David G. Schatz, PhD - US grants

DESCRIPTION (provided by applicant): V(D)J recombination assembles the variable portions of immunoglobulin and T cell receptor genes and is essential for lymphocyte development. Errors in the recombination process can lead to chromosomal translocations and the development of human malignancies, particularly childhood leukemias. The causes of such translocations are thought to be improper targeting of the recombination machinery and the premature release of broken chromosomal ends before they have been properly rejoined. The proteins encoded by the recombination activating genes, RAG1 and RAG2, play central roles in targeting and DNA end rejoining during V(D)J recombination and are therefore important in the generation of translocations. In the first phase of V(D)J recombination, DNA substrate recognition and the production of double strand breaks take place in highly organized nucleoprotein complexes whose integrity and specificity are in large part determined by RAG1 and RAG2, probably in conjunction with the DNA bending protein HMGB1. Little is known about the structure of these complexes or the conformational changes that occur during DNA binding and cleavage. The hypothesis underlying this proposal is that the sequential, properly regulated formation of these complexes requires an orchestrated series of structural changes in both the substrate DNA and the RAG/HMGB1 proteins. We have demonstrated that RAG1 undergoes a conformational change when it binds DNA and have used fluorescence resonance energy transfer (FRET) assays to characterize DNA organization and bending in single substrate complexes as well as in higher order synaptic complexes. We propose to use a combination of biochemical and fluorescence methods to elucidate protein and DNA structural changes that occur during assembly of RAG protein-DNA complexes and to construct initial three- dimensional models of the DNA in these complexes. The structural information will be connected to function (catalysis of DNA cleavage) through the use of altered DNA substrates, mutated RAG proteins, and fluorescently labeled HMGB1. We are particularly interested in understanding the structural underpinnings of the requirement for asymmetric DNA substrates and the molecular rules governing recently discovered restrictions on the rearrangements of endogenous gene segments. We anticipate that these studies will provide insights into how the DNA and proteins communicate with and influence one another to create a complex within which properly coordinated cleavage events can occur. Public health relevance: These experiments study V(D)J recombination, an essential process that occasionally makes mistakes and generates aberrations in human chromosomes during the development of white blood cells known as lymphocytes. These aberrations contribute to the development of certain blood cancers and it is hoped that this work will provide insights into the causes of these mistakes.

9453447 David G. Schatz This research focuses on understanding how vertebrate organisms change the structure of their DNA, particularly through site specific recombination. Specifically, the PI will examine the process by which lymphocytes assemble their antigen receptor genes, studying both the enzymology and molecular basis of this site specific recombination reaction. This work has implications beyond immunology, because the PI will analyze what other cell types besides lymphocytes may make use of such site specific mechanisms to encode genetic information. In particular, he will also study processes in the nervous system. Dr. Schatz Proposes to create transgenic mice in which the expression, but none has been adapted successfully for use in a living mammal. Such a system would provide a powerful new tool for the study of development and gene function. Analyses of these mice will provide a variety of insights into several different biological realms: (1) the immune system; (2) the enzymology of site specific recombination in vertebrates; (3) the integrity of the genome; and (4) the discovery of other site specific recombination processes in vertebrates. These experiments will provide a new vantage point from which to view the vertebrate genome, the forces that protect it, and the processes that alter it. ***

DESCRIPTION (provided by applicant): The goal of the Yale Interdisciplinary Immunology Training Program (YIITP) is to equip predoctoral and postdoctoral trainees with the intellectual and research foundations necessary to become independent scientists/educators investigating the immune system and its roles in human disease. The YIITP combines rigorous research training in a highly collaborative, interactive environment with a thorough academic program of instruction in immunology, microbiology, and related disciplines. The program offers training in virtually all aspects of immunology as well as host-pathogen interactions and a variety of autoimmune and inflammatory disorders. Areas of particular strength include innate immune recognition and function, lymphocyte development, immunological tolerance and memory, antigen presentation, immune cell signaling, the immune response to infectious organisms, vascular endothelial cells, cancer immunology, and autoimmune diseases such as Type 1 diabetes, multiple sclerosis, and systemic lupus erythematosus. The 35 YIITP mentors, who have primary appointments in 8 different Yale departments, have an outstanding record of research accomplishment and training and many are leaders in their fields. The YIITP is overseen by the Program Director, David Schatz, and an Executive Committee of four additional faculty members. The principal training entity is the Department of Immunobiology, whose graduate program was the top ranked immunology graduate program in the United States in a 2010 National Research Council study. All students admitted to the YIITP have at least a Bachelor's degree in a relevant field and enter via application to the Yale interdepartmental program in Biological and Biomedical Sciences (BBS). Postdocs enter from the labs of YIITP faculty, and hold a Ph.D. and/or M.D. degree. Predoctoral training leading to the Ph.D. degree involves formal course work in Immunology and other areas of biology, research rotations, teaching, and the qualifying exam in the first two years, with dissertation research beginning late in year one and becoming the primary focus of activity after completion of the qualifying exam. Postdoctoral training focuses intensively on research in the laboratory of one or more of the mentors. Both predoctoral and postdoctoral training are enriched by intensive training in the methods, logic, and responsible conduct of research, and by the many opportunities for collaboration and interaction. The vast majority of YIITP trainees go on obtain independent research and teaching positions at academic institutions or research positions in biotechnology companies. Extensive efforts are made by YIITP mentors and Yale Graduate and Medical Schools to attract and retain trainees from diverse backgrounds, particularly under-represented minority groups. This proposal requests continued support for 10 predoctoral and 3 postdoctoral trainees who will be supported by this grant for a maximum of 2 years. A major recent improvement in the program comes in the form of full institutional support from Yale for first year graduate students, allowing YIITP funding to be restricted to 2nd and 3rd year students.

! SUMMARY Errors made by the RAG1/RAG2 endonuclease during V(D)J recombination can lead to genome instability and the development of leukemia and lymphoma. One important cause of such instability is improper action of RAG at cryptic recombination signal sequences (RSSs) that are present abundantly in the genome. Our prior work provided mechanistic and structural insights into RAG protein-DNA complexes and revealed that RAG1 and RAG2 bind to numerous sites in the genome outside of the antigen receptor loci, virtually all of which are active promoters or enhancers. We further demonstrated that such off-target RAG binding occurs through two modes, one driven by a histone code reader function of RAG2 (largely at promoters) and the other dependent on regulatory portions of RAG1 (largely at enhancers). The thousands of off-target RAG binding sites and the millions of cryptic RSSs in the genome raise fundamental questions about how the genome is protected from devastating instability caused by RAG. The central objective of our proposed experiments is to determine the rules that govern RAG off-target activity by understanding the interactions that dictate RAG localization in the genome and the mechanisms that determine which cryptic RSSs are cleaved by RAG and which are spared. We will use complementary biochemical, biophysical, genetic, and genomic approaches to achieve the following aims: Aim 1. Determine the rules governing the selection of cryptic RSS targets by RAG. We and others have identified intriguing sequence and topological features of the cryptic RSSs cleaved by RAG, but it is unknown to what extent, or how, these features dictate RAG activity. We will systematically determine how cryptic RSS orientation, location, and sequence influence RAG off-target activity and in the process, test a provocative new hypothesis that RAG acquires its targets in part through linear tracking along DNA. Aim 2. Determine the domains and interactions directing RAG1 to active enhancers. Much off-target cutting by RAG occurs in enhancers, but the molecular interactions that dictate localization of RAG to these regions are not known. We will identify the portions of RAG1 and the RAG1-binding partners that specify enhancer binding and test the hypothesis that RAG1, like RAG2, is a reader of the histone code. Aim 3. Determine how retargeting of RAG alters cRSS selection and off-target cleavage patterns. We will use RAG mutants and a novel, inducible in vivo targeting system to direct RAG to new sites in the genome and determine the spectrum of new cleavage sites that arise. We will also reconstitute RAG- mediated cleavage at strong cryptic RSSs normally ignored by RAG to uncover the epigenetic and chromatin architectural parameters that specify RAG off-target activity. Together, our proposed studies have a dual significance, both for basic mechanisms of RAG function and for the causes of cancer.

SUMMARY Multiple Myeloma (MM) is a malignancy of bone marrow plasma cells preceded by a series of premalignant and transitional stages. Cells at all stages exhibit significant genomic aberrations, the sources of which are not well understood. This has left a major gap in our understanding of the mechanisms that drive MM initiation and progression, a gap that this proposal is designed to fill. Recent evidence suggests that cytidine deaminases?enzymes that convert cytosine to uracil in DNA?are important culprits in genomic instability in MM. One important example is the activation induced deaminase, AID, which initiates somatic hypermutation and class switch recombination but which also mutates many regions of the B cell genome and causes translocations similar to those found in MM. The related APOBEC3 family of cytidine deaminases can also mutate DNA, and numerous findings link AID and APOBEC3 enzymes to genome instability and mutations in MM. Our preliminary data demonstrate that MM cells express APOBEC3B, C, D, F and G, with APOBEC3B being expressed particularly strongly. Interestingly, APOBEC3B has recently been implicated in genome instability in breast cancer. We further find that expression of AID and certain APOBEC3 enzymes increases levels of DNA strand breaks in MM cells. Based on this, we hypothesize that AID and APOBEC3 family enzymes are a major cause of genomic aberrations and disease progression in MM. Recent findings provide a strong link between lipid disregulation, immune activation, and MM, with as much as a third of the clonal gammopathies found in MM patients reacting to lipids. Hence, an important overall guiding hypothesis of our work is that chronic B cell activation arising from elevated levels of inflammatory lipids contributes to increased activity of AID/APOBEC3 and MM disease progression. We systematically test these hypotheses in three Aims. In Aim 1, we determine the extent to which AID/APOBEC3 enzymes, alone and in concert, contribute to biochemical and molecular measures of nuclear deaminase activity and genomic damage. In Aim 2, we determine how these deaminases are regulated, revealing those regions of the MM genome that are susceptible to mutation, DNA breaks, and translocations due to their action. Finally, in Aim 3 we use established and novel mouse model systems and MM and its premalignant stage propagated in humanized mice to assess the in vivo effects of inflammatory lipids on the expression/activity of AID/APOBEC3 factors, DNA damage and mutation of the MM genome, and clonal evolution of MM. Together, the proposed experiments will provide a comprehensive picture of the activity, targeting, and outcome of cytidine deaminase action in MM, with broad implications for disease pathogenesis.

SUMMARY During the humoral immune response, somatic hypermutation (SHM) introduces point mutations in rearranged immunoglobulin (Ig) genes of activated germinal center (GC) B cells. SHM is essential for the fine-tuning of antibody affinity, the generation of B cells expressing high-affinity antibody, and the efficacy of many vaccines. Mistargeted SHM activities can lead to mutations and chromosomal translocations that contribute to the development of B cell lymphoma. Recent studies suggest that the three-dimensional (3D) organization of the genome regulates SHM targeting and mistargeting. However, it is largely unknown how the genome is spatially organized across multiple length scales in GC B cell development and lymphoma, and how 3D genome architecture mechanistically affects the targeting and mistargeting of SHM. Conventional approaches cannot address these questions in the primary GC tissue environment due to technical limitations. Here, we propose to apply a new method recently developed by our team, termed Multiplexed Imaging of Nucleome Architectures (MINA), to primary human tonsil tissue samples and malignant GC-derived human B cell lymphomas. We will investigate and test the association between SHM susceptibility and a variety of 3D nucleome architectures, including topologically associating domain (TAD) architecture, phase separation, and nuclear positioning of genomic regions relative to nuclear lamina, nucleoli, and nuclear pores. Through targeted genomic perturbations in human B cell lymphomas, we will test specific hypotheses linking SHM targeting elements to elevated chromatin looping interactions, TAD phase separation, nuclear pore proximity, and mutation vulnerability. Our study will significantly advance our understanding of the role of 3D genome architecture and nuclear organization in GC B cells undergoing SHM in both the developmental and tumorigenesis contexts. We expect this study to establish a new research paradigm and transform 3D nucleome investigations in immunobiology.