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Ursula Storb

Publications and source records attributed to Ursula Storb.

16 recordsLinked to original sources
PubMed2006

Somatic hypermutation and class switch recombination in Msh6(-/-)Ung(-/-) double-knockout mice.

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytosine deaminase (AID). The uracil, and potentially neighboring bases, are processed by error-prone base excision repair and mismatch repair. Deficiencies in Ung, Msh2, or Msh6 affect SHM and CSR. To determine whether Msh2/Msh6 complexes which recognize single-base mismatches and loops were the only mismatch-recognition complexes required for SHM and CSR, we analyzed these processes in Msh6(-/-)Ung(-/-) mice. SHM and CSR were affected in the same degree and fashion as in Msh2(-/-)Ung(-/-) mice; mutations were mostly C,G transitions and CSR was greatly reduced, making Msh2/Msh3 contributions unlikely. Inactivating Ung alone reduced mutations from A and T, suggesting that, depending on the DNA sequence, varying proportions of A,T mutations arise by error-prone long-patch base excision repair. Further, in Msh6(-/-)Ung(-/-) mice the 5' end and the 3' region of Ig genes was spared from mutations as in wild-type mice, confirming that AID does not act in these regions. Finally, because in the absence of both Ung and Msh6, transition mutations from C and G likely are "footprints" of AID, the data show that the activity of AID is restricted drastically in vivo compared with AID in cell-free assays.

Animals↗
PubMed2006

Scarcity of lambda 1 B cells in mice with a single point mutation in C lambda 1 is due to a low BCR signal caused by misfolded lambda 1 light chain.

The presence of valine-154 instead of glycine in the constant region of lambda1 causes a severe lambda1 B cell defect in SJL and lambda1-valine knock-in mice with a compensatory increase in lambda2,3 B cells. The defect is due to low signaling by the lambda1-valine BCR. lambda1-Valine B cells deficient in the SHP-1 phosphatase survive better than lambda2,3 B cells in these mice, or lambda1 B cells in lambda1 wildtype mice. Low signaling is apparently due to misfolding of the lambda1-valine light chain as demonstrated by the absence of a regular beta-sheet structure determined by circular dichroism, the sedimentation of the light chain in solution, and the association of valine-valine constant regions in a yeast two-hybrid assay. lambda1-Valine B cells that survive apparently have a higher BCR signal, presumably because of their specific lambda1-heavy chain combination or having encountered a high-affiniy antigen. lambda1-Valine mice have increased B1 cells which were shown by others to have a higher signaling potential. Valine mice crossed with non-conventional gamma2b transgenic mice, in which B cell development is accelerated and in which B1 cells and high signaling cells are greatly reduced, have essentially no, lambda2,3 B cells, but increased numbers of lambda1-valine B cells. This supports the conclusion that the major defect in lambda1-valine mice is the inability of valine-preB cells to produce a threshold signal for B cell development. The reduction of lambda2,3 B cells in valine mice with a gamma2b transgene shows that the majority of their compensatory increase is almost entirely of the B1 cell type.

Animals↗
PubMed2006

AID in somatic hypermutation and class switch recombination.

Somatic hypermutation and class-switch-recombination are initiated by the deamination of deoxycytosine in DNA by activation-induced-deaminase, AID. Recently, there has been much research into how AID targets double-stranded DNA in sub-regions of Ig genes, the involvement of co-factors and posttranslational modifications in this process, the co-option of DNA 'repair' mechanisms and AID evolution.

Animals↗
PubMed2005

The very 5' end and the constant region of Ig genes are spared from somatic mutation because AID does not access these regions.

Somatic hypermutation (SHM) is restricted to VDJ regions and their adjacent flanks in immunoglobulin (Ig) genes, whereas constant regions are spared. Mutations occur after about 100 nucleotides downstream of the promoter and extend to 1-2 kb. We have asked why the very 5' and most of the 3' region of Ig genes are unmutated. Does the activation-induced cytosine deaminase (AID) that initiates SHM not gain access to these regions, or does AID gain access, but the resulting uracils are repaired error-free because error-prone repair does not gain access? The distribution of mutations was compared between uracil DNA glycosylase (Ung)-deficient and wild-type mice in endogenous Ig genes and in an Ig transgene. If AID gains access to the 5' and 3' regions that are unmutated in wild-type mice, one would expect an "AID footprint," namely transition mutations from C and G in Ung-deficient mice in the regions normally devoid of SHM. We find that the distribution of total mutations and transitions from C and G is indistinguishable in wild-type and Ung-deficient mice. Thus, AID does not gain access to the 5' and constant regions of Ig genes. The implications for the role of transcription and Ung in SHM are discussed.

5' Untranslated Regions↗
PubMed2005

The contested role of uracil DNA glycosylase in immunoglobulin gene diversification.

Class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes are initiated by the activation-induced cytosine deaminase AID. The resulting uracils in Ig genes were believed to be removed by the uracil glycosylase (UNG) and the resulting abasic sites treated in an error-prone fashion, creating breaks in the Ig switch regions and mutations in the variable regions. A recent report suggests that UNG does not act as a glycosylase in CSR and SHM but rather has unknown activity subsequent to DNA breaks that were created by other mechanisms.

Animals↗
PubMed2005

DNA methylation precedes chromatin modifications under the influence of the strain-specific modifier Ssm1.

Ssm1 is responsible for the mouse strain-specific DNA methylation of the transgene HRD. In adult mice of the C57BL/6 (B6) strain, the transgene is methylated at essentially all CpGs. However, when the transgene is bred into the DBA/2 (D2) strain, it is almost completely unmethylated. Strain-specific methylation arises during differentiation of embryonic stem (ES) cells. Here we show that Ssm1 causes striking chromatin changes during the development of the early embryo in both strains. In undifferentiated ES cells of both strains, the transgene is in a chromatin state between active and inactive. These states are still observed 1 week after beginning ES cell differentiation. However, 4 weeks after initiating differentiation, in B6, the transgene has become heterochromatic, and in D2, the transgene has become euchromatic. HRD is always expressed in D2, but in B6, it is expressed only in early embryos. The transgene is already more methylated in B6 ES cells than in D2 ES cells and becomes increasingly methylated during development in B6, until essentially all CpGs in the critical guanosine phosphoribosyl transferase core are methylated. Clearly, DNA methylation of HRD precedes chromatin compaction and loss of expression, suggesting that the B6 form of Ssm1 interacts with DNA to cause strain-specific methylation that ultimately results in inactive chromatin.

Animals↗
PubMed2005

Targeting of the activation-induced cytosine deaminase is strongly influenced by the sequence and structure of the targeted DNA.

Activation-induced deaminase (AID) initiates immunoglobulin somatic hypermutation (SHM). Since in vitro AID was shown to deaminate cytosines on single-stranded DNA or the nontranscribed strand, it remained a puzzle how in vivo AID targets both DNA strands equally. Here we investigate the roles of transcription and DNA sequence in cytosine deamination. Strikingly different results are found with different substrates. Depending on the target sequence, the transcribed DNA strand is targeted as well as or better than the nontranscribed strand. The preferential targeting is not related to the frequency of AID hot spots. Comparison of cytosine deamination by AID and bisulfite shows different targeting patterns suggesting that AID may locally unwind the DNA. We conclude that somatic hypermutation on both DNA strands is the natural outcome of AID action on a transcribed gene; furthermore, the DNA sequence or structure and topology play major roles in targeting AID in vitro and in vivo. On the other hand, the lack of mutations in the first approximately 100 nucleotides and beyond about 1 to 2 kb from the promoter of immunoglobulin genes during SHM must be due to special conditions of transcription and chromatin in vivo.

Ampicillin Resistance↗
PubMed2004

Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled.

The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination of Ig genes. It has been shown that in vitro, AID protein deaminates C in single-stranded DNA or the coding-strand DNA that is being transcribed but not in double-stranded DNA. However, in vivo, both DNA strands are mutated equally during SHM. We show that AID efficiently deaminates C on both DNA strands of a supercoiled plasmid, acting preferentially on SHM hotspot motifs. However, this DNA is not targeted by AID when it is relaxed after treatment with topoisomerase I, and thus, supercoiling plays a crucial role for AID targeting to this DNA. Most of the mutations are in negatively supercoiled regions, suggesting a mechanism of AID targeting in vivo. During transcription the DNA sequences upstream of the elongating RNA polymerase are negatively supercoiled, and this transient change in DNA topology may allow AID to access both DNA strands.

Base Sequence↗
PubMed2003

A novel cytidine deaminase AIDs in the delivery of error-prone polymerases to immunoglobulin genes.

Immunoglobulin genes undergo somatic hypermutation in activated B lymphocytes. The mutations are targeted to the variable region of the gene, apparently by co-transcriptional deposition of a mutation complex. The novel activation induced deaminase (AID) gene is required for this process and recent work indicates that it is the cytidine deaminase activity of the AID protein rather than an associated RNA editing function that is required [Curr. Biol. 12 (2002) 1748; Nature 419 (2002) 1; Nature 418 (2002) 99]. The authors review the implications of this work and argue that deamination of deoxy-cytidine to deoxy-uracil seems to initiate several pathways in which error-prone polymerases are postulated to create mutations.

Animals↗
PubMed2003

The transcription factor Spi-B is not required for somatic hypermutation.

Mice with a homozygous inactivation of the transcription factor gene Spi-B(-)/(-) have abnormal B cell functions and a defect in germinal center (GC) formation. We report here that somatic hypermutation (SHM) of VH1 and VH11 genes is not diminished in Peyer's patches of Spi-B(-)/(-) mice. However, the mutation pattern shows an increase in the ratio of replacement to silent mutations in the framework sequences of the variable regions, suggesting that selection of mutated B cells based on functionality is affected. In support of this, two of the six sequences from Spi-B mutant mice have a point mutation in the framework which introduces predicted steric clashes with another amino acid in the variable region. This mutation (Leu81Phe) has not been observed in 120 mutated VH1 or VH11 genes of germinal center B cells from Spi-B wildtype mice. The mutation also does not exist in any of 136 published heavy chain proteins of the same VH family. The mutations causing the change to Phe are transitions which are favored by the SHM process over transversions. Clearly, Phe-81 must arise relatively frequently, but is not selected in Spi-B wildtype mice.

Animals↗
PubMed2003

The E box motif CAGGTG enhances somatic hypermutation without enhancing transcription.

The frequency of somatic hypermutations of an Ig kappa transgene with an artificial test insert, RS, is at least 4-fold higher than that of three related transgenes. The four transgenes differ only in the sequence of a 96 bp insert within the variable region. RS is hypermutable over the total 625 nucleotides of the variable/joining region. The RS insert contains two CAGGTG sequences, potential binding sites for basic helix-loop-helix proteins. Changing CAGGTG to AAGGTG reduces the mutability to that of the non-RS transgenes without altering the mutation pattern. The CAGGTG motif enhances somatic hypermutation without enhancing transcription. A DNA probe containing the two CAGGTG sites, but not AAGGTG, binds E47 and gives rise to two specific EMSA bands with nuclear extracts from mutating cells. Possible actions of this enhancer of somatic hypermutation are discussed.

Animals↗
PubMed2003

Ig gene somatic hypermutation in mice defective for DNA polymerase delta proofreading.

This study is an investigation of the possible role of DNA polymerase (pol) delta with an inactivated exonuclease (exo) in somatic hypermutation (SHM). Analysis of endogenous heavy chain transcripts revealed no difference in mutation frequency and pattern between exo(-/-), exo(+/-) and exo(+/+) mice. The lack of an effect of the pol delta exo mutation on SHM could be due to: (i) normally pol delta is used in SHM, but the exo is prevented from proofreading, (ii) normally pol delta is used, but the decrease in fidelity of the exo(-) pol does not increase hypermutation frequency enough to be detected, and (iii) pol delta is not used in SHM. Based on the finding in the exo(-/-) mice and the current understanding of the process of SHM, it is concluded that pol delta is not normally involved in creating the mutations. The majority of the mutated sequences obtained in this study, including many from the exo(-/-) mice, were from genes which had switched to a gamma heavy chain class. Thus, the pol delta proofreading activity is not required for class switch recombination (CSR). Genealogical trees observed with multiple mutated sequences of various Ig classes show that CSR and SHM occur intermingled during expansion of a cell clone, raising the possibility that they may occur at the same time.

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

Effects of sequence and structure on the hypermutability of immunoglobulin genes.

Somatic hypermutation (SHM) is investigated in related immunoglobulin transgenes that differ in a short artificial sequence designed to vary the content of hotspot motifs and the potential to form RNA or DNA secondary structures. Mutability depends on hotspots, not secondary structure. Hotspot motifs predict about 50% of the mutations; the rest are in neutral and coldspots. Clusters of mutations and the sequential addition of mutations found in cell pedigrees suggest epigenetic attributes of SHM. Sometime in SHM, an essential factor seems to become limiting. Particular error-prone DNA polymerases appear to create mutations in hotspots on the top and bottom DNA strands throughout the target and the SHM process. One transgene is superhypermutable in all regions, suggesting the presence of a cis-element that enhances SHM.

Animals↗
PubMed2002

A point mutation in the constant region of Ig lambda1 prevents normal B cell development due to defective BCR signaling.

Surface expression of B cell antigen receptors (BCRs) containing Ig and Igalpha/Igbeta generates signals required to transit discrete developmental checkpoints. The mechanism by which BCR components collaborate to initiate signals is still unclear. The expression of Iglambda1 in SJL mice is 50-fold lower than in other strains. Here, we demonstrate by gene targeting that a point mutation, which changes a glycine to a valine in the lambda1 constant region, is responsible for this defect. In vitro experiments show that Ig receptors bearing this mutation, while expressed normally, are deficient in signaling. These findings reveal a direct involvement of the Ig light chain (IgL) in B cell signaling and development beyond the requirement of light chains for BCR assembly.

Animals↗