Transcription, beta-like DNA polymerases and hypermutation.

This paper discusses two aspects of immunoglobulin (Ig) gene hypermutation. In the first approach, a transcription termination signal is introduced in an Ig light chain transgene acting as a mutation substrate, and transgenic lines are generated with control and mutant transgenes integrated in tandem. Analysis of transcription levels and mutation frequencies between mutant and control transgenes clearly dissociates transcription elongation and mutation, and therefore argues against models whereby specific pausing of the RNA polymerase during V gene transcription would trigger an error-prone repair process. The second part reports the identification of two novel beta-like DNA polymerases named Pol lambda and Pol mu, one of which (Pol mu) represents a good candidate for the Ig mutase due to its higher lymphoid expression and its similarity with the lymphoid enzyme terminal deoxynucleotidyl transferase. Peculiar features of the expression of this gene, including an unusual splicing variability and a splicing inhibition in response to DNA-damaging agents, are discussed.

CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversification pathway in humans.

Somatically mutated IgM(+)-only and IgM(+)IgD(+)CD27(+) B lymphocytes comprise approximately 25% of the human peripheral B cell pool. These cells phenotypically resemble class-switched B cells and have therefore been classified as postgerminal center memory B cells. X-linked hyper IgM patients have a genetic defect characterized by a mutation of the CD40L gene. These patients, who do not express a functional CD40 ligand, cannot switch Ig isotypes and do not form germinal centers and memory B cells. We report here that an IgM(+)IgD(+)CD27(+) B cell subset with somatically mutated Ig receptors is generated in these patients, implying that these cells expand and diversify their Ig receptors in the absence of classical cognate T-B collaboration. The presence of this sole subset in the absence of IgM(+)-only and switched CD27(+) memory B cells suggests that it belongs to a separate diversification pathway.

Two novel human and mouse DNA polymerases of the polX family.

We describe here two novel mouse and human DNA polymerases: one (pol lambda) has homology with DNA polymerase beta while the other one (pol mu) is closer to terminal deoxynucleotidyltransferase. However both have DNA polymerase activity in vitro and share similar structural organization, including a BRCT domain, helix-loop-helix DNA-binding motifs and polymerase X domain. mRNA expression of pol lambda is highest in testis and fetal liver, while expression of pol mu is more lymphoid, with highest expression both in thymus and tonsillar B cells. An unusually large number of splice variants is observed for the pol mu gene, most of which affect the polymerase domain. Expression of mRNA of both polymerases is down-regulated upon treatment by DNA damaging agents (UV light, gamma-rays or H(2)O(2)). This suggests that their biological function may differ from DNA translesion synthesis, for which several DNA polymerase activities have been recently described. Possible functions are discussed.

A targeted deletion of a region upstream from the Jkappa cluster impairs kappa chain rearrangement in cis in mice and in the 103/bcl2 cell line.

We have shown previously that a mutation of the KI-KII site immediately 5' to J(kappa)1 on the mouse immunoglobulin light chain kappa locus reduces the rearrangement level in cis, although it does not affect transcription. Here we deleted by homologous recombination in mouse embryonic stem cells a 4-kb DNA fragment, located immediately upstream of the KI-KII element, which contains the promoter of the long germline transcript. Analysis of gene-targeted heterozygous mouse splenic B cells showed a strong decrease in rearrangement for the allele bearing the deletion. When both the KI-KII mutation and the 4-kb deletion were present on the same allele, the overall reduction in rearrangement was stronger than with the 4-kb deletion alone underlying the role of these two elements in the regulation of rearrangement. The same deletion was performed by homologous recombination on one allele of the rearrangement-inducible mouse 103/bcl2-hygro(R) pre-B cell line, and resulted in a similar reduction in the induction of rearrangement of the mutated allele. This result validates this cell line as an in vitro model for studying the incidence of gene-targeted modifications of the kappa locus on the regulation of rearrangement.

Negative regulation of Ig gene rearrangement by a 150-bp transcriptional silencer.

We previously showed that the V-J intervening sequence of the chicken lambda immunoglobulin locus contains a strong silencer that acts both on transcription and rearrangement. We show here that the transcriptional silencer activity can be ascribed to a minimal 150-bp fragment. The rearrangement silencing activity was previously shown by the replacement of the V-J intervening sequence with a neutral DNA fragment that dramatically increased the rate of rearrangement of the transgene. Insertion of the minimal silencer in this neutral fragment is shown here to result in a marked decrease in rearrangement of the transgenic construct. Strikingly, deletion of 28 bp from the 150-bp fragment abolished most of the transcriptional silencing activity and had a similar effect on rearrangement. These results conclusively correlate the silencing activity on both rearrangement and transcription.

Defect in IgV gene somatic hypermutation in common variable immuno-deficiency syndrome.

Common Variable Immuno-Deficiency (CVID) is the most common symptomatic primary antibody-deficiency syndrome, but the basic immunologic defects underlying this syndrome are not well defined. We report here that among eight patients studied (six CVID and two hypogammaglobulinemic patients with recurrent infections), there is in two CVID patients a dramatic reduction in Ig V gene somatic hypermutation with 40-75% of IgG transcripts totally devoid of mutations in the circulating memory B cell compartment. Functional assays of the T cell compartment point to an intrinsic B cell defect in the process of antibody affinity maturation in these two cases.

Probing immunoglobulin gene hypermutation with microsatellites suggests a nonreplicative short patch DNA synthesis process.

As the rate of Ig gene hypermutation approximates the level of nucleotide discrimination of DNA polymerases (10(-3) to 10(-4)), a local inhibition of proofreading and mismatch repair during semiconservative replication could generate the mutations introduced by the process. To address this question, we have constructed transgenic mice that carry a hypermutation substrate containing a "polymerase slippage trap": an Ig gene with a mono or dinucleotide tract inserted in its V region. The low amount of slippage events as compared to the number of mutations, the absence of transient misalignment mutations at the border of the repeats, and the dissociation between the amount of frameshifts and mutations when the transgene is put on mismatch repair-deficient genetic backgrounds, suggest that Ig gene hypermutation occurs by an error-prone short patch DNA synthesis taking place outside global DNA replication.

Mismatch repair deficiency interferes with the accumulation of mutations in chronically stimulated B cells and not with the hypermutation process.

Primary responses to the hapten phenyloxazolone and chronic responses to environmental antigens occurring in Peyer's patches were analyzed in two different mismatch repair-deficient backgrounds. Paradoxically, whereas primary responses were found normal in MSH2- and only slightly diminished in PMS2-deficient mice, mutations in Peyer's patch B cells from both k.o. animals were reduced three times, the subset of Peyer's patch B cells with highly mutated sequences being specifically missing in the mismatch repair-deficient context. Strikingly, germinal center B cells from Peyer's patches of k.o. animals showed microsatellite instability at an unprecedented level. We thus propose that the amount of DNA damages generated prevents these cells from recycling in germinal centers and that mismatch repair deficiency is only the indirect cause of the lower mutation incidence observed.

Generation of diversity in mammalian gut-associated lymphoid tissues: restricted V gene usage does not preclude complex V gene organization.

The sheep genome contains 60 to 90 V lambda genes distributed in least 6 different families, whereas 37 V lambda genes and 10 families exist in humans. Comparison with human V lamda sequences indicates that sheep V lambda genes display less overall sequence divergence, but are closer to the genes most frequently used in the human peripheral repertoire. In both species, 2 to 3 genes contribute half of the expressed sequences. Therefore, similar large combinatorial potential and restriction of the expressed repertoire can exist in two species whose strategy of diversification differs widely (ongoing rearrangement throughout life in human bone marrow vs postrearrangement diversification during early development in sheep ileal Peyer's patches).

Rearrangement-enhancing element upstream of the mouse immunoglobulin kappa chain J cluster.

Transcriptional regulatory elements have been shown to be necessary but not sufficient for the developmental regulation of immunoglobulin gene rearrangement in mouse precursor B cells. In the chicken lambda light chain locus, additional elements in the V-J intervening sequence are involved in negative and positive regulation of rearrangement. Here, mutation of the mouse homolog of a chicken element, located in the V(K)-J(K) intervening sequence upstream of the J(K) cluster, was shown to significantly decrease rearrangement. This cis-acting recombination-enhancing element affects the rearrangement process without being involved in regulating transcription.

Rearrangement/hypermutation/gene conversion: when, where and why?

Diversification strategies for immunoglobulins vary widely in different species. Here, Jean-Claude Weill and Claude-Agnès Reynaud argue that V(D)J recombination arose as a means for achieving allelic exclusion rather than diversity, and postulate that the choice of a diversification strategy is selected along with a specific site of B-cell differentiation. They propose that somatic mutation and gene conversion represent analogous molecular strategies occurring in specific chromatin accessibility contexts.

Hypermutation generating the sheep immunoglobulin repertoire is an antigen-independent process.

Somatic hypermutation of light chain V genes during development of B cells in sheep ileal Peyer's patches was studied in three experimental conditions: in sterile fragments of the ileum surgically isolated from the gut during fetal life, in germ-free sheep, and in animals thymectomized during early fetal life. The somatic mutation pattern was found identical to control tissues in all three experiments. The same age-dependent amount of mutations, a higher than theoretical R/S ratio in complementarity-determining regions (CDRs), and a similar clustering of mutations in CDRs were observed. The mechanism, as estimated from the silent mutation pattern, appears to target mutations to CDRs; moreover, the major V lambda genes have a specific codon usage with a high purine content at the first two bases of the codons and a low content at the third position, which, together with a specific targeting of mutations to purines, favors replacement mutations in CDRs.

Rearrangement of the chicken lambda light chain locus: a silencer/antisilencer regulation.

Chicken immunoglobulin lambda light chain rearrangement is regulated by different controlling elements. One negative control element, acting as a strong transcriptional silencer, is located in the V-J intervening sequence which is excised during the rearrangement process. Positive control elements include the V lambda promoter, the enhancer of transcription (3' of C lambda), and one (or two) putative antisilencer element(s) located on each side of the silencer. It is proposed that these antisilencer elements counteract transiently the silencer thus allowing rearrangement of one allele in chicken B cell progenitors. The implications of this regulation for mouse B cell development are discussed.

Promoter, enhancer and silencer elements regulate rearrangement of an immunoglobulin transgene.

The chicken Ig lambda light chain locus is composed of a single V gene closely linked (1.8 kb) to a single J-C unit in its natural configuration. In mice transgenic for this locus, the transgene becomes rearranged in B cells and to a much lesser extent in T cells. Modifications were introduced in the transgene in order to characterize elements which target the recombinase to the Ig loci. In the absence of either the promoter or the enhancer located 3' of C lambda, rearrangement of the transgene is reduced 20- to 100-fold. Moreover, rearrangement is increased 5-fold when the DNA segment between V lambda and J lambda ('Uo segment'), which is deleted during the joining process, is replaced by a neutral DNA segment of equal length. The Uo segment behaved as a strong transcriptional silencer when tested in a CAT assay in vitro. Control transgenic mice harbouring only the two 3 bp mutations that introduced restriction sites at both ends of the Uo segment to allow for its replacement were also analysed. Rearrangement was reduced 10- to 100-fold in B cells from such transgenic lines. A model is proposed whereby the sites of these two mutations would function by counteracting transiently the repressing effect of the silencer, thus giving access of the chicken light chain locus to the recombinase.