP41/∆/AIDER CH12F3-2A cells. We found that JP41 expression strongly suppressed not only CSR but also Igh/c-Myc chromosomal translocations by AIDER. We showed that the dominant negative effect is not evident at the DNA cleavage step but obvious at both deletional and inversional recombination steps. We also confirmed the dominant negative effect of other C-terminal mutants, JP8Bdel (R183X) and P20 (34-aa insertion at residue 182) in AID-deficient spleen B cells. Finally, we showed that the expression of JP41 reduced the binding of AIDER with its cofactors (hnRNP L, SERBP1 and hnRNP U). Together, these data indicate that dominant negative effect of JP41 on CSR is likely due to the depletion of the CSR-specific RNA-binding proteins from WT AID.

Identification of DNA cleavage- and recombination-specific hnRNP cofactors for activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) is essential for antibody class switch recombination (CSR) and somatic hypermutation (SHM). AID originally was postulated to function as an RNA-editing enzyme, based on its strong homology with apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC1), the enzyme that edits apolipoprotein B-100 mRNA in the presence of the APOBEC cofactor APOBEC1 complementation factor/APOBEC complementation factor (A1CF/ACF). Because A1CF is structurally similar to heterogeneous nuclear ribonucleoproteins (hnRNPs), we investigated the involvement of several well-known hnRNPs in AID function by using siRNA knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated disruption. We found that hnRNP K deficiency inhibited DNA cleavage and thereby induced both CSR and SHM, whereas hnRNP L deficiency inhibited only CSR and somewhat enhanced SHM. Interestingly, both hnRNPs exhibited RNA-dependent interactions with AID, and mutant forms of these proteins containing deletions in the RNA-recognition motif failed to rescue CSR. Thus, our study suggests that hnRNP K and hnRNP L may serve as A1CF-like cofactors in AID-mediated CSR and SHM.

Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase.

Activation-induced cytidine deaminase (AID) is essential to class-switch recombination (CSR) and somatic hypermutation (SHM) in both V region SHM and S region SHM (s-SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair (BER) complex, is required for CSR. Strikingly, however, UNG deficiency causes augmentation of SHM, suggesting involvement of distinct functions of UNG in SHM and CSR. Here, we show that noncanonical scaffold functions of UNG regulate s-SHM negatively and CSR positively. The s-SHM suppressive function of UNG is attributed to the recruitment of faithful BER components at the cleaved DNA locus, with competition against error-prone polymerases. By contrast, the CSR-promoting function of UNG enhances AID-dependent S-S synapse formation by recruiting p53-binding protein 1 and DNA-dependent protein kinase, catalytic subunit. Several loss-of-catalysis mutants of UNG discriminated CSR-promoting activity from s-SHM suppressive activity. Taken together, the noncanonical function of UNG regulates the steps after AID-induced DNA cleavage: error-prone repair suppression in s-SHM and end-joining promotion in CSR.

C-terminal region of activation-induced cytidine deaminase (AID) is required for efficient class switch recombination and gene conversion.

Activation-induced cytidine deaminase (AID) introduces single-strand breaks (SSBs) to initiate class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM). CSR is mediated by double-strand breaks (DSBs) at donor and acceptor switch (S) regions, followed by pairing of DSB ends in two S regions and their joining. Because AID mutations at its C-terminal region drastically impair CSR but retain its DNA cleavage and SHM activity, the C-terminal region of AID likely is required for the recombination step after the DNA cleavage. To test this hypothesis, we analyzed the recombination junctions generated by AID C-terminal mutants and found that 0- to 3-bp microhomology junctions are relatively less abundant, possibly reflecting the defects of the classical nonhomologous end joining (C-NHEJ). Consistently, the accumulation of C-NHEJ factors such as Ku80 and XRCC4 was decreased at the cleaved S region. In contrast, an SSB-binding protein, poly (ADP)-ribose polymerase1, was recruited more abundantly, suggesting a defect in conversion from SSB to DSB. In addition, recruitment of critical DNA synapse factors such as 53BP1, DNA PKcs, and UNG at the S region was reduced during CSR. Furthermore, the chromosome conformation capture assay revealed that DNA synapse formation is impaired drastically in the AID C-terminal mutants. Interestingly, these mutants showed relative reduction in GC compared with SHM in chicken DT40 cells. Collectively, our data indicate that the C-terminal region of AID is required for efficient generation of DSB in CSR and GC and thus for the subsequent pairing of cleaved DNA ends during recombination in CSR.

Accumulation of the FACT complex, as well as histone H3.3, serves as a target marker for somatic hypermutation.

Somatic hypermutation (SHM) requires not only the expression of activation-induced cytidine deaminase, but also transcription in the target regions. However, how transcription guides activation-induced cytidine deaminase in targeting SHM to the Ig genes is not fully understood. Here, we found that the "facilitates chromatin transcription" (FACT) complex promotes SHM by RNAi screening of transcription elongation factors. Furthermore, FACT and histone H3.3, a hallmark of transcription-coupled histone turnover, are enriched at the V(D)J region, 5' flanking sequence of the Sµ switch region and the light chain Jκ 5 segment region in the Ig loci. The regions with the most abundant deposition of FACT and H3.3 were also the most efficient targets of SHM. These results demonstrate the importance of histone-exchanging dynamics at the chromatin of SHM targets, especially in Ig genes.

Opinion: uracil DNA glycosylase (UNG) plays distinct and non-canonical roles in somatic hypermutation and class switch recombination.

Activation-induced cytidine deaminase (AID) is essential to class switch recombination (CSR) and somatic hypermutation (SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair complex, is required for CSR. The role of UNG in CSR and SHM is extremely controversial. AID deficiency in mice abolishes both CSR and SHM, while UNG-deficient mice have drastically reduced CSR but augmented SHM raising a possibility of differential functions of UNG in CSR and SHM. Interestingly, UNG has been associated with a CSR-specific repair adapter protein Brd4, which interacts with acetyl histone 4, γH2AX and 53BP1 to promote non-homologous end joining during CSR. A non-canonical scaffold function of UNG, but not the catalytic activity, can be attributed to the recruitment of essential repair proteins associated with the error-free repair during SHM, and the end joining during CSR.

The DSIF subunits Spt4 and Spt5 have distinct roles at various phases of immunoglobulin class switch recombination.

Class-switch recombination (CSR), induced by activation-induced cytidine deaminase (AID), can be divided into two phases: DNA cleavage of the switch (S) regions and the joining of the cleaved ends of the different S regions. Here, we show that the DSIF complex (Spt4 and Spt5), a transcription elongation factor, is required for CSR in a switch-proficient B cell line CH12F3-2A cells, and Spt4 and Spt5 carry out independent functions in CSR. While neither Spt4 nor Spt5 is required for transcription of S regions and AID, expression array analysis suggests that Spt4 and Spt5 regulate a distinct subset of transcripts in CH12F3-2A cells. Curiously, Spt4 is critically important in suppressing cryptic transcription initiating from the intronic Sµ region. Depletion of Spt5 reduced the H3K4me3 level and DNA cleavage at the Sα region, whereas Spt4 knockdown did not perturb the H3K4me3 status and S region cleavage. H3K4me3 modification level thus correlated well with the DNA breakage efficiency. Therefore we conclude that Spt5 plays a role similar to the histone chaperone FACT complex that regulates H3K4me3 modification and DNA cleavage in CSR. Since Spt4 is not involved in the DNA cleavage step, we suspected that Spt4 might be required for DNA repair in CSR. We examined whether Spt4 or Spt5 is essential in non-homologous end joining (NHEJ) and homologous recombination (HR) as CSR utilizes general repair pathways. Both Spt4 and Spt5 are required for NHEJ and HR as determined by assay systems using synthetic repair substrates that are actively transcribed even in the absence of Spt4 and Spt5. Taken together, Spt4 and Spt5 can function independently in multiple transcription-coupled steps of CSR.

Nonimmunoglobulin target loci of activation-induced cytidine deaminase (AID) share unique features with immunoglobulin genes.

Activation-induced cytidine deaminase (AID) is required for both somatic hypermutation and class-switch recombination in activated B cells. AID is also known to target nonimmunoglobulin genes and introduce mutations or chromosomal translocations, eventually causing tumors. To identify as-yet-unknown AID targets, we screened early AID-induced DNA breaks by using two independent genome-wide approaches. Along with known AID targets, this screen identified a set of unique genes (SNHG3, MALAT1, BCL7A, and CUX1) and confirmed that these loci accumulated mutations as frequently as Ig locus after AID activation. Moreover, these genes share three important characteristics with the Ig gene: translocations in tumors, repetitive sequences, and the epigenetic modification of chromatin by H3K4 trimethylation in the vicinity of cleavage sites.

Smoking cessation advice by rheumatologists: results of an international survey.

OBJECTIVE: The aim of this study was to understand practices regarding smoking cessation among rheumatologists for patients with inflammatory rheumatic diseases. METHODS: A survey was sent to the rheumatologists participating in the multinational Quantitative Standard Monitoring of Patients with Rheumatoid Arthritis (QUEST-RA) group. The survey inquired about the clinical practice characteristics and practices regarding smoking cessation (proportion of smokers with inflammatory rheumatic diseases given smoking cessation advice, specific protocols and written advice material, availability of dedicated smoking cessation clinic). RESULTS: Rheumatologists from 44 departments in 25 countries (16 European) completed the survey. The survey involved 395 rheumatologists, of whom 25 (6.3%) were smokers, and 199 nurses for patient education, of whom 44 (22.1%) were smokers. Eight departments (18.1 %) had a specific protocol for smoking cessation; 255 (64.5%) rheumatologists reported giving smoking cessation advice to all or almost all smokers with inflammatory diseases. In a regression model, early arthritis clinics (P = 0.01) and high gross domestic product countries (P = 0.001) were both independently associated with advice by the rheumatologist. Nurse gives advice to most patients in 11 of the 36 (30.5%) departments with nurses for patient education. CONCLUSION: Advice for smoking cessation within rheumatology departments is not homogeneous. In half of the departments, most doctors give advice to quit smoking to all or almost all patients with inflammatory diseases. However, only one in five departments have a specific protocol for smoking cessation. Our data highlight the need to improve awareness of the importance of and better practice implementation of smoking cessation advice for inflammatory rheumatic disease patients.

The association of fatigue, comorbidity burden, disease activity, disability and gross domestic product in patients with rheumatoid arthritis. Results from 34 countries participating in the Quest-RA program.

OBJECTIVES: The aim is to assess the prevalence of comorbidities and to further analyse to which degree fatigue can be explained by comorbidity burden, disease activity, disability and gross domestic product (GDP) in patients with rheumatoid arthritis (RA). METHODS: Nine thousands eight hundred seventy-four patients from 34 countries, 16 with high GDP (>24.000 US dollars [USD] per capita) and 18 low-GDP countries (<24.000 USD) participated in the Quantitative Standard monitoring of Patients with RA (QUEST-RA) study. The prevalence of 31 comorbid conditions, fatigue (0-10 cm visual analogue scale [VAS] [10=worst]), disease activity in 28 joints (DAS28), and physical disability (Health Assessment Questionnaire score [HAQ]) were assessed. Univariate and multivariate linear regression analyses were performed to assess the association between fatigue and comorbidities, disease activity, disability and GDP. RESULTS: Overall, patients reported a median of 2 comorbid conditions of which hypertension (31.5%), osteoporosis (17.6%), osteoarthritis (15.5%) and hyperlipidaemia (14.2%) were the most prevalent. The majority of comorbidities were more common in high-GDP countries. The median fatigue score was 4.4 (4.8 in low-GDP countries and 3.8 in high-GDP countries, p<0.001). In low-GDP countries 25.4% of the patients had a high level of fatigue (>6.6) compared with 23.0% in high-GDP countries (p<0.001). In univariate analysis, fatigue increased with increasing number of comorbidities, disease activity and disability in both high- and low-GDP countries. In multivariate analysis of all countries, these 3 variables explained 29.4% of the variability, whereas GDP was not significant. CONCLUSIONS: Fatigue is a widespread problem associated with high comorbidity burden, disease activity and disability regardless of GDP.

An evolutionary view of the mechanism for immune and genome diversity.

An ortholog of activation-induced cytidine deaminase (AID) was, evolutionarily, the first enzyme to generate acquired immune diversity by catalyzing gene conversion and probably somatic hypermutation (SHM). AID began to mediate class switch recombination (CSR) only after the evolution of frogs. Recent studies revealed that the mechanisms for generating immune and genetic diversity share several critical features. Meiotic recombination, V(D)J recombination, CSR, and SHM all require H3K4 trimethyl histone modification to specify the target DNA. Genetic instability related to dinucleotide or triplet repeats depends on DNA cleavage by topoisomerase 1, which also initiates DNA cleavage in both SHM and CSR. These similarities suggest that AID hijacked the basic mechanism for genome instability when AID evolved in jawless fish. Thus, the risk of introducing genome instability into nonimmunoglobulin loci is unavoidable but tolerable compared with the advantage conferred on the host of being protected against pathogens by the enormous Ig diversification.