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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 the programmed genomic DNA alterations that occur in lymphocytes, as well as general processes that maintain stability of the mammalian genome and suppress cancer. This report focuses primarily on work aimed at elucidating the mechanism of immunoglobulin (Ig) heavy-chain (IgH) class-switch recombination (CSR).

B lymphocytes secrete antibodies composed of IgH and Ig light (IgL) chains. The amino-terminal portion of Ig chains binds antigens and is referred to as the variable (V) region because of its great diversity. 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 RAG (recombination-activating gene) endonuclease, which introduces breaks in both strands of the duplex chromosomal DNA (DSBs) precisely between short, conserved recombination sequences and adjacent V-, D-, or J-coding sequences. V(D)J recombination is completed by joining broken V, D, and J gene segments to create a V-region exon. Our lab discovered that RAG-generated DNA ends are joined by repair proteins that are expressed in all cells to repair DSBs in a process that requires no homologies to pair the sequences and that is referred to as nonhomologous DNA end joining (NHEJ).

	Dicentric translocation involving chromosomes 12 and 15... Genomic instability...

We have generated mice deficient for various NHEJ factors and found deficiencies for each lead to lack of mature B and T lymphocytes (severe combined immune deficiency) owing to inability to complete V(D)J recombination. Cells from NHEJ-deficient mice are hypersensitive to agents that generate DSBs and have dramatic genomic instability. All NHEJ-deficient mice, in the context of deficiency for the p53 cell cycle checkpoint factor, rapidly develop immature B lymphomas with RAG-initiated complex translocations of the IgH locus that lead to amplification of c-Myc or N-myc oncogenes. Deficiency for certain NHEJ factors, including the XRCC4 factor discovered by our lab, also leads to p53-dependent apoptosis of newly generated neurons. In this context, elimination of XRCC4 in developing neurons of p53-deficient mice causes brain tumors (medulloblastomas) that, like the immature B lymphomas, harbor recurrent translocations and frequent amplification of Myc-family oncogenes. Thus, NHEJ suppresses genomic instability and oncogenic transformation of multiple cell types.

The carboxyl-terminal portion of IgH and IgL chains is referred to as the constant (C) region. The IgH C region (C_H) determines the antibody class (IgM, IgG, IgA, etc.) and prescribes antigen-eliminating effector 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. There are seven additional sets of IgH exons (termed C_H genes) lying from 100 to 200 kb downstream of Cμ. CSR replaces Cμ with a downstream C_H gene (e.g., Cγ, Cε, or Cα) via recombination between very large (1–10 kb), repetitive "switch" (S) regions that lie just upstream of each C_H gene. CSR appears to be initiated by DSBs in two S regions, followed by their joining to complete CSR. There are several known requirements for CSR. First, we and others have shown, by gene-targeted deletion/replacement, that S regions are essential for normal CSR. Second, we have shown that CSR is targeted to specific S regions via B cell activation pathways that induce transcripts through particular unrearranged S regions. Finally, Tasuku Honjo (Kyoto University) discovered a protein termed activation-induced cytidine deaminase (AID) that is absolutely required for CSR and for the somatic hypermutation (SHM) process that introduces point mutations into IgH and IgL V-region exons to allow generation of B cells that secrete higher affinity antibodies. Recently, we have elucidated a mechanistic relationship between S regions, transcription, and AID with respect to initiation of CSR and discovered that factors used to carry out the general response to DSBs also join broken S regions during CSR.

Both RNA editing and DNA deamination have been argued to explain AID function in initiating CSR and SHM. Genetic evidence from Michael Neuberger (Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.) suggested that AID deaminates cytidines in DNA, followed by their further processing via nonstandard actions of general DNA repair pathways to yield V-region exon mutations (SHM) or S-region DSBs (CSR). In support of this model, we showed that purified AID from activated B cells efficiently deaminates cytidines in single-strand (ss), but not double-stranded (ds), DNA. As most DNA in cells is duplex, we sought to elucidate the mechanism by which AID accesses dsDNA. We first showed that mammalian S regions, when transcribed in vitro, form stable RNA-DNA hybrid structures, termed R loops, in which the looped out DNA strand serves as an effective AID substrate, providing one mechanism for AID access to transcribed S regions. Transcribed V regions do not, however, form R loops, indicating that another mechanism must operate during SHM.

We employed biochemical approaches to elucidate a mechanism by which AID accesses sequences that do not form R loops. This work led us to identify replication protein A (RPA), an ssDNA-binding protein that functions in replication and repair, as a cofactor that allows AID to deaminate in vitro transcribed V-region sequences. Both V-region exons and S regions are rich in four-nucleotide sequences termed RGYW motifs, which are known hot spots for SHM. We found that phosphorylated AID from activated B cells interacts specifically with RPA and that this complex deaminates in vitro transcribed substrates that are RGWY rich. We proposed that RPA promotes AID access to transcribed DNA by stabilizing ssDNA loops in the context of transcription bubbles on RGYW-rich substrates. As S regions are rich in RGYW motifs, we considered that AID also might target S regions via the RPA/transcription mechanism. Although amphibian S regions are AT rich and do not form R loops, they are rich in palindromic AGCT sequences, a subset of the RGYW motif. To test whether such sequences could mediate mammalian CSR, we replaced the mouse Sγ1 region with an amphibian S region and found the latter to mediate CSR effectively in activated mouse B cells, with the high-density AGCT patches serving as AID/RPA targets in vitro and CSR hot spots in vivo. Thus, AID appears to access transcribed mammalian S regions via both R-loop–dependent (RPA-independent) and RPA-dependent mechanisms. We showed that a portion of AID in activated B cells is phosphorylated on serine 38 by protein kinase A, and we provided evidence that such phosphorylation is necessary for RPA interaction in vitro and for full CSR activity in vivo. Thus, AID phosphorylation appears to augment AID activity in CSR, likely through an RPA-mediated mechanism for accessing transcribed S regions.

We showed that NHEJ suppresses translocations by joining chromosomal DSBs back together rather than joining them to separate DSBs, suggesting chromatin components hold DSB ends in proximity for rejoining via NHEJ. Work from William Bonner (National Institutes of Health) and others showed that, in response to DSBs, histone H2AX is phosphorylated over megabase flanking regions by ATM and related kinases. 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 chromatin complexes between H2AX and these other proteins contributes to tethering broken DNA ends for proper religation via NHEJ. In this context, work from our lab and others showed that normal CSR requires all of these DSB response factors. We also found that mice deficient for both H2AX and p53 had dramatically increased tumor onset, including mature B cell lymphomas with translocations that linked IgH S regions to the c-Myc oncogene, suggesting that H2AX may suppress oncogenic translocations associated with aberrant CSR.

Most recently, we discovered that AID-dependent IgH locus chromosome breaks occur at high frequency in H2AX-deficient primary B cells activated in culture for CSR, clearly showing that H2AX functions in end joining of DSBs during CSR and by extension, likely functions similarly in general DSB repair. Moreover, a substantial portion of these AID-initiated chromosomal breaks participated in chromosomal translocations. Consistent with our model, we further found that activated B cells deficient for ATM, 53BP1, and MDC1 had a similar chromosome breakage and translocation phenotype. Thus, our findings support a general role for DSB response factors in DSB repair via end joining, a function required to prevent DNA DSBs from prematurely separating and leading to chromosomal breaks and translocations.

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

Last updated: August 22, 2007