Janet Stavnezer

Mechanism and Regulation of Class Switch Recombination

Antibody class switching occurs in mature B cells in response to antigen stimulation and costimulatory signals. It occurs by a unique type of intrachromosomal deletional recombination within special G-rich tandem repeated DNA sequences [called switch, or S, regions located upstream of each of the heavy chain constant (C(H)) region genes, except Cdelta]. The recombination is initiated by the B cell-specific activation-induced cytidine deaminase (AID), which deaminates cytosines in both the donor and acceptor S regions. AID activity converts several dC bases to dU bases in each S region, and the dU bases are then excised by the uracil DNA glycosylase UNG; the resulting abasic sites are nicked by apurinic/apyrimidinic endonuclease (APE). AID attacks both strands of transcriptionally active S regions, but how transcription promotes AID targeting is not entirely clear. Mismatch repair proteins are then involved in converting the resulting single-strand DNA breaks to double-strand breaks with DNA ends appropriate for end-joining recombination. Proteins required for the subsequent S-S recombination include DNA-PK, ATM, Mre11-Rad50-Nbs1, gammaH2AX, 53BP1, Mdc1, and XRCC4-ligase IV. These proteins are important for faithful joining of S regions, and in their absence aberrant recombination and chromosomal translocations involving S regions occur.

Interaction and Functional Cooperation of PEBP2/CBF with Smads SYNERGISTIC INDUCTION OF THE IMMUNOGLOBULIN GERMLINE Cα PROMOTER

Smads are signal transducers for members of the transforming growth factor-β (TGF-β) superfamily. Upon ligand stimulation, receptor-regulated Smads (R-Smads) are phosphorylated by serine/threonine kinase receptors, form complexes with common-partner Smad, and translocate into the nucleus, where they regulate the transcription of target genes together with other transcription factors. Polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is a transcription factor complex composed of α and β subunits. The α subunits of PEBP2/CBF, which contain the highly conserved Runt domain, play essential roles in hematopoiesis and osteogenesis. Here we show that three mammalian α subunits of PEBP2/CBF form complexes with R-Smads that act in TGF-β/activin pathways as well as those acting in bone morphogenetic protein (BMP) pathways. Among them, PEBP2αC/CBFA3/AML2 forms a complex with Smad3 and stimulates transcription of the germline Ig Cα promoter in a cooperative manner, for which binding of both factors to their specific binding sites is essential. PEBP2 may thus be a nuclear target of TGF-β/BMP signaling.

Immunoglobulin class switching

Antibody class switching is induced by B-cell activators in the presence of cytokines. The identity of the heavy-chain class to which a B cell is switched is regulated by cytokines and B-cell activators at the level of transcription of unrearranged heavy chain constant genes. Gene-targeting experiments in mice have proved the essential role of these transcripts in switch recombination. Their possible functions are discussed in the context of a model for the mechanisms of class switching.

Interaction of stat6 and NF-kappaB: direct association and synergistic activation of interleukin-4-induced transcription

ABSTRACT Signal transducer and activator of transcription 6 (Stat6) and NF-κB are widely distributed transcription factors which are induced by different stimuli and bind to distinct DNA sequence motifs. Interleukin-4 (IL-4), which activates Stat6, synergizes with activators of NF-κB to induce IL-4-responsive genes, but the molecular mechanism of this synergy is poorly understood. Using glutathioneS-transferase pulldown assays and coimmunoprecipitation techniques, we find that NF-κB and tyrosine-phosphorylated Stat6 can directly bind each other in vitro and in vivo. An IL-4-inducible reporter gene containing both cognate binding sites in the promoter is synergistically activated in the presence of IL-4 when Stat6 and NF-κB proteins are coexpressed in human embryonic kidney 293 (HEK 293) cells. The same IL-4-inducible reporter gene is also synergistically activated by the endogenous Stat6 and NF-κB proteins in IL-4-stimulated I.29μ B lymphoma cells. Furthermore, Stat6 and NF-κB bind cooperatively to a DNA probe containing both sites, and the presence of a complex formed by their cooperative binding correlates with the synergistic activation of the promoter by Stat6 and NF-κB. We conclude that the direct interaction between Stat6 and NF-κB may provide a basis for synergistic activation of transcription by IL-4 and activators of NF-κB.

Inducible DNA breaks in Ig S regions are dependent on AID and UNG

Class switch recombination (CSR) occurs by an intrachromosomal deletion whereby the IgM constant region gene (Cμ) is replaced by a downstream constant region gene. This unique recombination event involves formation of double-strand breaks (DSBs) in immunoglobulin switch (S) regions, and requires activation-induced cytidine deaminase (AID), which converts cytosines to uracils. Repair of the uracils is proposed to lead to DNA breaks required for recombination. Uracil DNA glycosylase (UNG) is required for most CSR activity although its role is disputed. Here we use ligation-mediated PCR to detect DSBs in S regions in splenic B cells undergoing CSR. We find that the kinetics of DSB induction corresponds with AID expression, and that DSBs are AID- and UNG-dependent and occur preferentially at G:C basepairs in WRC/GYW AID hotspots. Our results indicate that AID attacks cytosines on both DNA strands, and staggered breaks are processed to blunt DSBs at the initiating ss break sites. We propose a model to explain the types of end-processing events observed.

Role for Mismatch Repair Proteins Msh2, Mlh1, and Pms2 in Immunoglobulin Class Switching Shown by Sequence Analysis of Recombination Junctions

B cells from mice deficient in mismatch repair (MMR) proteins show decreased ability to undergo class switch recombination in vitro and in vivo. The deficit is not accompanied by any reduction in cell viability or alterations in the cell cycle in B cells cultured in vitro. To assess the role of MMR in switching we examined the nucleotide sequences of Sμ-Sγ3 recombination junctions in splenic B cells induced in culture to switch to IgG3. The data demonstrate clear differences in the sequences of switch junctions in wild-type B cells in comparison with Msh2-, Mlh1-, and Pms2-deficient B cells. Sequences of switch junctions from Msh2-deficient cells showed decreased lengths of microhomology between Sμ and Sγ3 relative to junctions from wild-type cells and an increase in insertions, i.e., nucleotides which do not appear to be derived from either the Sμ or Sγ3 parental sequence. By contrast, 23% of junctions from Mlh1- and Pms2-deficient cells occurred at unusually long stretches of microhomology. The data indicate that MMR proteins are directly involved in class switching and that the role of Msh2 differs from that of Mlh1 and Pms2.

Activation-Induced Cytidine Deaminase-Dependent DNA Breaks in Class Switch Recombination Occur during G1 Phase of the Cell Cycle and Depend upon Mismatch Repair

Ab class switching occurs by an intrachromosomal recombination and requires generation of double-strand breaks (DSBs) in Ig switch (S) regions. Activation-induced cytidine deaminase (AID) converts cytosines in S regions to uracils, which are excised by uracil DNA glycosylase (UNG). Repair of the resulting abasic sites would yield single-strand breaks (SSBs), but how these SSBs are converted to DSBs is unclear. In mouse splenic B cells, we find that AID-dependent DSBs occur in Sμ mainly in the G1 phase of the cell cycle, indicating they are not created by replication across SSBs. Also, G1 phase cells express AID, UNG, and mismatch repair (MMR) proteins and possess UNG activity. We find fewer S region DSBs in MMR-deficient B cells than in wild-type B cells, and still fewer in MMR-deficient/SμTR−/− B cells, where targets for AID are sparse. These DSBs occur predominantly at AID targets. We also show that nucleotide excision repair does not contribute to class switching. Our data support the hypothesis that MMR is required to convert SSBs into DSBs when SSBs on opposite strands are too distal to form DSBs spontaneously.

Complex regulation and function of activation-induced cytidine deaminase

Activation-induced cytidine deaminase (AID) instigates mutations and DNA breaks in Ig genes that undergo somatic hypermutation and class switch recombination during B cell activation in response to immunization and infection. This review discusses how AID expression and activity are regulated, including recent discoveries of AID-interacting proteins that might recruit AID to Ig genes, and allow it to target both DNA strands. Also discussed is the accumulating evidence that AID binds to, mutates, and creates breaks at numerous non-Ig sites in the genome, which initiates cell transformation and malignancies.

Regulation of transcription of immunoglobulin germ-line gamma 1 RNA: analysis of the promoter/enhancer.

Antibody class switching is achieved by recombinations between switch (S) regions which consist of tandemly repeated sequences located 5′ to Ig heavy chain constant (CH) region genes. RNA transcripts from specific unrearranged or germ‐line Ig CH genes are induced in IgM+ B cells prior to their undergoing class switch recombination to the same CH genes. Thus, the antibody class switch appears to be directed by induction of accessibility, as assayed by transcription of germ line CH genes. For example, IL‐4 induces transcripts from the mouse germ‐line C gamma 1 and C epsilon genes to which it also directs switch recombination. We report here that the 150 bp region upstream of the first initiation site of RNA transcribed from the murine germ‐line C gamma 1 gene, contains promoter and enhancer elements responsible for basal level transcription and inducibility by anti‐Ig phorbol myristate acetate (PMA) and for synergy of these inducers with IL‐4 in a surface IgM+ B cell line, L10A6.2 and a surface IgG2a+ B cell line, A20.3. Linker‐scanning mutations demonstrated that multiple interdependent elements are required for inducibility by PMA and also for synergy with IL‐4. Within the 150 bp region are several consensus sequences that bind known or putative transcription factors, including a C/EBP binding site‐‐IL‐4 responsive element, four CACCC boxes, a PU box, a TGF beta inhibitory element (TIE), an alpha beta‐interferon response element (alpha beta‐IRE) and an AP‐3 site. The relationship between transcription regulated by these elements and the regulation of endogenous germ‐line gamma 1 transcripts and switching to IgG1 is discussed.

Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig loci in activated B cells

After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). We asked if, during B cell activation, AID also induces DNA breaks at genes other than IgH genes. Using a nonbiased genome-wide approach, we have identified hundreds of reproducible, AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. Unlike AID-induced DSBs in Ig genes, genome-wide AID-dependent DSBs are not restricted to transcribed regions and frequently occur within repeated sequence elements, including CA repeats, non-CA tandem repeats, and SINEs.