USP7 promotes IgA class switching through stabilizing RUNX3 for germline transcription activation.

Class switch recombination (CSR) diversifies the effector functions of antibodies and involves complex regulation of transcription and DNA damage repair. Here, we show that the deubiquitinase USP7 promotes CSR to immunoglobulin A (IgA) and suppresses unscheduled IgG switching in mature B cells independent of its role in DNA damage repair, but through modulating switch region germline transcription. USP7 depletion impairs Sα transcription, leading to abnormal activation of Sγ germline transcription and increased interaction with the CSR center via loop extrusion for unscheduled IgG switching. Rescue of Sα transcription by transforming growth factor ß (TGF-ß) in USP7-deleted cells suppresses Sγ germline transcription and prevents loop extrusion toward IgG CSR. Mechanistically, USP7 protects transcription factor RUNX3 from ubiquitination-mediated degradation to promote Sα germline transcription. Our study provides evidence for active transcription serving as an anchor to impede loop extrusion and reveals a functional interplay between USP7 and TGF-ß signaling in promoting RUNX3 expression for efficient IgA CSR.

CD9 shapes glucocorticoid sensitivity in pediatric B-cell precursor acute lymphoblastic leukemia.

Resistance to glucocorticoids (GCs), the common agents for remission induction in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), poses a significant therapeutic hurdle. Therefore, dissecting the mechanisms shaping GC resistance could lead to new treatment modalities. Here, we showed that CD9- BCP-ALL cells were preferentially resistant to prednisone and dexamethasone over other standard cytotoxic agents. Concordantly, we identified significantly more poor responders to the prednisone prephase among BCP-ALL patients with a CD9- phenotype, especially for those with adverse presenting features including older age, higher white cell count and BCR-ABL1. Furthermore, gain- and loss-of-function experiments dictated a definitive functional linkage between CD9 expression and GC susceptibility, as demonstrated by the reversal and acquisition of relative GC resistance in CD9low and CD9high BCP-ALL cells, respectively. Despite physical binding to the GC receptor NR3C1, CD9 did not alter its expression, phosphorylation or nuclear translocation but potentiated the induction of GC-responsive genes in GCresistant cells. Importantly, the MEK inhibitor trametinib exhibited higher synergy with GCs against CD9- than CD9+ lymphoblasts to reverse drug resistance in vitro and in vivo. Collectively, our results elucidate a previously unrecognized regulatory function of CD9 in GC sensitivity, and inform new strategies for management of children with resistant BCP-ALL.

A Catalytic Three-Component Aminofluorination of Unactivated Alkenes with Electron-Rich Amino Sources.

We present herein a copper-catalyzed three-component aminofluorination of unactivated alkenes with N-bromodialkylamines and readily available nucleophilic fluoride under the assistance of a bidentate auxiliary. This protocol exhibits excellent functional group tolerance toward a wide range of unactivated alkenes and N-bromodialkylamines to furnish the corresponding ß-fluoroalkylamines in a highly regio- and diastereoselective manner. The appropriate choice of nucleophilic fluoro source is essential to make this reaction a reality. Further DFT calculations show that the exothermic ion exchange between external fluoride ion and Cu(II) intermediate provides additional driving force to the irreversible migratory insertion, which offsets the unfavorable reaction energetics associated with the subsequent C(sp3)-F reductive elimination. This finding offers a new avenue to catalytic intermolecular aminofluorination of unactivated alkenes with electron-rich amino sources via a remarkable reductive elimination of Cu(III) species to forge the C(sp3)-F bonds.

C-to-G editing generates double-strand breaks causing deletion, transversion and translocation.

Base editors (BEs) introduce base substitutions without double-strand DNA cleavage. Besides precise substitutions, BEs generate low-frequency 'stochastic' byproducts through unclear mechanisms. Here, we performed in-depth outcome profiling and genetic dissection, revealing that C-to-G BEs (CGBEs) generate substantial amounts of intermediate double-strand breaks (DSBs), which are at the centre of several byproducts. Imperfect DSB end-joining leads to small deletions via end-resection, templated insertions or aberrant transversions during end fill-in. Chromosomal translocations were detected between the editing target and off-targets of Cas9/deaminase origin. Genetic screenings of DNA repair factors disclosed a central role of abasic site processing in DSB formation. Shielding of abasic sites by the suicide enzyme HMCES reduced CGBE-initiated DSBs, providing an effective way to minimize DSB-triggered events without affecting substitutions. This work demonstrates that CGBEs can initiate deleterious intermediate DSBs and therefore require careful consideration for therapeutic applications, and that HMCES-aided CGBEs hold promise as safer tools.

Unique fluorophilic pores engineering within porous aromatic frameworks for trace perfluorooctanoic acid removal.

Perfluorooctanoic acid (PFOA), a representative of per/polyfluorinated alkyl substances, has become a persistent water pollutant of widespread concern due to its biological toxicity and refractory property. In this work, we design and synthesize two porous aromatic frameworks (PAF) of PAF-CF3 and PAF-C2F5 using fluorine-containing alkyl based monomers in tetrahedral geometry. Both PAFs exhibit nanosized pores (∼1.0 nm) of high surface areas (over 800 m2 g-1) and good fluorophilicity. Remarkable adsorption capacity (˃740 mg g-1) and superior efficiency (˃24 g mg-1 h-1) are achieved toward the removal of PFOA with 1 µg L-1 concentration owing to unique C-F···F-C interactions. In particular, PAF-CF3 and PAF-C2F5 are able to reduce the PFOA concentration in water to 37.9 ng L-1 and 43.3 ng L-1, below EPA regulations (70 ng L-1). The reusability and high efficiency give both PAFs a great potential for sewage treatment.

Ultramicroporous Covalent Organic Framework Nanosheets with Functionality Pair for Membrane C2 H2 /C2 H4 Separation.

Gas separation efficiency of covalent organic framework (COF) membrane can be greatly elevated through precise functionalization. A pair-functionalized COF membrane of 1,3,5-triformylphloroglucinol (TP) and isoquinoline-5,8-diamine (IQD) monomers in two and three nodes is designed and synthesized. TP-IQD is crystallized in a two-dimensional structure with a pore size of 6.5 Šand a surface area of 289 m2 g-1 . This COF possesses N-O paired groups which cooperatively interact with C2 H2 instead of C2 H4 . TP-IQD nanosheets of ≈10 µm in width and ≈4 nm in thickness are prepared by mechanical exfoliation; they are further processed with 6FDA-ODA polymer into a hybrid membrane. High porosity and functionality pair of TP-IQD offer the membrane with significantly increased C2 H2 permeability and C2 H2 /C2 H4 selectivity which are 160 % and 430 % higher of pure 6FDA-ODA. The boosted performance demonstrates high efficiency of the pair-functionality strategy for the synthesis of separation-led COFs.

Alterations in cellular metabolisms after TKI therapy for Philadelphia chromosome-positive leukemia in children: A review.

Incidence rates of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) are lower but more aggressive in children than in adults due to different biological and host factors. After the clinical application of tyrosine kinase inhibitor (TKI) blocking BCR/ABL kinase activity, the prognosis of children with CML and Ph+ ALL has improved dramatically. Yet, off-target effects and drug tolerance will occur during the TKI treatments, contributing to treatment failure. In addition, compared to adults, children may need a longer course of TKIs therapy, causing detrimental effects on growth and development. In recent years, accumulating evidence indicates that drug resistance and side effects during TKI treatment may result from the cellular metabolism alterations. In this review, we provide a detailed summary of the current knowledge on alterations in metabolic pathways including glucose metabolism, lipid metabolism, amino acid metabolism, and other metabolic processes. In order to obtain better TKI treatment outcomes and avoid side effects, it is essential to understand how the TKIs affect cellular metabolism. Hence, we also discuss the relevance of cellular metabolism in TKIs therapy to provide ideas for better use of TKIs in clinical practice.

[Construction of Nalm6-Cas9 Cell Line for Genome-Wide Translocation Sequencing].

OBJECTIVE: In order to conduct high-throughput genome-wide translocation sequencing based on CRISPR/Cas9, Nalm6-cas9 monoclonal cell line expressing Cas9 protein was constructed by lentivirus transduction. METHODS: Lentiviral vectors LentiCas9-Blast, pSPAX2, and pMD2.G were used to co-transfect HEK293T cells to obtain recombinant lentivirus. After Nalm6 cells were infected with the recombinant lentivirus, the cells were screened by Blasticidin, and multiple monoclonal cell lines expressing Cas9 protein were obtained by limited dilution. Western blot was used to detect the expression level of Cas9 protein in monoclonal cell lines, and cell count analysis was used to detect the proliferation activity of monoclonal cell lines. LentiCRISPRV2GFP-Δcas9, LentiCRISPRV2GFP-Δcas9-AF4, LentiCRISPRV2GFP-Δ cas9-MLL plasmids were constructed, and transfected with pSPAX2 and pMD2.G, respectively. T vector cloning was used to detect the function of Cas9 protein in Nalm6-Cas9 monoclonal cell line infected with virus. RESULTS: Western blot showed that Nalm6-Cas9_1-6 monoclonal cell line had high expression of Cas9 protein. Cell count analysis showed that high expression of Cas9 protein in Nalm6-Cas9_1-6 monoclonal cell line did not affect cell proliferation activity. The Nalm6-Cas9_1-6 monoclonal cell line had high cleavage activity, and the editing efficiency of AF4 and MLL genes was more than 90% which was determined by T vector cloning. CONCLUSION: Nalm6-Cas9_1-6 monoclonal cell line stably expressing highly active Cas9 protein was obtained, which provided a basis for exploring the translocation of MLL in therapy-related leukemias based on CRISPR/Cas9 genome-wide high-throughput genome-wide translocation sequencing.

Alternative end-joining in BCR gene rearrangements and translocations.

Programmed DNA double-strand breaks (DSBs) occur during antigen receptor gene recombination, namely V(D)J recombination in developing B lymphocytes and class switch recombination (CSR) in mature B cells. Repair of these DSBs by classical end-joining (c-NHEJ) enables the generation of diverse BCR repertoires for efficient humoral immunity. Deletion of or mutation in c-NHEJ genes in mice and humans confer various degrees of primary immune deficiency and predisposition to lymphoid malignancies that often harbor oncogenic chromosomal translocations. In the absence of c-NHEJ, alternative end-joining (A-EJ) catalyzes robust CSR and to a much lesser extent, V(D)J recombination, but the mechanisms of A-EJ are only poorly defined. In this review, we introduce recent advances in the understanding of A-EJ in the context of V(D)J recombination and CSR with emphases on DSB end processing, DNA polymerases and ligases, and discuss the implications of A-EJ to lymphoid development and chromosomal translocations.

Multiple DSB Resection Activities Redundantly Promote Alternative End Joining-Mediated Class Switch Recombination.

Alternative end joining (A-EJ) catalyzes substantial level of antibody class switch recombination (CSR) in B cells deficient for classical non-homologous end joining, featuring increased switch (S) region DSB resection and junctional microhomology (MH). While resection has been suggested to initiate A-EJ in model DSB repair systems using engineered endonucleases, the contribution of resection factors to A-EJ-mediated CSR remains unclear. In this study, we systematically dissected the requirement for individual DSB resection factors in A-EJ-mediated class switching with a cell-based assay system and high-throughput sequencing. We show that while CtIP and Mre11 both are mildly required for CSR in WT cells, they play more critical roles in mediating A-EJ CSR, which depend on the exonuclease activity of Mre11. While DNA2 and the helicase/HRDC domain of BLM are required for A-EJ by mediating long S region DSB resection, in contrast, Exo1's resection-related function does not play any obvious roles for class switching in either c-NHEJ or A-EJ cells, or mediated in an AID-independent manner by joining of Cas9 breaks. Furthermore, ATM and its kinase activity functions at least in part independent of CtIP/Mre11 to mediate A-EJ switching in Lig4-deficient cells. In stark contrast to Lig4 deficiency, 53BP1-deficient cells do not depend on ATM/Mre11/CtIP for residual joining. We discuss the roles for each resection factor in A-EJ-mediated CSR and suggest that the extent of requirements for resection is context dependent.

The 3'-flap endonuclease XPF-ERCC1 promotes alternative end joining and chromosomal translocation during B cell class switching.

Robust alternative end joining (A-EJ) in classical non-homologous end joining (c-NHEJ)-deficient murine cells features double-strand break (DSB) end resection and microhomology (MH) usage and promotes chromosomal translocation. The activities responsible for removing 3' single-strand overhangs following resection and MH annealing in A-EJ remain unclear. We show that, during class switch recombination (CSR) in mature mouse B cells, the structure-specific endonuclease complex XPF-ERCC1SLX4, although not required for normal CSR, represents a nucleotide-excision-repair-independent 3' flap removal activity for A-EJ-mediated CSR. B cells deficient in DNA ligase 4 and XPF-ERCC1 exhibit further impaired class switching, reducing joining to the resected S region DSBs without altering the MH pattern in S-S junctions. In ERCC1-deficient A-EJ cells, 3' single-stranded DNA (ssDNA) flaps that are generated predominantly in S/G2 phase of the cell cycle are susceptible to nuclease resolution. Moreover, ERCC1 promotes c-myc-IgH translocation in Lig4-/- cells. Our study reveals an important role of the flap endonuclease XPF-ERCC1 in A-EJ and oncogenic translocation in mouse B cells.

Copper-Catalyzed Diamination of Unactivated Alkenes With Electron-Rich Amino Sources.

The catalytic intermolecular diamination of unactivated alkenes with electron-rich amino sources is a challenge. Herein, by employing a directing-group strategy, a copper-catalyzed diamination of unactivated alkenes was realized. Symmetrical diamines were efficiently produced in a highly diastereoselective manner with readily available dialkylamines as amino sources, while a one-pot and two-step operation was necessary to produce the unsymmetrical diamines. These reactions were proposed to proceed through aziridinium intermediates.

ATR kinase activity promotes antibody class switch recombination in B cells through cell cycle regulation without suppressing DSB resection and microhomology usage.

Class switch recombination (CSR) changes the effector functions of antibodies and is carried out by classical and alternative nonhomologous end joining (c-NHEJ and A-EJ) of repetitive switch (S) region double-strand breaks (DSBs). The master DNA damage response (DDR) kinase ataxia-telangiectasia mutated (ATM) is critical for CSR in part by suppressing S region DSB resection. However, whether another related DDR kinase ATM- and Rad3-related (ATR) plays similar role in CSR remains elusive. In this study, we investigated the requirement for ATR kinase activity on CSR in both c-NHEJ competent and deficient B cell lines with high-throughput sequencing of S-S junctions. We found that ATR kinase inhibition efficiently blocked both c-NHEJ- and A-EJ-mediated CSR without affecting germline transcription and activation-induced cytosine deaminase expression. In contrast to ATM, ATR does not suppress S region DSB resection and microhomology usage. In addition, ATR kinase inhibition did not affect Cas9-generated DSB end joining by either c-NHEJ and A-EJ. ATR kinase-inhibited stimulated B cells proliferate much slower than controls and exhibited altered cell cycle profile with increased G1 and G2/M phase cells. In summary, our data revealed a role for ATR in promoting both c-NHEJ- and A-EJ-mediated CSR through regulating cell proliferation upon damage without negatively influencing DSB end-joining features.

Presence of Tertiary Lymphoid Organ in Nasal Inverted Papilloma Is Correlated with Eosinophil Infiltration and Local Immunoglobulin Production.

INTRODUCTION: Nasal inverted papilloma (NIP) is a benign tumour with multiple inflammatory cell infiltration. Tertiary lymphoid organs (TLOs) support local antibody production and play important roles in airway inflammation. However, the evidence of TLOs and local immunoglobulins in NIP has not been reported yet. We investigated the presence of TLOs and immunoglobulins in NIP tissues and their association with the clinical-pathological characteristics of NIPs. METHODS: We analyzed the occurrence and composition of TLOs and local immunoglobulins by immunohistochemistry and evaluated the lymph organogenesis associated genes and cytokines by quantitative qPCR and Luminex assays, respectively, in papilloma tissues from 84 NIP cases. RESULTS: TLOs were present in 54% (45/84) of the NIP patients but not in control subjects. TLOs were composed of T cells, B cells, follicular dendritic cells, macrophages, and natural killer cells. Compared to NIP tissues without TLOs, tissues with TLOs showed significantly higher eosinophil infiltration levels (3.5-fold), elevation of lymphorganogenic genes (CXCL12, CXCL13, CCL20, CCL21, CD21L, and lymphotoxin alpha and beta), and increased Th17 (IL-21, IL-22, and GM-CSF) and Th2 (IL-5 and IL-13) cytokine production. Moreover, NIP with TLOs demonstrated a higher number of follicular T helper cells and immunoglobulin-producing plasma cells (CD138+ IgA+, CD138+ IgM+, CD138+ IgE+, and CD138+ IgG+) than those without TLOs, and these antibody-producing cells were positively correlated with the eosinophil number. CONCLUSION: The high frequency of TLOs and excess local immunoglobulin production are associated with an eosinophilic and Th2 skew microenvironment in the NIP mucosa, which would contribute to an important immunopathogenic response during NIP pathogenesis.

REV7 is required for processing AID initiated DNA lesions in activated B cells.

Activation-induced cytidine deaminase (AID) initiates both antibody class switch recombination (CSR) and somatic hypermutation (SHM) in antibody diversification. DNA double-strand break response (DSBR) factors promote rearrangement in CSR, while translesion synthesis (TLS) polymerases generate mutations in SHM. REV7, a component of TLS polymerase zeta, is also a downstream effector of 53BP1-RIF1 DSBR pathway. Here, we study the multi-functions of REV7 and find that REV7 is required for the B cell survival upon AID-deamination, which is independent of its roles in DSBR, G2/M transition or REV1-mediated TLS. The cell death in REV7-deficient activated B cells can be fully rescued by AID-deficiency in vivo. We further identify that REV7-depedent TLS across UNG-processed apurinic/apyrimidinic sites is required for cell survival upon AID/APOBEC deamination. This study dissects the multiple roles of Rev7 in antibody diversification, and discovers that TLS is not only required for sequence diversification but also B cell survival upon AID-initiated lesions.

ERCC6L2 promotes DNA orientation-specific recombination in mammalian cells.

Programmed DNA recombination in mammalian cells occurs predominantly in a directional manner. While random DNA breaks are typically repaired both by deletion and by inversion at approximately equal proportions, V(D)J and class switch recombination (CSR) of immunoglobulin heavy chain gene overwhelmingly delete intervening sequences to yield productive rearrangement. What factors channel chromatin breaks to deletional CSR in lymphocytes is unknown. Integrating CRISPR knockout and chemical perturbation screening we here identify the Snf2-family helicase-like ERCC6L2 as one such factor. We show that ERCC6L2 promotes double-strand break end-joining and facilitates optimal CSR in mice. At the cellular levels, ERCC6L2 rapidly engages in DNA repair through its C-terminal domains. Mechanistically, ERCC6L2 interacts with other end-joining factors and plays a functionally redundant role with the XLF end-joining factor in V(D)J recombination. Strikingly, ERCC6L2 controls orientation-specific joining of broken ends during CSR, which relies on its helicase activity. Thus, ERCC6L2 facilitates programmed recombination through directional repair of distant breaks.

IRF-7 Is a Critical Regulator of Type 2 Innate Lymphoid Cells in Allergic Airway Inflammation.

Allergic asthma is a highly prevalent airway disease triggered by hyperresponsiveness to inhaled allergens. Interferon regulatory factor 7 (IRF7) has been shown to be highly expressed in nasal aspirates from children with asthma. Type 2 innate lymphoid cells (ILC2s) represent the major player in allergic airway inflammation. The role of IRF7 in ILC2-driven asthma remains to be explored. Here, we report that IRF7 expression in murine lung ILC2s is dramatically induced upon papain or interleukin-33 (IL-33) stimulation. ILC2s from asthma patients display a much higher level of IRF7 than those from healthy donors. Deficiency of IRF7 in mice significantly impairs the expansion and function of lung ILC2s in multiple models of allergic asthma. Furthermore, the regulation of ILC2s by IRF7 is cell intrinsic and mediated by the transcription factor Bcl11b. These observations identify IRF7 as a regulator of lung ILC2s, which may have immunotherapeutic value in allergic asthma.

Rapid generation and selection of Cas9-engineering TRP53 R172P mice that do not have off-target effects.

BACKGROUND: Genetic mutations cause severe human diseases, and suitable animal models to study the regulatory mechanisms involved are required. The CRISPR/Cas9 system is a powerful, highly efficient and easily manipulated tool for genetic modifications. However, utilization of CRISPR/Cas9 to introduce point mutations and the exclusion of off-target effects in mice remain challenging. TP53-R175 is one of the most frequently mutated sites in human cancers, and it plays crucial roles in human diseases, including cancers and diabetes. RESULTS: Here, we generated TRP53-R172P mutant mice (C57BL/6 J, corresponding to TP53-R175P in humans) using a single microinjection of the CRISPR/Cas9 system. The optimal parameters comprised gRNA selection, donor designation (silent mutations within gRNA region), the concentration of CRISPR components and the cellular sites of injection. TRP53-R172P conversion was genetically and functionally confirmed. Combination of TA cloning and Sanger sequencing helped identify the correctly targeted mice as well as the off-target effects in the engineered mice, which provide us a strategy to select the on-target mice without off-target effects quickly and efficiently. CONCLUSIONS: A single injection of the this optimized CRISPR/Cas9 system can be applied to introduce particular mutations in the genome of mice without off-target effects to model various human diseases.

Directed Copper-Catalyzed Intermolecular Aminative Difunctionalization of Unactivated Alkenes.

A diverse collection of copper-catalyzed intermolecular aminative difunctionalizations of unactivated alkenes with N-halodialkylamines as the terminal dialkylamino source is reported. A bidentate auxiliary tethered on the alkene substrates is crucial, which can promote the migratory insertion of nonactivated alkenes into the aminyl radical-metal complex and stabilize the resultant high-valent copper intermediate to allow for further transformations. By employing this strategy, the intermolecular aminohalogenation reactions and a three-component aminoazidation reaction of unactivated alkenes with dialkylamino source were successively achieved in a remarkable regio- and stereoselective manner. These reactions were performed under neutral conditions and maintained excellent functional group tolerance toward a wide range of N-halodialkylamines and unactivated alkenes. Further mechanistic studies and DFT calculations supported a concerted migratory insertion of the C-C double bond into the aminyl radical-metal complex to form a Cu(III) intermediate.

DNA double-strand break response factors influence end-joining features of IgH class switch and general translocation junctions.

Ig heavy chain (IgH) class switch recombination (CSR) in B lymphocytes switches IgH constant regions to change antibody functions. CSR is initiated by DNA double-strand breaks (DSBs) within a donor IgH switch (S) region and a downstream acceptor S region. CSR is completed by fusing donor and acceptor S region DSB ends by classical nonhomologous end-joining (C-NHEJ) and, in its absence, by alternative end-joining that is more biased to use longer junctional microhomologies (MHs). Deficiency for DSB response (DSBR) factors, including ataxia telangiectasia-mutated (ATM) and 53BP1, variably impair CSR end-joining, with 53BP1 deficiency having the greatest impact. However, studies of potential impact of DSBR factor deficiencies on MH-mediated CSR end-joining have been technically limited. We now use a robust DSB joining assay to elucidate impacts of deficiencies for DSBR factors on CSR and chromosomal translocation junctions in primary mouse B cells and CH12F3 B-lymphoma cells. Compared with wild-type, CSR and c-myc to S region translocation junctions in the absence of 53BP1, and, to a lesser extent, other DSBR factors, have increased MH utilization; indeed, 53BP1-deficient MH profiles resemble those associated with C-NHEJ deficiency. However, translocation junctions between c-myc DSB and general DSBs genome-wide are not MH-biased in ATM-deficient versus wild-type CH12F3 cells and are less biased in 53BP1- and C-NHEJ-deficient cells than CSR junctions or c-myc to S region translocation junctions. We discuss potential roles of DSBR factors in suppressing increased MH-mediated DSB end-joining and features of S regions that may render their DSBs prone to MH-biased end-joining in the absence of DSBR factors.