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During immune responses, B cells engaging a cognate antigen are recruited to GCs in secondary lymphoid organs where they will diversify their BCR to generate highly specific and adapted humoral responses. They do so, by inducing the expression of activation-induced cytidine deaminase (AID), which initiates somatic hypermutation (SHM) and class switch recombination (CSR). AID deaminates cytosines in ss DNA, generating U:G mismatches that are processed to induce ds DNA break intermediates during CSR that result in the expression of a different antibody isotype. Interestingly, hypoxia regions have been reported in GCs and suggesting that hypoxia could modulate the humoral response. Furthermore, hypoxia inducible transcription factor (HIF) can bind to the AID promoter and induce AID expression in a non-B-cell setting, suggesting that it might be involved in the transcriptional induction of AID in B cells, hence, regulating SHM and CSR. We, thus, hypothesized that HIF could regulate the efficiency of CSR. Here, we show that the inactivation of both the HIF-1α and HIF-1β subunits of the HIF transcription factor in murine CH12 B cells results in defective CSR and that this is due to the suboptimal induction of AID expression., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Background & Aims: Malnutrition is associated with adverse clinical outcomes in patients with cirrhosis. Accurate assessment of energy requirements is needed to optimize dietary intake. Resting energy expenditure (REE), the major component of total energy expenditure, can be measured using indirect calorimetry (mREE) or estimated using prediction equations (pREE). This study assessed the usefulness of predicted estimates of REE in this patient population., Methods: Individual mREE data were available for 900 patients with cirrhosis (mean [±1 SD] age 55.7±11.6 years-old; 70% men; 52% south-east Asian) and 282 healthy controls (mean age 36.0±12.8 years-old; 52% men; 18% south-east Asian). Metabolic status was classified using thresholds based on the mean ± 1 SD of the mREE in the healthy controls. Comparisons were made between mREE and pREE estimates obtained using the Harris-Benedict, Mifflin, Schofield and Henry equations. Stepwise regression was used to build 3 new prediction models which included sex, ethnicity, body composition measures, and model for end-stage liver disease scores., Results: The mean mREE was significantly higher in patients than controls when referenced to dry body weight (22.4±3.8 cf. 20.8±2.6 kcal/kg/24 hr; p <0.001); there were no significant sex differences. The mean mREE was significantly higher in Caucasian than Asian patients (23.1±4.4 cf. 21.7±2.9 kcal/kg/24 hr; p <0.001). Overall, 37.1% of Caucasian and 25.3% of Asian patients were classified as hypermetabolic. The differences between mREE and pREE were both statistically and clinically relevant; in the total patient population, pREE estimates ranged from 501 kcal/24 hr less to 548 kcal/24 hr more than the mREE. Newly derived prediction equations provided better estimates of mREE but still had limited clinical utility., Conclusions: Prediction equations do not provide useful estimates of REE in patients with cirrhosis. REE should be directly measured., Lay Summary: People with cirrhosis are often malnourished and this has a detrimental effect on outcome. Provision of an adequate diet is very important and is best achieved by measuring daily energy requirements and adjusting dietary intake accordingly. Prediction equations, which use information on age, sex, weight, and height can be used to estimate energy requirements; however, the results they provide are not accurate enough for clinical use, particularly as they vary according to sex and ethnicity., Competing Interests: Conflicts of interest None of the authors report a conflict of interest in relation to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Centrioles are formed by microtubule triplets in a ninefold symmetric arrangement. In flagellated protists and animal multiciliated cells, accessory structures tethered to specific triplets render the centrioles rotationally asymmetric, a property that is key to cytoskeletal and cellular organization in these contexts. In contrast, centrioles within the centrosome of animal cells display no conspicuous rotational asymmetry. Here, we uncover rotationally asymmetric molecular features in human centrioles. Using ultrastructure expansion microscopy, we show that LRRCC1, the ortholog of a protein originally characterized in flagellate green algae, associates preferentially to two consecutive triplets in the distal lumen of human centrioles. LRRCC1 partially co-localizes and affects the recruitment of another distal component, C2CD3, which also has an asymmetric localization pattern in the centriole lumen. Together, LRRCC1 and C2CD3 delineate a structure reminiscent of a filamentous density observed by electron microscopy in flagellates, termed the 'acorn.' Functionally, the depletion of LRRCC1 in human cells induced defects in centriole structure, ciliary assembly, and ciliary signaling, supporting that LRRCC1 cooperates with C2CD3 to organizing the distal region of centrioles. Since a mutation in the LRRCC1 gene has been identified in Joubert syndrome patients, this finding is relevant in the context of human ciliopathies. Taken together, our results demonstrate that rotational asymmetry is an ancient property of centrioles that is broadly conserved in human cells. Our work also reveals that asymmetrically localized proteins are key for primary ciliogenesis and ciliary signaling in human cells., Competing Interests: NG, PM, MB, DE, CP, MB, QD, LM, TP, VH, BR, JA No competing interests declared, (© 2022, Gaudin et al.)

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID) 1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins 1-3 . Although uracils arising in DNA are accurately repaired 1-4 , how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown 1-3 . Here we performed a genome-wide CRISPR-Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2) 5 and is overexpressed in several cancers 5 . We show that the FAM72A-UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a -/- B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a -/- B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Multidisciplinary care has the potential to improve outcomes among patients with cirrhosis, yet its impact on this population remains unclear, with existing studies demonstrating discrepant results. Using data from the multidisciplinary outpatient Cirrhosis Care Clinic (CCC) at the University of Alberta Hospital, we aimed to evaluate acute care utilization and survival outcomes of patients followed by the CCC compared with those receiving standard care (SC)., Methods: We performed a retrospective chart review of 212 patients with cirrhosis admitted to University of Alberta Hospital between 2014 and 2015. CCC patients ( n = 36) were followed through the CCC before index admission. SC patients ( n = 176) were managed outside of the CCC. Readmission time in hospital was collected until 1 year, death, or liver transplant., Results: CCC patients had more advanced liver disease (higher prevalence of ascites, encephalopathy, and varices). Despite this, acute care utilization was significantly lower among CCC patients (adjusted length of stay lower by 3 days, p = 0.03, and adjusted survival days spent in hospital lower by 9%, p = 0.02). CCC patients also had improved 1-year transplant-free survival, with an adjusted 1-year relative risk reduction of 53% ( p = 0.03). Total mean cost of care was lower in the CCC group by $2,280 per patient-month of life., Discussion: For patients admitted with cirrhosis, specialized post-discharge multidisciplinary outpatient care is associated with decreased acute care utilization, improved 1-year transplant-free survival probability, and the potential for cost savings to the system., Competing Interests: The authors have nothing to disclose., (Copyright © 2021 Canadian Association for the Study of the Liver.)

The autosomal recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a genetically heterogeneous disorder. Despite the identification of the underlying gene defects, it is unclear how mutations in any of the four known ICF genes cause a primary immunodeficiency. Here we demonstrate that loss of ZBTB24 in B cells from mice and ICF2 patients affects nonhomologous end-joining (NHEJ) during immunoglobulin class-switch recombination and consequently impairs immunoglobulin production and isotype balance. Mechanistically, we found that ZBTB24 associates with poly(ADP-ribose) polymerase 1 (PARP1) and stimulates its auto-poly(ADP-ribosyl)ation. The zinc-finger in ZBTB24 binds PARP1-associated poly(ADP-ribose) chains and mediates the PARP1-dependent recruitment of ZBTB24 to DNA breaks. Moreover, through its association with poly(ADP-ribose) chains, ZBTB24 protects them from degradation by poly(ADP-ribose) glycohydrolase (PARG). This facilitates the poly(ADP-ribose)-dependent assembly of the LIG4/XRCC4 complex at DNA breaks, thereby promoting error-free NHEJ. Thus, we uncover ZBTB24 as a regulator of PARP1-dependent NHEJ and class-switch recombination, providing a molecular basis for the immunodeficiency in ICF2 syndrome., Competing Interests: Disclosure: The authors declare no competing interests exist., (© 2020 Helfricht et al.)

Background: Drowning is still a major cause of accidental death worldwide. In 1997, Szpilman proposed a classification of drowning that has become the reference. As considerable efforts have been made to improve prevention and care, it seemed appropriate to reassess the prognosis and clinical presentation of drowning patients more than 20 years after this first publication. The aim of this study is to provide a reappraisal of patients who need advanced health care and a precise description of their respective neurologic, respiratory, and hemodynamic profiles., Methods: This retrospective study was conducted over four consecutive summer periods between 2014 and 2017 in ICUs located in France, French Polynesia, and the French Antilles. Patients were classified according to the drowning classification system proposed by Szpilman., Results: During the study period, 312 drowning patients were admitted with severe clinical presentation (grades 2-6). All patients benefited from rapid extraction from the water (< 10 min for all) and specialized care (emergency medical services), starting from the prehospital period. Although the global hospital mortality was similar to that previously reported (18.5%), great differences existed among the severity grades. Respective grade mortalities were low for grades 2 through 5 (grade 2, 0%; grade 3, 3%; grade 4, 0%; grade 5, 2%), and the mortality for grade 6 remained similar to that previously reported (54%). These results confirmed that the occurrence of cardiac arrest after drowning is still bad prognosis. Conversely, for other grades, this study strengthens the importance of specialized intervention to interrupt the drowning process., Conclusions: On the basis of these results, drowning-related cardiac arrest is still the prognosis cornerstone. For other victims, the prognosis was better than previously expected, which strengthens the importance of specialized intervention to interrupt the drowning process., (Copyright © 2020 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

Background: The centrosome regulates cell spatial organisation by controlling the architecture of the microtubule (MT) cytoskeleton. Conversely, the position of the centrosome within the cell depends on cytoskeletal networks it helps organizing. In mammalian cells, centrosome positioning involves a population of MT stably anchored at centrioles, the core components of the centrosome. An MT-anchoring complex containing the proteins ninein and Cep170 is enriched at subdistal appendages (SAP) that decorate the older centriole (called mother centriole) and at centriole proximal ends. Here, we studied the role played at the centrosome by hVFL3/CCDC61, the human ortholog of proteins required for anchoring distinct sets of cytoskeletal fibres to centrioles in unicellular eukaryotes., Results: We show that hVFL3 co-localises at SAP and at centriole proximal ends with components of the MT-anchoring complex, and physically interacts with Cep170. Depletion of hVFL3 increased the distance between mother and daughter centrioles without affecting the assembly of a filamentous linker that tethers the centrioles and contains the proteins rootletin and C-Nap1. When the linker was disrupted by inactivating C-Nap1, hVFL3-depletion exacerbated centriole splitting, a phenotype also observed following depletion of other SAP components. This supported that hVFL3 is required for SAP function, which we further established by showing that centrosome positioning is perturbed in hVFL3-depleted interphase cells. Finally, we found that hVFL3 is an MT-binding protein., Conclusions and Significance: Together, our results support that hVFL3 is required for anchoring MT at SAP during interphase and ensuring proper centrosome cohesion and positioning. The role of the VFL3 family of proteins thus appears to have been conserved in evolution despite the great variation in the shape of centriole appendages in different eukaryotic species., (© 2019 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

PARP3 has been shown to be a key driver of TGFβ-induced epithelial-to-mesenchymal transition (EMT) and stemness in breast cancer cells, emerging as an attractive therapeutic target. Nevertheless, the therapeutic value of PARP3 inhibition has not yet been assessed. Here we investigated the impact of the absence of PARP3 or its inhibition on the tumorigenicity of BRCA1-proficient versus BRCA1-deficient breast cancer cell lines, focusing on the triple-negative breast cancer subtype (TNBC). We show that PARP3 knockdown exacerbates centrosome amplification and genome instability and reduces survival of BRCA1-deficient TNBC cells. Furthermore, we engineered PARP3 -/- BRCA1-deficient or BRCA1-proficient TNBC cell lines using the CRISPR/nCas9 D10A gene editing technology and demonstrate that the absence of PARP3 selectively suppresses the growth, survival and in vivo tumorigenicity of BRCA1-deficient TNBC cells, mechanistically via effects associated with an altered Rictor/mTORC2 signaling complex resulting from enhanced ubiquitination of Rictor. Accordingly, PARP3 interacts with and ADP-ribosylates GSK3β, a positive regulator of Rictor ubiquitination and degradation. Importantly, these phenotypes were rescued by re-expression of a wild-type PARP3 but not by a catalytic mutant, demonstrating the importance of PARP3's catalytic activity. Accordingly, reduced survival and compromised Rictor/mTORC2 signaling were also observed using a cell-permeable PARP3-specific inhibitor. We conclude that PARP3 and BRCA1 are synthetic lethal and that targeting PARP3's catalytic activity is a promising therapeutic strategy for BRCA1-associated cancers via the Rictor/mTORC2 signaling pathway.

A large number of genetic studies in yeast rely on the use of expression vectors. To facilitate the experimental approach of these studies, several collections of expression vectors have been generated (YXplac, pRS series, etc.). Subsequently, these collections have been expanded by adding more diversity to many of the plasmid features, including new selection markers and new promoter sequences. However, the ever growing number of plasmid features makes it unrealistic for research labs to maintain an up-to-date collection of plasmids. Here, we developed the COSPLAY toolbox: a Golden Gate approach based on the scheme of a simple modular plasmid that recapitulates and completes all the properties of the pRS plasmids. The COSPLAY toolbox contains a basal collection of individual functional modules. Moreover, we standardized a simple and rapid, software-assisted protocol which facilitates the addition of new personalized modules. Finally, our toolbox includes the possibility to select a genomic target location and to perform a single copy integration of the expression vector., Competing Interests: The authors have declared that no competing interests exist.

Malnutrition is associated with significant morbidity and mortality in cirrhosis. An accurate nutrition prescription is an essential component of care, often estimated using time-efficient predictive equations. Our aim was to compare resting energy expenditure (REE) estimated using predictive equations (predicted REE, pREE) versus REE measured using gold-standard, indirect calorimetry (IC) (measured REE, mREE). We included full-text English language studies in adults with cirrhosis comparing pREE versus mREE. The mean differences across studies were pooled with RevMan 5.3 software. A total of 17 studies (1883 patients) were analyzed. The pooled cohort was comprised of 65% men with a mean age of 53 ± 7 years. Only 45% of predictive equations estimated energy requirements to within 90⁻110% of mREE using IC. Eighty-three percent of predictive equations underestimated and 28% overestimated energy needs by ±10%. When pooled, the mean difference between the mREE and pREE was lowest for the Harris⁻Benedict equation, with an underestimation of 54 (95% CI: 30⁻137) kcal/d. The pooled analysis was associated with significant heterogeneity (I 2 = 94%). In conclusion, predictive equations calculating REE have limited accuracy in patients with cirrhosis, most commonly underestimating energy requirements and are associated with wide variations in individual comparative data.

Semantic transparency has been in the focus of psycholinguistic research for decades, with the controversy about the time course of the application of morpho-semantic information during the processing of morphologically complex words not yet resolved. This study reports two masked priming studies with English - ness and Russian - ost' nominalisations, investigating how semantic transparency modulates native speakers' morphological priming effects at short and long stimulus onset asynchronies (SOAs). In both languages, we found increased morphological priming for nominalisations at the transparent end of the scale (e.g. paleness - pale) in comparison to items at the opaque end of the scale (e.g. business - busy) but only at longer prime durations. The present findings are in line with models that posit an initial phase of morpho-orthographic (semantically blind) decomposition.

Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VANIMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells., (© 2018 Conic et al.)

Systemic lupus erythematosus (SLE) is a severe and heterogeneous autoimmune disease with a complex genetic etiology, characterized by the production of various pathogenic autoantibodies, which participate in end-organ damages. The majority of human SLE occurs in adults as a polygenic disease, and clinical flares interspersed with silent phases of various lengths characterize the usual evolution of the disease in time. Trying to understand the mechanism of the different phenotypic traits of the disease, and considering the central role of B cells in SLE, we previously performed a detailed wide analysis of gene expression variation in B cells from quiescent SLE patients. This analysis pointed out an overexpression of TRIB1 . TRIB1 is a pseudokinase that has been implicated in the development of leukemia and also metabolic disorders. It is hypothesized that Trib1 plays an adapter or scaffold function in signaling pathways, notably in MAPK pathways. Therefore, we planned to understand the functional significance of TRIB1 overexpression in B cells in SLE. We produced a new knock-in model with B-cell-specific overexpression of Trib1 . We showed that overexpression of Trib1 specifically in B cells does not impact B cell development nor induce any development of SLE symptoms in the mice. By contrast, Trib1 has a negative regulatory function on the production of immunoglobulins, notably IgG1, but also on the production of autoantibodies in an induced model. We observed a decrease of Erk activation in BCR-stimulated Trib1 overexpressing B cells. Finally, we searched for Trib1 partners in B cells by proteomic analysis in order to explore the regulatory function of Trib1 in B cells. Interestingly, we find an interaction between Trib1 and CD72, a negative regulator of B cells whose deficiency in mice leads to the development of autoimmunity. In conclusion, the overexpression of Trib1 could be one of the molecular pathways implicated in the negative regulation of B cells during SLE.

DNA breaks are complex DNA lesions that can be repaired by two alternative mechanisms: non-homologous end-joining and homologous recombination. The decision between them depends on the activation of the DNA resection machinery, which blocks non-homologous end-joining and stimulates recombination. On the other hand, post-translational modifications play a critical role in DNA repair. We have found that the SUMO E3 ligase CBX4 controls resection through the key factor CtIP. Indeed, CBX4 depletion impairs CtIP constitutive sumoylation and DNA end processing. Importantly, mutating lysine 896 in CtIP recapitulates the CBX4-depletion phenotype, blocks homologous recombination and increases genomic instability. Artificial fusion of CtIP and SUMO suppresses the effects of both the non-sumoylatable CtIP mutant and CBX4 depletion. Mechanistically, CtIP sumoylation is essential for its recruitment to damaged DNA. In summary, sumoylation of CtIP at lysine 896 defines a subpopulation of the protein that is involved in DNA resection and recombination.The choice between non-homologous end-joining and homologous recombination to repair a DNA double-strand break depends on activation of the end resection machinery. Here the authors show that SUMO E3 ligase CBX4 sumoylates subpopulation of CtIP to regulate recruitment to breaks and resection.

Repetitive DNA is packaged into heterochromatin to maintain its integrity. We use CRISPR/Cas9 to induce DSBs in different mammalian heterochromatin structures. We demonstrate that in pericentric heterochromatin, DSBs are positionally stable in G1 and recruit NHEJ factors. In S/G2, DSBs are resected and relocate to the periphery of heterochromatin, where they are retained by RAD51. This is independent of chromatin relaxation but requires end resection and RAD51 exclusion from the core. DSBs that fail to relocate are engaged by NHEJ or SSA proteins. We propose that the spatial disconnection between end resection and RAD51 binding prevents the activation of mutagenic pathways and illegitimate recombination. Interestingly, in centromeric heterochromatin, DSBs recruit both NHEJ and HR proteins throughout the cell cycle. Our results highlight striking differences in the recruitment of DNA repair factors between pericentric and centromeric heterochromatin and suggest a model in which the commitment to specific DNA repair pathways regulates DSB position., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation., (© 2016 Thomas-Claudepierre et al.)

Background: For patients awaiting liver transplantation, we aimed to determine the prevalence and predictors of insufficient protein intake as well as to determine whether very low protein intake was an independent predictor of malnutrition and mortality., Materials and Methods: Adults with cirrhosis who were activated on our local liver transplant waiting list between January 2000 and October 2009 were included. Estimated protein intake was derived from dietary records. Patients with incomplete dietary records were excluded. Multivariable logistic regression and competing risk analysis were used., Results: Of 742 potential patients, 112 were excluded due to insufficient data, leaving 630 patients for evaluation. Mean protein intake was 1.0 ± 0.36 g/kg/d and only 24% of patients met the expert consensus recommended threshold of > 1.2 g/kg of protein per day. Very low protein intake (< 0.8 g/kg/d) was associated with worse liver disease severity (as measured by Child-Pugh or MELD). Protein intake below 0.8 g/kg/d was an independent predictor both of malnutrition as measured by the subjective global assessment (adjusted odds ratio [95% confidence interval (CI)]: 2.0 [1.3-3.0]) and of transplant waiting list mortality (adjusted hazard ratio [95% CI]: 1.8 [1.2-2.7])., Conclusion: In this large cohort of liver transplant waitlisted patients, very low protein intake was prevalent and independently associated with malnutrition and mortality. Unlike many other prognostic factors, protein intake is potentially modifiable. Prospective studies are warranted to evaluate the effect of targeted protein repletion on clinically relevant outcomes such as muscle mass, muscle function, immune function, and mortality., (© 2015 American Society for Parenteral and Enteral Nutrition.)

To generate highly specific and adapted immune responses, B cells diversify their antibody repertoire through mechanisms involving the generation of programmed DNA damage. Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by the recruitment of activation-induced cytidine deaminase (AID) to immunoglobulin loci and by the subsequent generation of DNA lesions, which are differentially processed to mutations during SHM or to double-stranded DNA break intermediates during CSR. The latter activate the DNA damage response and mobilize multiple DNA repair factors, including Parp1 and Parp2, to promote DNA repair and long-range recombination. We examined the contribution of Parp3 in CSR and SHM. We find that deficiency in Parp3 results in enhanced CSR, while SHM remains unaffected. Mechanistically, this is due to increased occupancy of AID at the donor (Sμ) switch region. We also find evidence of increased levels of DNA damage at switch region junctions and a bias towards alternative end joining in the absence of Parp3. We propose that Parp3 plays a CSR-specific role by controlling AID levels at switch regions during CSR.

Faithful DNA repair is essential to avoid chromosomal rearrangements and promote genome integrity. Nuclear organization has emerged as a key parameter in the formation of chromosomal translocations, yet little is known as to whether DNA repair can efficiently occur throughout the nucleus and whether it is affected by the location of the lesion. Here, we induce DNA double-strand breaks (DSBs) at different nuclear compartments and follow their fate. We demonstrate that DSBs induced at the nuclear membrane (but not at nuclear pores or nuclear interior) fail to rapidly activate the DNA damage response (DDR) and repair by homologous recombination (HR). Real-time and superresolution imaging reveal that DNA DSBs within lamina-associated domains do not migrate to more permissive environments for HR, like the nuclear pores or the nuclear interior, but instead are repaired in situ by alternative end-joining. Our results are consistent with a model in which nuclear position dictates the choice of DNA repair pathway, thus revealing a new level of regulation in DSB repair controlled by spatial organization of DNA within the nucleus., (© 2014 Lemaître et al.; Published by Cold Spring Harbor Laboratory Press.)

We report results from an eye-tracking during listening study examining English-speaking adults' online processing of reflexive pronouns, and specifically whether the search for an antecedent is restricted to syntactically appropriate positions. Participants listened to a short story where the recipient of an object was introduced with a reflexive, and were asked to identify the object recipient as quickly as possible. This allowed for the recording of participants' offline interpretation of the reflexive, response times, and eye movements on hearing the reflexive. Whilst our offline results show that the ultimate interpretation for reflexives was constrained by binding principles, the response time, and eye-movement data revealed that during processing participants were temporarily distracted by a structurally inappropriate competitor antecedent when this was prominent in the discourse. These results indicate that in addition to binding principles, online referential decisions are also affected by discourse-level information.

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to switch regions and by the subsequent generation of double-stranded DNA breaks (DSBs). These DNA breaks are ultimately resolved through the nonhomologous end joining (NHEJ) pathway. We show that during CSR, AID associates with subunits of cohesin, a complex previously implicated in sister chromatid cohesion, DNA repair, and the formation of DNA loops between enhancers and promoters. Furthermore, we implicate the cohesin complex in the mechanism of CSR by showing that cohesin is dynamically recruited to the Sμ-Cμ region of the IgH locus during CSR and that knockdown of cohesin or its regulatory subunits results in impaired CSR and increased usage of microhomology-based end joining.

Antibody diversification requires the DNA deaminase AID to induce DNA instability at immunoglobulin (Ig) loci upon B cell stimulation. For efficient cytosine deamination, AID requires single-stranded DNA and needs to gain access to Ig loci, with RNA pol II transcription possibly providing both aspects. To understand these mechanisms, we isolated and characterized endogenous AID-containing protein complexes from the chromatin of diversifying B cells. The majority of proteins associated with AID belonged to RNA polymerase II elongation and chromatin modification complexes. Besides the two core polymerase subunits, members of the PAF complex, SUPT5H, SUPT6H, and FACT complex associated with AID. We show that AID associates with RNA polymerase-associated factor 1 (PAF1) through its N-terminal domain, that depletion of PAF complex members inhibits AID-induced immune diversification, and that the PAF complex can serve as a binding platform for AID on chromatin. A model is emerging of how RNA polymerase II elongation and pausing induce and resolve AID lesions.

Immunoglobulin class switch recombination (CSR) is initiated by double-stranded DNA breaks (DSBs) in switch regions triggered by activation-induced cytidine deaminase (AID). Although CSR correlates with epigenetic modifications at the IgH locus, the relationship between these modifications and AID remains unknown. In this study, we show that during CSR, AID forms a complex with KAP1 (KRAB domain-associated protein 1) and HP1 (heterochromatin protein 1) that is tethered to the donor switch region (Sμ) bearing H3K9me3 (trimethylated histone H3 at lysine 9) in vivo. Furthermore, in vivo disruption of this complex results in impaired AID recruitment to Sμ, inefficient DSB formation, and a concomitant defect in CSR but not in somatic hypermutation. We propose that KAP1 and HP1 tether AID to H3K9me3 residues at the donor switch region, thus providing a mechanism linking AID to epigenetic modifications during CSR.