SUMMARY Class switch recombination (CSR) requires activation-induced deaminase (AID) to instigate double-stranded DNA breaks at the immunoglobulin locus. DNA breaks activate the DNA damage response (DDR) by inducing phosphorylation of histone H2AX followed by non-homologous end joining (NHEJ) repair. We carried out a genome-wide screen to identify CSR factors. We found that Usp22, Eny2, and Atxn7, members of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitination module, are required for deubiquitination of H2BK120ub following DNA damage, are critical for CSR, and function downstream of AID. The SAGA deubiquitinase activity was required for optimal irradiation-induced γH2AX formation, and failure to remove H2BK120ub inhibits ATM- and DNAPK-induced γH2AX formation. Consistent with this effect, these proteins were found to function upstream of various double-stranded DNA repair pathways. This report demonstrates that deubiquitination of histone H2B impacts the early stages of the DDR and is required for the DNA repair phase of CSR.

It is estimated that in 2017 there were 451 million people with diabetes worldwide. These figures are expected to increase to 693 million by 2045; thus, innovative preventative programs and treatments are a necessity to fight this escalating pandemic disorder. Caveolin-1 (CAV1), an integral membrane protein, is the principal component of caveolae in membranes and is involved in multiple cellular functions such as endocytosis, cholesterol homeostasis, signal transduction, and mechanoprotection. Previous studies demonstrated that CAV1 is critical for insulin receptor-mediated signaling, insulin secretion, and potentially the development of insulin resistance. Here, we summarize the recent progress on the role of CAV1 in diabetes and diabetic complications.

COVID-19 is challenging healthcare preparedness, world economies, and livelihoods. The infection and death rates associated with this pandemic are strikingly variable in different countries. To elucidate this discrepancy, we analyzed 2431 early spread SARS-CoV-2 sequences from GISAID. We estimated continental-wise admixture proportions, assessed haplotype block estimation, and tested for the presence or absence of strains’ recombination. Herein, we identified 1010 unique missense mutations and seven different SARS-CoV-2 clusters. In samples from Asia, a small haplotype block was identified, whereas samples from Europe and North America harbored large and different haplotype blocks with nonsynonymous variants. Variant frequency and linkage disequilibrium varied among continents, especially in North America. Recombination between different strains was only observed in North American and European sequences. In addition, we structurally modelled the two most common mutations, Spike_D614G and Nsp12_P314L, which suggested that these linked mutations may enhance viral entry and replication, respectively. Overall, we propose that genomic recombination between different strains may contribute to SARS-CoV-2 virulence and COVID-19 severity and may present additional challenges for current treatment regimens and countermeasures. Furthermore, our study provides a possible explanation for the substantial second wave of COVID-19 presented with higher infection and death rates in many countries.

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to switch from IgM expression to the expression of downstream isotypes. CSR is preceded by inducible germline (GL) transcription of the constant genes and is controlled by the 3′ regulatory region (3′RR) in a stimulus-dependent manner. Why the 3′RR-mediated upregulation of GL transcription is delayed to the mature B-cell stage is presently unknown. Here we show that mice devoid of an inducible CTCF binding element, located in the α constant gene, display a marked isotype-specific increase of GL transcription in developing and resting splenic B cells and altered CSR in activated B cells. Moreover, insertion of a GL promoter downstream of the CTCF insulator led to premature activation of the ectopic promoter. This study provides functional evidence that the 3′RR has a developmentally controlled potential to constitutively activate GL promoters but that this activity is delayed, at least in part, by the CTCF insulator, which borders a transcriptionally active domain established by the 3′RR in developing B cells. E xpression of complex loci is developmentally programmed or induced by specific stimuli and is often controlled by distant regulatory elements within relatively large chromatin domains. Transcriptional and architectural factors play an important role in the establishment and maintenance of these domains and facilitate long-range interactions between regulatory elements and target promoters (1, 2). The Ig heavy chain (IgH) locus is expressed in a lineage-and developmental stage-dependent manner. Various cis-acting elements including promoters, enhancers, and insulators control IgH locus expression and are engaged in multiple long-range interactions (3, 4).Factors such as YY1, PAX5, IKAROS, CTCF, and Cohesin play important roles in various aspects of long-range events at the IgH locus, including V(D)J recombination, CSR, and promoter/enhancer and enhancer/enhancer interactions (3-6). Multiple CTCF binding elements (CBEs) were reported along the IgH locus. The majority of these CBEs lie within the variable domain (7), and two CBEs were identified within the V H -D intergenic region (7-9). At the 3′ end of the locus, ∼10 CBEs were identified downstream of the 3′RR and are thought to delineate the 3′ border of the IgH locus (10). More recently, a discrete CBE was identified within the α constant gene (11), but its role in vivo is presently unknown.Upon antigen challenge, mature B cells can undergo CSR that allows B cells to change the heavy-chain constant domain of an IgM to IgG, IgE, or IgA. CSR to a particular isotype is induced by specific external stimuli, including antigens, mitogens, cytokines, and intercellular interactions. CSR is mediated by highly repetitive sequences called switch (S) sequences located upstream of the constant exons and is preceded by germline (GL) transcription of the S sequences that originates from GL promoters, named I promoters (12).The 3′RR is composed of four enhance...

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The severity of the new COVID-19 pandemic caused by the SARS-CoV-2 virus is strikingly variable in different global populations. SARS-CoV-2 uses ACE2 as a cell receptor, TMPRSS2 protease, and FURIN peptidase to invade human cells. Here, we investigated 1,378 whole-exome sequences of individuals from the Middle Eastern populations (Kuwait, Qatar, and Iran) to explore natural variations in the ACE2, TMPRSS2, and FURIN genes. We identified two activating variants (K26R and N720D) in the ACE2 gene that are more common in Europeans than in the Middle Eastern, East Asian, and African populations. We postulate that K26R can activate ACE2 and facilitate binding to S-protein RBD while N720D enhances TMPRSS2 cutting and, ultimately, viral entry. We also detected deleterious variants in FURIN that are frequent in the Middle Eastern but not in the European populations. This study highlights specific genetic variations in the ACE2 and FURIN genes that may explain SARS-CoV-2 clinical disparity. We showed structural evidence of the functionality of these activating variants that increase the SARS-CoV-2 aggressiveness. Finally, our data illustrate a significant correlation between ACE2 variants identified in people from Middle Eastern origins that can be further explored to explain the variation in COVID-19 infection and mortality rates globally.

d Activation-induced deaminase (AID) converts DNA cytosines to uracils in immunoglobulin genes, creating antibody diversification. It also causes mutations and translocations that promote cancer. We examined the interplay between uracil creation by AID and its removal by UNG2 glycosylase in splenocytes undergoing maturation and in B cell cancers. The genomic uracil levels remain unchanged in normal stimulated B cells, demonstrating a balance between uracil generation and removal. In stimulated UNG ؊/؊ cells, uracil levels increase by 11-to 60-fold during the first 3 days. In wild-type B cells, UNG2 gene expression and enzymatic activity rise and fall with AID levels, suggesting that UNG2 expression is coordinated with uracil creation by AID. Remarkably, a murine lymphoma cell line, several human B cell cancer lines, and human B cell tumors expressing AID at high levels have genomic uracils comparable to those seen with stimulated UNG ؊/؊ splenocytes. However, cancer cells express UNG2 gene at levels similar to or higher than those seen with peripheral B cells and have nuclear uracil excision activity comparable to that seen with stimulated wild-type B cells. We propose that more uracils are created during B cell cancer development than are removed from the genome but that the uracil creation/excision balance is restored during establishment of cell lines, fixing the genomic uracil load at high levels. When B lymphocytes are activated through antigen presentation, they acquire hypermutations in the immunoglobulin (Ig) genes, facilitating affinity maturation of antibodies. An enzyme, activation-induced deaminase (AID), initiates these events by converting cytosines in DNA to uracil (1-4). The introduction of this rare base into DNA leads to a very high frequency of base substitution mutations in the Ig variable domain (known as somatic hypermutations [SHMs]; reviewed in references 5 and 6). Generation of uracils is also the first step in the creation of strand breaks in the switch regions of Ig genes, leading to the replacement of the constant domain with other domains such as ␥, in a process called class-switch recombination (CSR; reviewed in reference 7). AID also binds near the transcription start sites of a large number of actively transcribed genes (8) and mutates a number of additional genes, including those encoding BCL-6, MYC, PAX-5, and PIM1 (9-12). The uracils generated by AID are thought to be removed by the nuclear form of the uracil-DNA glycosylase, UNG2, creating abasic sites that are repaired by error-prone copying mechanisms causing hypermutations (13,14). Another uracil-DNA glycosylase, SMUG1, is normally considered the backup enzyme for UNG2 (15), but overproduction of SMUG1 is required for it to complement an UNG Ϫ/Ϫ mutant during CSR (16). Additionally, DNA mismatch repair (MMR) also plays a role in shaping the mutation spectrum in SHM (17).There is a strong connection between expression of AID and cancers in animals. Constitutive expression of AID in mice causes T cell cancers (18). Many human...

Advances in sequencing technologies are giving unprecedented insights into the spectrum of somatic mutations underlying acute myeloid leukaemia with a normal karyotype (AML–NK). It is clear that the prognosis of individual patients is strongly influenced by the combination of mutations in their leukaemia and that many leukaemias are composed of multiple subclones, with differential susceptibilities to treatment. Here, we describe a method, employing targeted capture coupled with next-generation sequencing and tailored bioinformatic analysis, for the simultaneous study of 24 genes recurrently mutated in AML–NK. Mutational analysis was performed using open source software and an in-house script (Mutation Identification and Analysis Software), which identified dominant clone mutations with 100% specificity. In each of seven cases of AML–NK studied, we identified and verified mutations in 2–4 genes in the main leukaemic clone. Additionally, high sequencing depth enabled us to identify putative subclonal mutations and detect leukaemia-specific mutations in DNA from remission marrow. Finally, we used normalised read depths to detect copy number changes and identified and subsequently verified a tandem duplication of exons 2–9 of MLL and at least one deletion involving PTEN. This methodology reliably detects sequence and copy number mutations, and can thus greatly facilitate the classification, clinical research, diagnosis and management of AML–NK.