Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 41
Filter
Add more filters










Publication year range
1.
Nucleic Acids Res ; 52(8): 4422-4439, 2024 May 08.
Article in English | MEDLINE | ID: mdl-38567724

ABSTRACT

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.


Subject(s)
Bromodomain Containing Proteins , DNA End-Joining Repair , Immunoglobulin Class Switching , Transcription Factors , Animals , Humans , Mice , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Bromodomain Containing Proteins/metabolism , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , DNA Breaks, Double-Stranded , DNA End-Joining Repair/genetics , DNA Repair , Immunoglobulin Class Switching/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Recombination, Genetic , Transcription Factors/metabolism
2.
Cell Rep ; 42(3): 112284, 2023 03 28.
Article in English | MEDLINE | ID: mdl-36943867

ABSTRACT

B cells generate functionally different classes of antibodies through class-switch recombination (CSR), which requires classical non-homologous end joining (C-NHEJ) to join the DNA breaks at the donor and acceptor switch (S) regions. We show that the RNA-binding protein HNRNPU promotes C-NHEJ-mediated S-S joining through the 53BP1-shieldin DNA-repair complex. Notably, HNRNPU binds to the S region RNA/DNA G-quadruplexes, contributing to regulating R-loop and single-stranded DNA (ssDNA) accumulation. HNRNPU is an intrinsically disordered protein that interacts with both C-NHEJ and R-loop complexes in an RNA-dependent manner. Strikingly, recruitment of HNRNPU and the C-NHEJ factors is highly sensitive to liquid-liquid phase separation inhibitors, suggestive of DNA-repair condensate formation. We propose that HNRNPU facilitates CSR by forming and stabilizing the C-NHEJ ribonucleoprotein complex and preventing excessive R-loop accumulation, which otherwise would cause persistent DNA breaks and aberrant DNA repair, leading to genomic instability.


Subject(s)
DNA-Binding Proteins , R-Loop Structures , DNA , DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA, Single-Stranded , DNA-Binding Proteins/metabolism , Immunoglobulin Class Switching , Immunoglobulin Isotypes/genetics , RNA , Heterogeneous-Nuclear Ribonucleoprotein U/metabolism
3.
Sci Adv ; 8(47): eadd1466, 2022 11 25.
Article in English | MEDLINE | ID: mdl-36427307

ABSTRACT

The transcriptional coactivator Med12 regulates gene expression through its kinase module. Here, we show a kinase module-independent function of Med12 in CSR. Med12 is essential for super-enhancer activation by collaborating with p300-Jmjd6/Carm1 coactivator complexes. Med12 loss decreases H3K27 acetylation and eRNA transcription with concomitant impairment of AID-induced DNA breaks, S-S synapse formation, and 3'RR-Eµ interaction. CRISPR-dCas9-mediated enhancer activation reestablishes the epigenomic and transcriptional hallmarks of the super-enhancer and fully restores the Med12 depletion defects. Moreover, 3'RR-derived eRNAs are critical for promoting S region epigenetic regulation, synapse formation, and recruitment of Med12 and AID to the IgH locus. We find that XLID syndrome-associated Med12 mutations are defective in both 3'RR eRNA transcription and CSR, suggesting that B and neuronal cells may have cell-specific super-enhancer dysfunctions. We conclude that Med12 is essential for IgH 3'RR activation/eRNA transcription and plays a central role in AID-induced antibody gene diversification and genomic instability in B cells.


Subject(s)
Immunoglobulin Class Switching , Immunoglobulin Isotypes , Humans , RNA , Epigenesis, Genetic , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/metabolism , Transcription Factors/metabolism , Syndrome , Chromatin/genetics , Mediator Complex/genetics , Mediator Complex/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism
4.
Vaccines (Basel) ; 9(10)2021 Sep 22.
Article in English | MEDLINE | ID: mdl-34696158

ABSTRACT

The availability and adequate balance of deoxyribonucleoside triphosphate (dNTP) is an important determinant of both the fidelity and the processivity of DNA polymerases. Therefore, maintaining an optimal balance of the dNTP pool is critical for genomic stability in replicating and quiescent cells. Since DNA synthesis is required not only in genomic replication but also in DNA damage repair and recombination, the abnormalities in the dNTP pool affect a wide range of chromosomal activities. The generation of antibody diversity relies on antigen-independent V(D)J recombination, as well as antigen-dependent somatic hypermutation and class switch recombination. These processes involve diverse sets of DNA polymerases, which are affected by the dNTP pool imbalances. This review discusses the role of the optimal dNTP pool balance in the diversification of antibody encoding genes.

5.
EMBO J ; 40(12): e106393, 2021 06 15.
Article in English | MEDLINE | ID: mdl-33938017

ABSTRACT

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.


Subject(s)
DNA Helicases/genetics , DNA Repair , DNA-Binding Proteins/genetics , Histones/genetics , RNA-Binding Proteins/genetics , Trans-Activators/genetics , Animals , B-Lymphocytes , Cell Line , Humans , Immunoglobulin Class Switching , Mice, Inbred C57BL , RNA, Small Interfering/genetics , Recombination, Genetic
6.
EMBO J ; 39(15): e102931, 2020 08 03.
Article in English | MEDLINE | ID: mdl-32511795

ABSTRACT

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.


Subject(s)
DNA Repair , Deoxyribonucleotides/metabolism , Immunoglobulin Class Switching , SAM Domain and HD Domain-Containing Protein 1/metabolism , Cell Line , Deoxyribonucleotides/genetics , Humans , SAM Domain and HD Domain-Containing Protein 1/genetics
7.
Proc Natl Acad Sci U S A ; 117(21): 11624-11635, 2020 05 26.
Article in English | MEDLINE | ID: mdl-32385154

ABSTRACT

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.


Subject(s)
Antibodies , Cytidine Deaminase , DNA Breaks , Heterogeneous-Nuclear Ribonucleoprotein K , RNA-Binding Motifs/genetics , Animals , Antibodies/chemistry , Antibodies/genetics , Antibodies/metabolism , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , Heterogeneous-Nuclear Ribonucleoprotein K/chemistry , Heterogeneous-Nuclear Ribonucleoprotein K/genetics , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Humans , Immunoglobulin Class Switching/genetics , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/metabolism , Mice , Somatic Hypermutation, Immunoglobulin/genetics
8.
Bio Protoc ; 10(23): e3837, 2020 Dec 05.
Article in English | MEDLINE | ID: mdl-33659486

ABSTRACT

Protein-protein interactions play key roles in nuclear processes including transcription, replication, DNA damage repair, and recombination. Co-immunoprecipitation (Co-IP) followed by western blot or mass spectrometry is an invaluable approach to identify protein-protein interactions. One of the challenges in the Co-IP of a protein localized to nucleus is the extraction of nuclear proteins from sub-nuclear fractions without losing physiologically relevant protein interactions. Here we describe a protocol for native Co-IP, which was originally used to successfully identify previously known as well novel topoisomerase 1 (TOP1) interacting proteins. In this protocol, we first extracted nuclear proteins by sequentially increasing detergent and salt concentrations, the extracted fractions were then diluted, pooled, and used for Co-IP. This protocol can be used to identify protein-interactome of other chromatin-associated proteins in a variety of mammalian cells.

9.
Int Immunol ; 29(11): 525-537, 2017 12 30.
Article in English | MEDLINE | ID: mdl-29136157

ABSTRACT

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.


Subject(s)
Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , Immunoglobulin Class Switching/genetics , Immunoglobulin Class Switching/immunology , Mutation , Animals , Cell Line , Cytidine Deaminase/immunology , Mice
10.
Proc Natl Acad Sci U S A ; 113(11): E1545-54, 2016 Mar 15.
Article in English | MEDLINE | ID: mdl-26929374

ABSTRACT

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.


Subject(s)
Cytidine Deaminase/chemistry , Cytidine Deaminase/metabolism , RNA-Binding Proteins/metabolism , Animals , Cytidine Deaminase/genetics , DNA/metabolism , Glycerol/chemistry , HEK293 Cells , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Mice, Mutant Strains , Mutation , Protein Interaction Mapping/methods , Protein Multimerization , RNA-Binding Proteins/chemistry , Recombination, Genetic , Structure-Activity Relationship
11.
Nat Commun ; 7: 10549, 2016 Feb 04.
Article in English | MEDLINE | ID: mdl-26842758

ABSTRACT

Topoisomerase 1, an enzyme that relieves superhelical tension, is implicated in transcription-associated mutagenesis and genome instability-associated with neurodegenerative diseases as well as activation-induced cytidine deaminase. From proteomic analysis of TOP1-associated proteins, we identify SMARCA4, an ATP-dependent chromatin remodeller; FACT, a histone chaperone; and H3K4me3, a transcriptionally active chromatin marker. Here we show that SMARCA4 knockdown in a B-cell line decreases TOP1 recruitment to chromatin, and leads to increases in Igh/c-Myc chromosomal translocations, variable and switch region mutations and negative superhelicity, all of which are also observed in response to TOP1 knockdown. In contrast, FACT knockdown inhibits association of TOP1 with H3K4me3, and severely reduces DNA cleavage and Igh/c-Myc translocations, without significant effect on TOP1 recruitment to chromatin. We thus propose that SMARCA4 is involved in the TOP1 recruitment to general chromatin, whereas FACT is required for TOP1 binding to H3K4me3 at non-B DNA containing chromatin for the site-specific cleavage.


Subject(s)
Chromatin/metabolism , DNA Helicases/genetics , DNA Topoisomerases, Type I/genetics , Genomic Instability/genetics , Histone Chaperones/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Animals , B-Lymphocytes , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/genetics , Chromatin Assembly and Disassembly/genetics , Chromatin Immunoprecipitation , DNA Breaks, Double-Stranded , DNA Cleavage , DNA Helicases/metabolism , DNA Topoisomerases, Type I/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Flow Cytometry , Gene Knockdown Techniques , Genes, Immunoglobulin Heavy Chain , Genes, myc , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Histone Chaperones/metabolism , Humans , Immunoprecipitation , Mice , Nuclear Proteins/metabolism , Proteome , Transcription Factors/metabolism , Transcription, Genetic , Transcriptional Elongation Factors/genetics , Transcriptional Elongation Factors/metabolism , Translocation, Genetic
12.
Proc Natl Acad Sci U S A ; 112(18): 5791-6, 2015 May 05.
Article in English | MEDLINE | ID: mdl-25902538

ABSTRACT

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.


Subject(s)
Cytidine Deaminase/chemistry , Cytidine Deaminase/metabolism , DNA/chemistry , Heterogeneous-Nuclear Ribonucleoproteins/chemistry , RNA-Binding Proteins/metabolism , APOBEC-1 Deaminase , Cell Line, Tumor , Cell Separation , Flow Cytometry , Genetic Complementation Test , HEK293 Cells , Heterogeneous-Nuclear Ribonucleoprotein K/chemistry , Heterogeneous-Nuclear Ribonucleoprotein L/chemistry , Humans , Immunoglobulin A/chemistry , Immunoglobulin Class Switching , Immunoglobulin Heavy Chains/chemistry , Mutation , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Small Interfering/metabolism , Recombination, Genetic , Somatic Hypermutation, Immunoglobulin
13.
Int Immunol ; 26(10): 575-8, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24994819

ABSTRACT

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.


Subject(s)
Immunoglobulin Class Switching , Somatic Hypermutation, Immunoglobulin , Uracil-DNA Glycosidase/metabolism , Animals , Catalysis , Cytidine Deaminase/metabolism , DNA Breaks, Single-Stranded , Mice , Uracil-DNA Glycosidase/genetics
14.
Mol Cell ; 55(1): 97-110, 2014 Jul 03.
Article in English | MEDLINE | ID: mdl-24954901

ABSTRACT

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.


Subject(s)
DNA End-Joining Repair , Immunoglobulin Class Switching/genetics , Nuclear Proteins/physiology , Transcription Factors/physiology , Animals , Cell Line , Chromatin/metabolism , DNA Damage , Mice , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Recombination, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism
15.
Proc Natl Acad Sci U S A ; 111(11): E1016-24, 2014 Mar 18.
Article in English | MEDLINE | ID: mdl-24591630

ABSTRACT

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.


Subject(s)
Cytidine Deaminase/metabolism , Immunoglobulin Class Switching/immunology , Immunoglobulin Switch Region/genetics , Models, Molecular , Somatic Hypermutation, Immunoglobulin/immunology , Uracil-DNA Glycosidase/metabolism , Animals , Chromatin Immunoprecipitation , Cytidine Deaminase/genetics , DNA End-Joining Repair/immunology , DNA Primers/genetics , Flow Cytometry , Fluorescence , Green Fluorescent Proteins/genetics , Immunoprecipitation , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Somatic Hypermutation, Immunoglobulin/genetics , Uracil-DNA Glycosidase/genetics
16.
Proc Natl Acad Sci U S A ; 111(6): 2253-8, 2014 Feb 11.
Article in English | MEDLINE | ID: mdl-24469810

ABSTRACT

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.


Subject(s)
Cytidine Deaminase/metabolism , Gene Conversion , Immunoglobulin Switch Region/genetics , Recombination, Genetic , Animals , Cells, Cultured , Chromatin Immunoprecipitation , Cytidine Deaminase/chemistry , Cytidine Deaminase/genetics , DNA End-Joining Repair , Flow Cytometry , Mice , Mice, Inbred C57BL , Mice, Knockout
17.
Proc Natl Acad Sci U S A ; 110(19): 7784-9, 2013 May 07.
Article in English | MEDLINE | ID: mdl-23610419

ABSTRACT

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.


Subject(s)
Chromatin/chemistry , DNA-Binding Proteins/metabolism , High Mobility Group Proteins/metabolism , Histones/genetics , Somatic Hypermutation, Immunoglobulin , Transcriptional Elongation Factors/metabolism , Cell Line, Tumor , Chromatin/metabolism , Cinnamates/metabolism , Cytidine Deaminase/genetics , DNA Mutational Analysis , DNA-Binding Proteins/genetics , Genes, Reporter , Genetic Markers , Green Fluorescent Proteins/metabolism , High Mobility Group Proteins/genetics , Humans , Hygromycin B/analogs & derivatives , Hygromycin B/metabolism , Immunoglobulin Class Switching , Immunoglobulins/genetics , RNA Interference , RNA, Small Interfering/metabolism , Sequence Analysis, DNA , Transcription, Genetic , Transcriptional Elongation Factors/genetics
18.
J Biol Chem ; 287(39): 32415-29, 2012 Sep 21.
Article in English | MEDLINE | ID: mdl-22843687

ABSTRACT

H3K4me3 plays a critical role in the activation-induced cytidine deaminase (AID)-induced DNA cleavage of switch (S) regions in the immunoglobulin heavy chain (IgH) locus during class-switch recombination (CSR). The histone chaperone complex facilitates chromatin transcription (FACT) is responsible for forming H3K4me3 at AID target loci. Here we show that the histone chaperone suppressor of Ty6 (Spt6) also participates in regulating H3K4me3 for CSR and for somatic hypermutation in AID target loci. We found that H3K4me3 loss was correlated with defects in AID-induced DNA breakage and reduced mutation frequencies in IgH loci in both S and variable regions and in non-IgH loci such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and small nucleolar RNA host gene 3 (SNHG3). Global gene expression analysis revealed that Spt6 can act as both a positive and negative transcriptional regulator in B cells, affecting ∼5% of the genes that includes suppressor of Ty4 (Spt4) and AID. Interestingly, Spt6 regulates CSR and AID expression through two distinct histone modification pathways, H3K4me3 and H3K36me3, respectively. Tandem SH2 domain of Spt6 plays a critical role in CSR and H3K4me3 regulation involving Set1 histone methyltransferase. We conclude that Spt6 is a unique histone chaperone capable of regulating the histone epigenetic state of both AID targets and the AID locus.


Subject(s)
Cytidine Deaminase/metabolism , Epigenesis, Genetic/physiology , Histones/metabolism , Molecular Chaperones/metabolism , Transcription Factors/metabolism , Cell Line, Tumor , Cytidine Deaminase/genetics , DNA Breaks , Genetic Loci/physiology , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/genetics , Humans , Immunoglobulin Class Switching/physiology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/metabolism , Methylation , Molecular Chaperones/genetics , Protein Processing, Post-Translational/physiology , RNA, Long Noncoding/biosynthesis , RNA, Long Noncoding/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Somatic Hypermutation, Immunoglobulin/physiology , Transcription Factors/genetics
19.
Biochemistry ; 51(26): 5243-56, 2012 Jul 03.
Article in English | MEDLINE | ID: mdl-22712724

ABSTRACT

It is generally assumed that the genetic mechanism for immune diversity is unique and distinct from that for general genome diversity, in part because of the high efficiency and strict regulation of immune diversity. This expectation was partially met by the discovery of RAG1 and -2, which catalyze V(D)J recombination to generate the immune repertoire of B and T lymphocyte receptors. RAG1 and -2 were later shown to be derived from a transposon. On the other hand, activation-induced cytidine deaminase (AID), which mediates both somatic hypermutation (SHM) and the class-switch recombination (CSR) of the immunoglobulin genes, evolved earlier than RAG1 and -2 in jawless vertebrates. This review compares immune diversity and general genome diversity from an evolutionary perspective, shedding light on the roles of DNA-cleaving enzymes and target recognition markers. This comparison revealed that AID-mediated SHM and CSR share the cleaving enzyme topoisomerase 1 with transcription-associated mutation (TAM) and triplet contraction, which is involved in many genetic diseases. These genome-altering events appear to target DNA with non-B structure, which is induced by the inefficient correction of the excessive supercoiling that is caused by active transcription. Furthermore, an epigenetic modification on chromatin (histone H3K4 trimethylation) is used as a mark for DNA cleavage sites in meiotic recombination, V(D)J recombination, CSR, and SHM. We conclude that acquired immune diversity evolved via the appearance of an AID orthologue that utilized a preexisting mechanism for genomic instability, such as TAM.


Subject(s)
Genomic Instability/genetics , Animals , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , DNA Cleavage , Evolution, Molecular , Humans , Immunoglobulin Class Switching/genetics , Somatic Hypermutation, Immunoglobulin/genetics
20.
PLoS Genet ; 8(4): e1002675, 2012.
Article in English | MEDLINE | ID: mdl-22570620

ABSTRACT

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.


Subject(s)
Chromatin , Chromosomal Proteins, Non-Histone , DNA Repair , Homologous Recombination , Immunoglobulin Class Switching , Immunoglobulins , Transcriptional Elongation Factors , Animals , Antigens, Nuclear/genetics , Antigens, Nuclear/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Culture Techniques , Chromatin/genetics , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/immunology , Cytidine Deaminase/genetics , DNA Cleavage , DNA End-Joining Repair/genetics , DNA Repair/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Rearrangement/immunology , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/genetics , Histones/metabolism , Homologous Recombination/genetics , Homologous Recombination/immunology , Immunoglobulin Class Switching/genetics , Immunoglobulin Class Switching/immunology , Immunoglobulins/genetics , Immunoglobulins/metabolism , Ku Autoantigen , Mice , Protein Processing, Post-Translational , Signal Transduction , Transcriptional Elongation Factors/genetics , Transcriptional Elongation Factors/immunology
SELECTION OF CITATIONS
SEARCH DETAIL
...