HomeResearchGene Rearrangement, DNA Repair, Genomic Stability, and Cancer

Our Scientists

Gene Rearrangement, DNA Repair, Genomic Stability, and Cancer

Research Summary

Frederick Alt is interested in the molecular mechanisms involved in the somatic assembly and modification of antigen receptor genes. He is also interested in elucidating mechanisms that suppress genomic instability and cancers of the immune system.

We study programmed genomic DNA alterations in lymphocytes, as well as general processes that maintain stability of the mammalian genome and suppress cancer. This report summarizes recent work in which we link these two areas of focus through studies of immunoglobulin (Ig) heavy-chain (IgH) class-switch recombination (CSR).

B lymphocyte–lineage cells secrete antibodies composed of IgH and Ig light (IgL) chains. The amino-terminal variable (V) region of Ig chains has great diversity and is involved in antigen binding. V-region exons are assembled from germline V, D (diversity), and J (joining) gene segments by V(D)J recombination. V(D)J recombination is initiated by the recombination-activating gene (RAG) endonuclease, which introduces breaks in both strands of the duplex chromosomal DNA, referred to as double-strand breaks (DSBs), between short, conserved signal sequences and adjacent V-, D-, or J-coding sequences. V(D)J recombination is completed by joining broken V, D, and J segments to create a V-region exon. We discovered that RAG-generated DNA ends are joined by repair proteins that are expressed in all cells, where they repair DSBs in a process that requires no homologies to pair the ends. This process is referred to as nonhomologous DNA end joining (NHEJ).

The carboxyl-terminal portion of IgH and IgL chains is referred to as the constant (C) region. The IgH C region (CH) determines antibody class (IgM, IgG, IgA, etc.) and antigen-eliminating functions. The IgH V(D)J exon is assembled upstream of the Cμ exons, which leads to generation of μ IgH chains and IgM antibodies. Additional sets of IgH exons (termed "CH genes") are located 100 to 200 kb downstream of Cμ. CSR replaces Cμ with a downstream CH gene (e.g., Cγ or Cα) via breakage and joining of large (1–10 kb), repetitive "switch" (S) regions that lie upstream of CH genes. For CSR, DSBs must be introduced into the donor Sμ and into a downstream acceptor S region. Targeted S regions also must be brought together over long distances ("synapsed") and joined. Our recent work has elucidated mechanistic aspects of these processes.

We and others have shown that S regions are essential for CSR and that CSR is targeted via B cell activation pathways that induce transcription through S regions prior to rearrangement. Tasuku Honjo (Kyoto University) discovered that the activation-induced cytidine deaminase (AID) protein initiates CSR, as well as the somatic hypermutation (SHM) process that introduces point mutations into V-region exons to generate higher affinity antibodies. Our work elucidated a unifying mechanism that explains the role of AID, S regions, and transcription in CSR.

How AID functions in CSR and SHM has been a contentious question. Honjo argued that AID functions via RNA editing, while Michael Neuberger (Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.) and others argued, based on genetic data, that DSBs in CSR and SHM of V-region exons both result from differential processing of AID-deaminated cytidines in DNA. We provided strong evidence for the DNA-based mechanism by showing purified mouse B cell AID (mAID) deaminates cytidines in single-strand DNA (ssDNA) and that AID is recruited to both donor and acceptor S regions in activated B cells. Because mAID lacked double-strand DNA (dsDNA) deamination activity and most DNA in cells is duplex, we sought mechanisms by which AID accesses dsDNA. We found that transcribed mouse S regions form stable RNA-DNA hybrid structures, termed "R loops," in which the looped-out DNA strand is a robust AID substrate, and we provided genetic evidence to support the role of R loops in mammalian CSR.

Transcribed V regions do not form R loops, indicating additional AID access mechanisms during SHM. Also, we found that amphibian S regions do not form R loops but function in mouse CSR, indicating additional AID access mechanisms during CSR. We used biochemical approaches to identify replication protein A (RPA), an ssDNA-binding protein known to function in replication and repair, as a cofactor that allows AID to deaminate V regions transcribed in vitro, amphibian S regions, and synthetic sequences rich in four-nucleotide sequences (RGYW motifs), which are hot spots for SHM. We further found that a portion of activated B cell AID is phosphorylated by protein kinase A (PKA) at serine 38 (S38). S38 phosphorylation, although not necessary for ssDNA deamination activity (basic catalytic activity), is necessary for RPA interaction, transcription-dependent dsDNA deamination, and normal CSR activity. We proposed that RPA promotes access of S38-phosphorylated AID to transcribed dsDNA rich in RGYW motifs, thereby augmenting CSR and SHM activity.

Bony fish carry out SHM but not CSR. However, zebrafish AID (zAID) has CSR activity in mouse B cells comparable to that of mAID, even though zAID lacks a PKA phosphorylation site, raising questions about the role of this modification in mAID function. In this context, we showed that zAID has particular amino acid differences from mAID that constitutively endow it with the biochemical and CSR properties of S38-phosphorylated mAID. DSBs are a most dangerous form of cellular DNA damage, leading to apoptosis or oncogenic genomic alterations if not properly repaired. Therefore, we propose that evolution of CSR, which relies on AID-induced DSBs, might have selected for fixation of post-translational control mechanisms to tightly regulate AID activities. Based on our zAID studies, we have engineered mAID proteins that robustly function in the absence of S38 phosphorylation, and which, via gene targeting, should allow physiologic evaluation of this mode of AID post-translational regulation.

We elucidated pathways that synapse and join S regions during CSR. We demonstrated that DSBs induced by a yeast endonuclease replace both S regions and AID to generate recombinational IgH class switching in activated B cells. Thus, S regions may function largely as AID targets to produce DSBs that subsequently are synapsed and joined over long range (e.g., 100 kb) by general cellular repair processes. The high efficiency of this long-range synapsis/joining process is remarkable, given findings of Maria Jasin (Memorial Sloan-Kettering Cancer Center) that similarly introduced DSBs on separate chromosomes are joined at far lower frequency. We are now studying whether such long-range DSB synapsis/joining mechanisms are specific to the IgH locus and activated B cells or reflect general processes that promote joining of DSBs within a chromosome. Of potential relevance to that latter possibility, our lab previously found that chromatin components prevent translocations by preferentially holding DSB ends in proximity for rejoining via NHEJ, thereby suppressing joining to DSBs on other chromosomes.

Our lab and the labs of Andre Nussenzweig (Nation Cancer Institute) and Michel Nussenzweig (HHMI, Rockefeller University) have further elucidated roles of chromatin components in NHEJ and CSR based on knowledge that histone H2AX is phosphorylated by the ataxia-telangiectasia-mutated kinase (ATM) and related kinases over megabase regions flanking DSBs. During the DSB response, these kinases also phosphorylate the MDC1, 53BP1, and NBS1 cell cycle/checkpoint proteins, which specifically bind phosphorylated H2AX. We proposed that formation of such complexes contributes to tethering broken DNA ends for proper ligation via NHEJ. Correspondingly, we demonstrated that these DSB response factors are required for the joining phase of CSR, where they prevent DNA breaks initiated by the AID enzyme from progressing to chromosomal breaks and translocations, providing direct evidence that such factors function in DNA end joining. Finally, we demonstrated the role of this process in tumor suppression when we found that mice deficient for both H2AX and p53 have a dramatic predisposition to cancer, including mature B cell lymphomas in which the IgH locus is linked to the c-myc oncogene via aberrant CSR.

Our lab previously showed that end joining during V(D)J recombination requires NHEJ and does not occur in its absence. In contrast, we recently have shown that joining during CSR is carried out both by classical NHEJ and by a distinct alternative end-joining pathway (A-EJ) that is strongly biased toward the use of DNA microhomologies to form joins. We have also shown that A-EJ can, in the absence of classical NHEJ, robustly catalyze chromosomal translocations. Elucidation of this novel A-EJ pathway is an ongoing goal.

Based on our discovery that DSB response and NHEJ factors suppress chromosomal breaks and translocations, we have generated several novel mouse models for lymphomas and solid tumors that are being used to elucidate mechanisms that suppress genomic stability and cancer.

Grants from the National Institutes of Health provided support for some of these studies.

As of August 07, 2008

Scientist Profile

Investigator
Boston Children's Hospital
Cancer Biology, Immunology