David G. Schatz, PhD

Generating 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 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.

We have used chromatin immunoprecipitation (ChIP) to demonstrate 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. We have also demonstrated that RAG1 and RAG2 bind to thousands of other sites in the genome, almost entirely at active promoters and enhancers. Computational analysis suggests that RAG binding is driven by interactions with chromatin proteins including modified histone tails, and that this is mediated by regulatory regions in both RAG1 and RAG2. We are now working to understand the molecular interactions that mediate RAG binding patterns and the implications of wide-spread RAG binding for genome stability in developing lymphocytes.

We have a long standing interest in the evolutionary origins of RAG1 and RAG2, beginning with our demonstration in 1998 that these proteins possess cut-and-paste DNA transposase activity. We are studying evolutionarily related transposases from a wide range of invertebrate species, which has led to significant progress in understanding the steps that led to domestication of the ancient RAG transposase and evolution of our adaptive immune system.

Somatic hypermutation (SHM) introduces point mutations into the variable regions of immunoglobulin genes 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.

SHM 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. SHM 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 immunoglobulin enhancer elements function as SHM targeting elements (which we refer to as DIVAC--diversification activator) and are very likely responsible for the high efficiency with which immunoglobulin genes undergo SHM. We are studying the protein factors that bind to DIVAC and attempting to understand the mechanism by which they enhance SHM. In addition, we have developed rapid cell based assays for SHM that have opened the door for CRISPR screens and degron approaches for discovery and analysis of factors involved in SHM. We make use of epigenetic, genome architecture, and computational approaches to determine the rules that dictate susceptibility to AID/SHM.