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Janet Stavnezer

Publications and source records attributed to Janet Stavnezer.

17 recordsLinked to original sources
PubMed2007

Stimuli that enhance IgA class switching increase histone 3 acetylation at S alpha, but poorly stimulate sequential switching from IgG2b.

Germ-line (GL) alpha transcription can be induced in mouse splenic B cells by LPS and TGF-beta. This stimulation results in approximately 1% IgA+ cells, which can be increased by IL-4, IL-5, and anti-IgD dextran (alpha delta Dex). To determine the mechanism of this increase, we asked whether IgA class switching correlates with acetylation of histone 3 at S alpha, the switch region for IgA. In the presence of the survival factor B lymphocyte stimulator (BLyS), acetylated histone 3 (AcH3) at S alpha was changed little by TGF-beta in LPS-stimulated mouse splenic B cell cultures, despite induction of GL alpha RNA. Compared with BLyS/LPS/TGF-beta alone, treatment with BLyS/LPS/TGF-beta/IL-4/IL-5/alpha delta Dex increased AcH3 at S alpha fourfold, and also increased GL alpha RNA levels more than eightfold. By contrast, IgG2b class switching was optimal in BLyS/LPS/TGF-beta alone, and was suppressed by IL-4/IL-5/alpha delta Dex. Thus, B cell activators that increase IgA class switching do not increase IgG2b class switching. Further investigation showed that in contrast to purified IgM+ cells, IgG2b+ cells switched poorly to IgA in response to BLyS/LPS/TGF-beta/IL-4/IL-5/ +/- alpha delta Dex. These results suggest that IgA class switching is unusual among isotypes in its requirement for multiple B cell activation signals in addition to LPS and the cytokine that initiates the corresponding GL transcription.

Acetylation↗
PubMed2006

Enhanced IgA class switching in marginal zone and B1 B cells relative to follicular/B2 B cells.

Mouse splenic marginal zone (MZ) B cells and B1 B cells enriched in the peritoneal cavity respond preferentially to T cell-independent Ags compared with follicular (FO)/B2 B cells. Despite the differential responses of B cell subsets to various stimuli, and despite the need for multiple stimuli to induce IgA class switching, the relative contribution of B cell subpopulations to IgA production is unknown. By culturing purified B cell populations, we find that MZ and peritoneal B1 cells switch more readily to IgA than do splenic FO or peritoneal B2 cells in BLyS/LPS/TGF-beta. Addition of IL-4, IL-5, and anti-IgD dextran to the cultures enhances IgA switching in FO/B2 and MZ B cells to a similar frequency, but this treatment suppresses IgA class switching in B1 cells. Thus, IgA switching differs among purified B cell subsets, suggesting that individual B cell populations could contribute differentially to IgA expression in vivo, depending on available stimuli.

Animals↗
PubMed2006

The histone methyltransferase Suv39h1 increases class switch recombination specifically to IgA.

Ab class (isotype) switching allows the humoral immune system to adaptively respond to different infectious organisms. Isotype switching occurs by intrachromosomal DNA recombination between switch (S) region sequences associated with C(H) region genes. Although isotype-specific transcription of unrearranged (germline) C(H) genes is required for switching, recent results suggest that isotype specificity is also determined by the sequences of downstream (acceptor) S regions. In the current study, we identify the histone methyltransferase Suv39h1 as a novel Salpha-specific factor that specifically increases IgA switching (Smu-Salpha recombination) in a transiently transfected plasmid S substrate, and demonstrate that this effect requires the histone methyltransferase activity of Suv39h1. Additionally, B cells from Suv39h1-deficient mice have an isotype-specific reduction in IgA switching with no effect on the level of germline Ialpha-Calpha transcripts. Taken together, our results suggest that Suv39h1 activity inhibits the activity of a sequence-specific DNA-binding protein that represses switch recombination to IgA.

Animals↗
PubMed2005

Mismatch repair converts AID-instigated nicks to double-strand breaks for antibody class-switch recombination.

Mismatch repair (MMR) proteins are important for antibody class-switch recombination (CSR), but their roles are unknown. We propose a model for the function of MMR in CSR in which MMR proteins convert single-strand nicks instigated by activation-induced cytidine deaminase (AID) into the double-strand breaks (DSBs) that are required for CSR. This model does not invoke any novel functions for MMR but simply posits that, owing to numerous single-strand nicks in the switch (S) regions of both DNA strands, when MMR proteins are recruited by U:G mismatches, they excise one strand of DNA and soon reach a nick on the opposite strand. This halts excision activity and creates a DSB. This model explains why B cells that lack either S mu and MSH2 or UNG and MSH2 cannot undergo CSR.

Animals↗
PubMed2005

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 (Cmu) 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.

Animals↗
PubMed2005

Shifts in targeting of class switch recombination sites in mice that lack mu switch region tandem repeats or Msh2.

The mechanisms that target class switch recombination (CSR) to antibody gene switch (S) regions are unknown. Analyses of switch site locations in wild-type mice and in mice that lack the Smu tandem repeats show shifts indicating that a 4-5-kb DNA domain (bounded upstream by the Imu promoter) is accessible for switching independent of Smu sequences. This CSR-accessible domain is reminiscent of the promoter-defined domains that target somatic hypermutation. Within the 4-5-kb CSR domain, the targeting of S site locations also depends on the Msh2 mismatch repair protein because Msh2-deficient mice show an increased focus of sites to the Smu tandem repeat region. We propose that Msh2 affects S site location because sequences with few activation-induced cytidine deaminase targets generate mostly switch DNA cleavages that require Msh2-directed processing to allow CSR joining.

Animals↗
PubMed2005

Analysis of transforming growth factor-beta1-induced Ig germ-line gamma2b transcription and its implication for IgA isotype switching.

Transforming growth factor (TGF)-beta1 directs class switch recombination (CSR) to IgG2b as well as to IgA. Smad3/4, Runx3 and p300 mediate TGF-beta1-induced germ-line (GL) alpha transcription leading to IgA expression. However, the molecular mechanisms by which TGF-beta1 induces IgG2b CSR are unknown. We used luciferase reporter plasmids to investigate how TGF-beta1 regulates the activity of the promoter for GL transcripts of IgG2b constant gene (GLgamma2b promoter). Similarly to the GLalpha promoter, overexpression of Smad3/4 and Runx3 enhances TGF-beta1-induced GLgamma2b promoter activity. Mutation analysis of the promoter identified likely Smad- and Runx3-binding sites. Also similar to the GLalpha promoter, overexpression of p300 enhances Smad3/4-mediated promoter activity, whereas E1A represses promoter activity. Since these regulation mechanisms underlying both GLalpha and GLgamma2b transcription are similar, we explored the possibility that TGF-beta1 induces IgA CSR via transitional IgG2b CSR. TGF-beta1 enhances the expression of both Ialpha-Cmu and Ialpha-Cgamma2b circle transcripts, indicative of direct (Smu-->Salpha) and sequential CSR (Smu-->Sgamma2b-->Salpha).

Animals↗
PubMed2004

Deletion of the nucleotide excision repair gene Ercc1 reduces immunoglobulin class switching and alters mutations near switch recombination junctions.

The structure-specific endonuclease ERCC1-XPF is an essential component of the nucleotide excision DNA repair pathway. ERCC1-XPF nicks double-stranded DNA immediately adjacent to 3' single-strand regions. Substrates include DNA bubbles and flaps. Furthermore, ERCC1 interacts with Msh2, a mismatch repair (MMR) protein involved in class switch recombination (CSR). Therefore, ERCC1-XPF has abilities that might be useful for antibody CSR. We tested whether ERCC1 is involved in CSR and found that Ercc1(-)(/)(-) splenic B cells show moderately reduced CSR in vitro, demonstrating that ERCC1-XPF participates in, but is not required for, CSR. To investigate the role of ERCC1 in CSR, the nucleotide sequences of switch (S) regions were determined. The mutation frequency in germline Smicro segments and recombined Smicro-Sgamma3 segments cloned from Ercc1(-)(/)(-) splenic B cells induced to switch in culture was identical to that of wild-type (WT) littermates. However, Ercc1(-)(/)(-) cells show increased targeting of the mutations to G:C bp in RGYW/WRCY hotspots and mutations occur at sites more distant from the S-S junctions compared with WT mice. The results indicate that ERCC1 is not epistatic with MMR and suggest that ERCC1 might be involved in processing or repair of DNA lesions in S regions during CSR.

Animals↗
PubMed2004

Evolution of isotype switching.

This review discusses evolution of the process of Ig heavy chain class switching, relating it to the first appearance of somatic hypermutation (SHM) of variable region genes. First, we discuss recent findings on the mechanism of class switch recombination (CSR) in mice and humans, and then review the mechanisms of expression of Ig heavy chain isotypes from fishes to mammals. Importantly, activation-induced cytidine deaminase (AID), which is essential for CSR and somatic hypermutation, is found in fishes. Although at least some fishes are likely to undergo SHM, CSR is highly unlikely to occur in this group. We discuss the first appearance of CSR in amphibians and how it differs in birds and mammals.

Animals↗
PubMed2004

Antibody class switching: uncoupling S region accessibility from transcription.

Immunogloblin class switch recombination (CSR) is a regulated process that changes antibody effector functions. Recently, Nambu et al. showed that histone acetylation is induced at switch (S) regions undergoing CSR; however, histone acetylation without accompanying S region transcription is insufficient to attract activation-induced cytidine deaminase (AID), which is required for CSR. They also show that AID can associate with RNA polymerase II. These results support the model that germline transcripts are required to form single-stranded DNA, the AID substrate and further suggest that AID is recruited to S regions by the transcriptional machinery.

Acetylation↗
PubMed2003

Mutations occur in the Ig Smu region but rarely in Sgamma regions prior to class switch recombination.

Nucleotide substitutions are found in recombined Ig switch (S) regions and also in unrecombined (germline, GL) Smicro segments in activated splenic B cells. Herein we examine whether mutations are also introduced into the downstream acceptor S regions prior to switch recombination, but find very few mutations in GL Sgamma3 and Sgamma1 regions in activated B cells. These data suggest that switch recombination initiates in the Smicro segment and secondarily involves the downstream acceptor S region. Furthermore, the pattern and specificity of mutations in GL and recombined Smicro segments differ, suggesting different repair mechanisms. Mutations in recombined Smicro regions show a strong bias toward G/C base pairs and WRCY/RGYW hotspots, whereas mutations introduced into the GL Smicro do not. Additionally, induction conditions affect mutation specificity within the GL Smicro segment. Mutations are most frequent near the S-S junctions and decrease rapidly with distance from the junction. Finally, we find that mice expressing a transgene for terminal deoxynucleotidyl transferase (TdT) have nucleotide insertions at S-S junctions, indicating that the recombining DNA ends are accessible to end-processing enzyme activities.

Animals↗
PubMed2003

Mlh1 can function in antibody class switch recombination independently of Msh2.

Mismatch repair proteins participate in antibody class switch recombination, although their roles are unknown. Previous nucleotide sequence analyses of switch recombination junctions indicated that the roles of Msh2 and the MutL homologues, Mlh1 and Pms2, differ. We now asked if Msh2 and Mlh1 function in the same pathway during switch recombination. Splenic B cells from mice deficient in both these proteins were induced to undergo switching in culture. The frequency of switching is reduced, similarly to that of B cells singly deficient in Msh2 or Mlh1. However, the nucleotide sequences of the Smu-Sgamma3 junctions resemble junctions from Mlh1- but not from Msh2-deficient cells, suggesting Mlh1 functions either independently of or before Msh2. The substitution mutations within S regions that are known to accompany switch recombination are increased in Msh2- and Mlh1 single-deficient cells and further increased in the double-deficient cells, again suggesting these proteins function independently in class switch recombination. The finding that MMR functions to reduce mutations in switch regions is unexpected since MMR proteins have been shown to contribute to somatic hypermutation of antibody variable region genes.

Adaptor Proteins, Signal Transducing↗
PubMed2003

The Smu tandem repeat region is critical for Ig isotype switching in the absence of Msh2.

Deficiencies of the Msh2 protein or the Smu tandem repeat (SmuTR) sequences each reduce isotype switching in mice by about 2- to 3-fold. We find that switching in mice deficient for both Msh2 and SmuTR is nearly ablated. We propose that the SmuTR provides closely spaced cleavage sites that can undergo switch recombination independent of Msh2, whereas cleavages in sequences flanking the SmuTR require Msh2 processing to allow recombinational joining. We also find that changes in Smu sequences alter the focus of switch junctions within Sgamma sequences, indicating that sequences of switch regions act together in the choice of switch recombination junctions. These findings help to explain the conservation of tandemly repeated switch regions associated with heavy chain constant genes in species capable of switching.

Animals↗
PubMed2002

Immunoglobulin genes: generating diversity with AID and UNG.

Somatic hypermutation and switch recombination of immunoglobulin genes require the activity of the activation-induced deaminase, AID. Recent studies of mice deficient for the uracil-DNA glycosylase UNG, which removes U from DNA, suggest that AID catalyses the deamination of dC to dU during antibody diversification.

Animals↗
PubMed2002

The ubiquitously expressed DNA-binding protein late SV40 factor binds Ig switch regions and represses class switching to IgA.

Ig heavy chain class switch recombination (CSR) determines the expression of Ig isotypes. The molecular mechanism of CSR and the factors regulating this process have remained elusive. Recombination occurs primarily within switch (S) regions, located upstream of each heavy chain gene (except Cdelta). These repetitive sequences contain consensus DNA-binding sites for the DNA-binding protein late SV40 factor (LSF) (CP2/leader-binding protein-1c). In this study, we demonstrate by EMSA that purified rLSF, as well as LSF within B cell extracts, directly binds both Smu and Salpha sequences. To determine whether LSF is involved in regulating CSR, two different LSF dominant negative variants were stably expressed in the mouse B cell line I.29 mu, which can be induced to switch from IgM to IgA. Overexpression of these dominant negative LSF proteins results in decreased levels of endogenous LSF DNA-binding activity and an increase in cells undergoing CSR. Thus, LSF represses class switching to IgA. In agreement, LSF DNA-binding activity was found to decrease in whole cell extracts from splenic B cells induced to undergo class switching. To elucidate the mechanism of CSR regulation by LSF, the interactions of LSF with proteins involved in chromatin modification were tested in vitro. LSF interacts with both histone deacetylases and the corepressor Sin3A. We propose that LSF represses CSR by histone deacetylation of chromatin within S regions, thereby limiting accessibility to the switch recombination machinery.

Amino Acid Substitution↗
PubMed2002

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 Smicro-Sgamma3 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 Smicro and Sgamma3 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 Smicro or Sgamma3 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.

Adaptor Proteins, Signal Transducing↗