Amy L. Kenter, PhDProfessor
Ph.D., Albert Einstein College of Medicine (Bronx, NY)
Molecular Characterization of Immunoglobulin Switch Recombination Introduction
We study the molecular events leading to the immunoglobulin (Ig) heavy chain (H) class switch. This is a developmentally controlled recombination event that occurs at the Ig locus in normal B lymphocytes. Stimulation of B lymphocytes by antigen and the appropriate lymphokines induces the production of a different Ig isotypes while maintaining the same antigen-binding specificity. This phenomenon provides for the expression of the same Ig variable (antigen binding) region with different constant regions. The IgH class switch results from an intrachromosomal ecombination event such that one of seven “new” constant region genes replaces the Cµ gene with a concomitant deletion of the intervening genes (fig.1). The recombination event focuses on the switch (S) DNA, the region of repetitive sequence near each gene, and results in a new hybrid DNA combination. The deletion-looping out model for switch recombination (SR) predicts that the intervening DNA between S regions will be excised as a circle. Circular excision products of SR have recently been isolated from LPS stimulated B cells.
Specific Protein-DNA Interactions in SR
Formation of synaptic nucleoprotein complexes in which recombination sites are aligned occurs frequently during site-specific recombination events. Ig switch recombination (SR) involves DNA rearrangements between sequences separated by 50-100 kb of genetic material. Since SR is highly specific for S regions, we hypothesize that some DNA-binding factor(s) might be involved in specifically recognizing and facilitating the alignment of S regions prior to recombination. We have identified 3 proteins which specifically interact with S DNA. The first is a DNA-binding activity termed SNUP, which specifically recognizes the Sµ tandem repeat (1). The Sg3 specific DNA-binding proteins, SNAP and SNIP interact with two discrete regions of the Sg3 tandem repeat (2). Recombination breakpoints in the Sg3 switch region were found to significantly correlate with the binding sites of the Sg3 switch-binding proteins (2). Moreover, we have found that the SNIP and SNAP binding sites are conserved in Sg2B and Sg1 DNA and are functional binding motifs for these proteins (3). SNIP was found to be identical with the well described transcription factor, NF-kB p50 and SNAP contains epitopes in common with a helix-loop-helix transcription factor, E47 (2,4). Recent in vivo footprinting studies indicate the presence of protein-DNA contacts at SNIP and SNAP sites in living cells (Wuerffel and Kenter, manuscript in preparation). These footprints are restricted to B cells and are dependent on the expression of NF-kB p50. However, the precise function of these protein-DNA interactions is not known.
The Mechanism of SR
The recombination mechanism mediating SR has not been defined. SR breakpoints are located within S regions but not at consensus recombination signal sequences suggesting that SR is not site-specific recombination. Switch junctions do not contain long stretches of homology arguing that homologous recombination does not contribute to this process. Thus, SR is unique in that it does not fall neatly into one of the well defined categories of recombination. Which mechanism of recombination is responsible for SR? To address this question I will consider each of the major forms of mitotic recombination and their possible relationship to SR (5,6). The similarity between S regions has led to the suggestion that short stretches of homology could participate in the recombination process and that SR is mediated through a homologous recombination mechanism.
Homologous recombination in mitotic cells occurs through the RAD52 pathway in Saccharomyces cerevisiae and higher eukaryotes. The RAD52 pathway is highly conserved from fungi to humans and includes RAD54. Analysis of the RAD54-/- mouse phenotype has revealed that homologous recombination is reduced. In contrast, V(D)J joining and SR are normal in B cells from RAD54-/- mice indicating that the major pathways for homologous recombination do not contribute to these processes.
A second pathway for recombination in mitotic cells is nonhomologous end joining (NHEJ) of double strand breaks (DSBs). There are two manifestations of this form of recombination, illegitimate recombination, as occurs in repair of damaged DNA, and specialized recombination, as is found for V(D)J joining of Ig V genes. Illegitimate recombination is stimulated by the introduction of DSBs through agents which damage DNA whereas V(D)J recombination is initiated through cleavage of DNA at specific sites catalyzed by the RAG1 and RAG2 proteins. In both cases, the DSB is repaired through recombination mediated by the DNA-dependent protein kinase (DNA-PK). DNA-PK is composed of DNA-PK catalytic subunit (DNA-PKCS) and the Ku70/Ku80 heterodimer which has DNA end-binding activity.
SR May Occur By Nonhomologous End Joining
Evidence has been accumulating that non-homologous end joining (NHEJ) of DSBs, mediates Ig class switching (for a review (see ref. 6). DSB repair by NHEJ is a process characterized by recombination junctions with distinct features and the involvement of specific proteins in both yeast and mammalian cells. I will first consider the recombination junctions formed by NHEJ and will then address the nature and identity of proteins which are involved in this process. Occasionally, DSBs repaired by NHEJ undergo blunt end ligation which uses no homology between the ligated ends. More frequently the recombination junctions contain microhomologies of 1-5 nucleotides and short deletions of DNA. In mammalian cells, 97% of the end-joining events are within 15 nucleotides of the original broken end. Repair of a site-specific DSB is associated with introduction of mutations in the flanking DNA. In murine cells, DSBs are frequently subjected to extensive single strand 5′->3′ or 3′->5′ exonuclease digestion during NHEJ. The gap is thought to be re-synthesized resulting in little net loss of genetic material. The resynthesis of DNA by an error-prone DNA polymerase could introduce nucleotide substitutions which would be detected as mutations in the regions flanking the DSB.
The recombination junctions in composite S/S regions conform to the classical features associated with DSB repair by NHEJ. There is either no homology or microhomologies of 1-8 nucleotides at the recombination breakpoints for S/S composite genes. Mutations flanking the S junctions have been frequently observed in recombined S regions. It has been difficult to assess the degree of deletion at broken DNA ends since the precise locations of most DNA breaks in S regions are not known. However, if DNA breaks occur at specific sites in the S region tandem repeat then the processing which leads to short deletions at the DNA ends is predicted to result in clustered recombination breakpoints for switch junctions. Clustered recombination breakpoints in switch junctions of Sm and Sg3, Sg1 or Sg2b have been reported. Taken together, these findings indicate that switch junctions contain the characteristic features associated with DSB repair by NHEJ.
Significance of DSBs and Ku to the Mechanism of SR
Information regarding the position and structure of the broken ends in switch DNA may provide information with which to infer mechanism. If isotype switching occurs through a non-homologous DNA end-joining mechanism then it is predicted to be dependent on the presence of DSBs in switch regions and on Ku for DSB repair during the recombination reaction. Using the sensitive ligation-mediated (LM) PCR based assay we have examined the Sg3 region in normal resting and mitogen activated B cells for the presence of DSBs in vivo. We have found inducible DSBs which are B cell specific, restricted to the S region and sequence specific (7).
NHEJ is a well regulated process mediated by DNA-dependent protein kinase (DNA-PK). DNA-PK is composed of the DNA-PK catalytic subunit (DNA-PKCS) and the Ku70/Ku80 heterodimer which has DNA end binding activity. The finding of DSBs in Sg3 DNA and the dependency of SR on the DSB repair proteins, Ku80 (8), Ku70 and DNA-PKCS strongly suggests that isotype switching occurs through a NHEJ mechanism (for review see ref. 5). Alternatively, it may be that DSBs in S DNA are not related to the switching process but are normally repaired in a Ku- dependent pathway. Thus, viability of Ku deficient B cells is poor in the presence of DSBs atS regions, making it appear as though SR is dependent on DNA-PK. Nonetheless,both the distinctive DNA sequence at switch junctions and the identification of a role for DSB repair proteins in SR is consistent with the preliminary conclusion that isotype switching is mediated by NHEJ.
A Plasmid Based Transient Transfection Assay For SR
Two forms of NHEJ have been described. Illegitimate recombination is involved in the repair of damaged DNA, including DSBs, through NHEJ. V(D)J joining is a site specific recombination which is initiated through the specific introduction of DSBs at recombination signal sequences and is dependent on NHEJ to finish the recombination reaction. If SR is similar to V(D)J joining then there may be specific switching activities which cleave S DNA. To address the hypothesis that SR is mediated by isotype specific factors, we have developed a plasmid based transient transfection assay for SR to test for the presence of trans-acting switch activities (9). The plasmids are unique in that they lack a eukaryotic origin of DNA replication (10). The recombination activity of these switch substrates is restricted to a subgroup of B cell lines which support isotype switching on their endogenous loci and to mitogen activated normal splenic B cells. Our studies indicate that two distinct switching activities exist which independently mediate m->a and m->g3 SR. These studies imply that SR may be similar to V(D)J joining in that specific factors are required to initiate the switch which is then completed through a NHEJ mechanism.
A model for the mechanism of SR is taking shape. Recent analyses suggest that isotype switching is mediated by NHEJ. The V(D)Jjoining reaction is also in part mediated by NHEJ. This implies that NHEJ, which is the minor pathway for DSB repair in yeast, is the major pathway facilitating the assembly and diversification of Ig genes. The availability of a plasmid based transient transfection assay for SR s an exciting development which is likely to spur important new insights into SR and genome stability.