Antibody Diversity; Antibody Formation; Genes, Immunoglobulin; Leukemia, Lymphoid; Lymphoma, Non-Hodgkin; Molecular Biology; Gene Rearrangement, B-Lymphocyte; Gene Rearrangement, T-Lymphocyte; Developmental Biology; Somatic Hypermutation, Immunoglobulin
Generating a Diverse and Effective Repertoire of Antibodies During an Immune Response:
The Schatz laboratory studies V(D)J recombination and somatic hypermutation, reactions that create and optimize antibody genes. Antibodies are blood proteins produced by B cells that are important for fighting infectious disease. V(D)J recombination puts antibody genes together from small pieces of chromosomal DNA, while somatic hypermutation makes mutations in antibody genes and allows for the generation of antibodies that bind viruses and bacteria very tightly. We study these reactions using a wide variety of molecular, genetic, cellular, and biochemical approaches. The focus of our research is understanding the underlying mechanisms of these reactions and how they are targeted specifically to antibody genes. We are also very interested in understanding why V(D)J recombination and somatic hypermutation sometimes affect the wrong genes, and how such mistakes contribute to the development of B cell cancers known as lymphomas and leukemias.
Extensive Research Description
a Diverse and Effective Repertoire of Antigen-Specific Receptors During
Development of the Immune System
The B and T lymphocytes that constitute the adaptive immune system make use of antigen receptor molecules, known as immunoglobulins and T cell receptors, to combat viral and bacterial infections. Each of the hundreds of millions of lymphocytes expresses a different antigen receptor on its surface, indicative of an extraordinary level of diversity in these receptors. The fundamental interest of our lab is to understand the two major processes that generate this diversity: V(D)J recombination and somatic hypermutation.
V(D)J recombination assembles immunoglobulin and T cell receptor genes from component V (variable), D (diversity), and J (joining) gene segments in developing B and T cells. In the first phase of the reaction, two DNA segments are bound by the recombination machinery, brought into close physical proximity, and the DNA is cleaved. In the second phase, the DNA ends are processed and joined by the cellular DNA repair machinery to form the reaction products.
One of our major interests is the enzymatic mechanism of the first phase of V(D)J recombination, which is catalyzed by the proteins encoded by the recombination-activating genes, RAG1 and RAG2. We are studying how the RAG proteins bend and twist the substrate DNA during DNA cleavage and have developed fluorescence resonance energy transfer (FRET) as a method to study the structure of protein-DNA complexes formed by the RAG proteins. We are using a variety of ensemble and single molecule biophysical assays to characterize the structure, composition, and stability of the DNA complexes formed by RAG1/RAG2.
Recently, we have used chromatin immunoprecipitation (ChIP) to determine the pattern of binding of RAG1 and RAG2 to antigen receptor loci in developing lymphocytes. We find that the RAG proteins associate with one small, discrete region of each antigen receptor locus. These regions, which we refer to as recombination centers, are rich in activating histone modifications and RNA polymerase II. We propose that recombination centers are specialized sites within which the RAG proteins bind one DNA segment and then capture a second to allow for recombination. Interestingly, each RAG protein has the ability to be recruited into recombination centers in the absence of the other, suggesting several distinct levels of regulation of RAG binding. We are currently using ChIP combined with parallel sequencing (ChIP-seq) to determine the pattern of binding of RAG1 and RAG2 through the genome in developing B and T lymphocytes. We hope that this study will shed light on the rules that dictate normal and aberrant binding of the RAG proteins and on how the RAG proteins contribute to genome instability and cancer.
Somatic hypermutation introduces point mutations into the variable regions of immunoglobulin genes (which encode antibodies) in B cells during an immune response. These mutations allow for the generation of B cells expressing antibodies with high affinity for an invading microorganism, a process known as affinity maturation. This process is important for the "memory" of the immune system, which helps protect individuals from recurrent infections with the same microorganism, and underlies the success of many vaccines.
Somatic hypermutation is initiated by an enzyme known as activation-induced deaminase (AID), which deaminates cytosine to create uracil in immunoglobulin genes. The uracil is then processed by the mismatch and base excision repair pathways to create mutations at the site of deamination and at nearby sites in the DNA. Somatic hypermutation has been linked to genomic instability and B cell cancers, and our lab is interested in understanding how the reaction is targeted to immunoglobulin loci and how the rest of the genome is protected from its deleterious effects. We have demonstrated that the immunoglobulin promoter plays a significant role in determining the efficiency of somatic hypermutation, and we are investigating the role of promoters and other DNA elements in immunoglobulin loci in the targeting of the reaction. A major focus of our work is to identify the DNA sequences that recruit AID preferentially to immunoglobulin genes.
We have discovered that the genome is protected from damage due to somatic hypermutation by two distinct mechanisms: specific targeting of AID and gene-specific, high-fidelity DNA repair. AID preferentially deaminates immunoglobulin genes, and our data demonstrate that many genes avoid the action of AID altogether. Surprisingly, however, a large number of non-immunoglobulin genes are hit by AID but fail to accumulate mutations because the uracils are repaired in a high-fidelity manner. These genes include many, such as Myc, that have been implicated in the development of B cell malignancies. It is clear that AID acts more widely in the genome than previously suspected. An important implication of these findings is that anything that undermines high-fidelity DNA repair would be expected to allow for widespread accumulation of mutations, with potentially disastrous consequences. We are testing the hypothesis that cellular stress, viral infection, or the early steps in malignant transformation perturb high-fidelity repair and thereby contribute to or hasten the development of cancer.
- Buerstedde, J.M., Alinikula, J., Arakawa, H., McDonald, J.J., and Schatz, D.G. (2014). Targeting of somatic hypermutation by immunoglobulin enhancer and enhancer-like sequences. PLoS Biol 12, e1001831.
- Ji, Y., Resch, W., Corbett, E., Yamane, A., Casellas, R., and Schatz, D.G. (2010). The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci. Cell 141, 419-431.
- Liu, M., Duke, J. L., Richter, D. J., Vinuesa, C. G., Goodnow, C. C., Kleinstein, S. H. and Schatz, D. G. Two levels of protection for the B cell genome during somatic hypermutation. Nature 451, 841-845 (2008)
- Yin, F.F., Bailey, S., Innis, C.A., Ciubotaru, M., Kamtekar, S., Steitz, T.A., and Schatz, D.G. (2009). Structure of the RAG1 nonamer binding domain with DNA reveals a dimer that mediates DNA synapsis. Nat Struct Mol Biol 16, 499-508.
- Ciubotaru, M., Kriatchko, A.N., Swanson, P.C., Bright, F.V., and Schatz, D.G. (2007). Fluorescence resonance energy transfer analysis of RSS configuration in the RAG1/2 synaptic complex. Mol. Cell. Biol. 27, 4745-4758.
- Yang, S.Y., Fugmann, S.D., and Schatz, D.G. (2006). Control of gene conversion and somatic hypermutation by immunoglobulin promoter and enhancer sequences. J. Exp. Med. 203:2919-2928.