Immunoglobulin (Ig) genes undergo three genetic modifications during B cell development, namely V(D)J recombination, somatic hypermutation, and class switching. For these reactions to occur, RAGs and the AID enzymes must gain access to recombination and hypermutation sites. The recombinases RAG1 and RAG2 are expressed in progenitor B and T cells and promote V(D)J recombination. The cytidine deaminase AID on the other hand mediates both somatic hypermutation and class switching by converting cytidines into uracils at Ig genes. ? Accessibility during V(D)J, switching, and hypermutation appears to be provided by gene transcription, presumably by way of chromatin remodeling, which exposes Ig genes to enzymatic activity. However, while the accessibility model provides a rationale to specific targeting, it is unclear how DNA lesions downstream of RAG and AID can be processed in the presence of active transcription. ? Last year we published data showing that a particular DNA repair pathway, led by the ATM enzyme, downregulates transcription at sites of DNA damage. This discovery was made using rRNA genes as a system, mainly because i) these genes are highly abundant in the cell (+400 copies per genome), ii) are compartmentalized in the cell nucleus in a highly defined microenvironment known as the nucleolus, and iii) their transcription is exclusively driven by DNA polymerase I. These three features make of rRNA genes an ideal system to address complex questions on cellular dynamics. However, because most genes in the cell (including immunoglobulin genes) are transcribed by polymerase II, it is still unclear whether our published results shed light to the mechanism of recombination or hypermutation in B lymphocytes. To extend our studies to polymerase II transcription we have now generated transgenic mice carrying at a particular genomic site a large number of copies (an array) of polymerase II genes. In addition, we have also developed another transgenic strain expressing a fluorescently labeled polymerase II. Out of several transgenic founders produced, we are currently in the process of selecting a single line that will best provide a means to visualize polymerase II transcription in vivo. Once this mouse is selected we will then isolate mouse embryonic fibroblasts to begin our polII confocal microscopy studies.? As previously mentioned, enzyme accessibility to sites of DNA recombination and hypermutation is also provided by chromatin remodeling. Nucleosomes, formed by the wrapping of 147 bp of DNA around a histone octamer, represent the first level of DNA compaction in the nucleus and an obstacle to DNA repair. To counteract chromatin condensation and facilitate repair, cells have evolved chromatin remodeling complexes and specialized repair enzymes that covalently modify core histones and disrupt DNA-chromatin structures. The evolutionarily conserved complex NuA4 has been shown to be involved in DNA repair via the histone acetyltransferase Tip60 and the ATP-dependent chromatin remodeler p400. Most of what is known about this complex's role in repair was revealed by indirect means because its genetic ablation leads to lethality, at least in mammals. Thus to determine whether the NuA4 complex plays a role in Ig gene recombination and hypermutation we have generated mouse models where the Tip60 enzyme is exclusively deleted in B or T cells. Our preliminary studies indicate that under specific pathogen free conditions these animals are largely normal but have deficiencies in T and B cell development. We are currently using confocal microscopy, flow cytometry, molecular biology techniques, and deep sequencing to dissect the phenotype of Tip60-/- cells. These studies will help us understand whether chromatin decompaction plays a role in the building of the antibody molecule.
