The AID enzyme initiates introduction of DNA double strand breaks (DSBs) during class switch recombination (CSR) thus exposing B cells to high chances of genomic instability. In fact, 95% of aggressive lymphomas are of B cell origin and translocations involving Igh during CSR are the main cause of diseases such as diffuse large B cell lymphoma. It is therefore crucial to have a complete understanding of this dangerous programed recombination event in order to better understand what conditions predispose to the occurrence of these cancers and how they can potentially be cured. Even though considerable progress has been made in understanding how DSBs are generated during CSR, mechanistic insight into the dynamics of DSBs introduction and repair is still unclear. In this proposal I will address some of these questions. To address these questions, I developed a single locus assay to investigate how DNA breaks are introduced during CSR. My preliminary data shows that of the two locations to which AID is recruited, the most upstream region is targeted first. The mechanism responsible for this ordered introduction of DNA breaks relies on 53BP1. This was surprising as 53BP1 is a protein known to play a role in DNA break repair and therefore a role in deciding where breaks are introduced was not expected. In addition, no other protein involved in DNA repair seems to be required for this process thus supporting the theory that 53BP1 is able to direct AID targeting, before DNA damage. This is of high relevance as 53BP1 has the most extreme defect of all proteins involved in DNA repair during CSR thus suggesting that ordered introduction of breaks is necessary for a successful recombination event. In this proposal I will study the mechanisms by which 53BP1 is recruited to DNA upstream of damage (AIM 1). I will then characterize how 53BP1 impacts order of break introduction by altering Igh chromatin architecture (AIM 2). In the independent phase I will extend these findings to other loci and cell types. Specifically, I will investigate whether 53BP1 is important, upstream of damage to silence transposons (AIM 3). This is highly important as transposable elements are estimated to be responsible for 10% of the de novo mutagenic activity in mouse and human genomes. The K99 phase of this award will be essential for me to finalize my postdoctoral training. It will allow me to continue to work under supervision of Dr. Jane Skok, which I will require to finalize Aim 1 where her knowledge of DNA FISH and DNA damage repair will be instrumental. Most importantly, training by the other 3 members of my advisory board will be required for the experiments I want to set up as an independent researcher. Dr Danny Reinberg will help me with the ChIP-seq and DNA methylation assays. Dr Jef Boeke will introduce me to the field of transposon biology. Dr. Richard Bonneau will guide me in the analysis and integration of the different types of genome-wide dataset analysis. Finally, the environment and opportunities (workshops, conferences, courses, etc) at NYU will provide the ideal setting for the final stages of my postdoctoral training.
When programmed DNA breaks generated during B cell development are not properly repaired, chromosomal translocations can occur that might lead to activation of oncogenes, malignant transformation and ultimately lymphomas. I will investigate how lymphocytes use epigenetic mechanisms to prepare for the introduction of these breaks to ensure successful recombination and avoid genomic instability. I will also study how these same mechanisms minimize the mutagenic potential of transposons, which are another source of genomic instability for B lymphocytes and other cell types.
