Regulation of recombination and further diversification of immunoglobulin genes by class switch recombination and somatic hypermutation is tightly controlled in a cell type, stage specific and allele specific manner. The precise mechanisms that regulate chromatin accessibility of antigen receptor genes during these events remain elusive. Our goal is to evaluate the role of RNAi in B cell development and function. We have previously demonstrated that miRNAs are essential for B cell survival. Specifically, ablation of miRNAs in B cells upon deletion of Dicer, Drosha or DGCR8 blocked the pro-B to pre-B cell transition due to an increase in apoptosis. By rescuing B cell development using a pro-survival Bcl2 transgene, we are able to investigate the role of short non-coding RNAs in V(D)J recombination as well as in later stages of B cell differentiation. Preliminary data from these studies reveals that ablation of Dicer impacts primary immune repertoire and that this does not happen when Drosha is deleted in a similar manner, suggesting a role for Dicer dependent non-coding RNAs in direct regulation of immunoglobulin loci. We will extend our studies to investigate the nature and role of these non-coding RNAs and examine the contribution of RNAi to immunoglobulin locus accessibility during the germinal center reaction. The control of the immunoglobulin loci by Dicer dependent, microprocessor complex independent RNAi mechanism represents a novel mechanism in mammalian locus regulation. Our goal is to understand the regulation of chromatin accessibility in this context. In addition, the ability to rescue B cell development in the absence of RNAi gives us an opportunity to examine the role of non-coding RNAs in terminal B cell differentiation and the role of miRNA in plasma cell generation as the regulation of the terminal B cell differentiation program remains poorly understood. Our work suffered a significant hit as a result of superstorm Sandy as all the animal models used for these studies were lost with the flooding of the animal facility. In addition, our laboratory was forced to relocate following the storm due to significant damage to our building. This application outlines our efforts to recover the complex animal models necessary for our research and to reproduce the exciting preliminary data that we need to submit a competitive RO1 application within the next one to two years. Specifically, we will generate inducible pluripotent cells (iPS) from the sorted B cells of stored bone marrow samples of our original mice to generate the necessary mouse models for this research. The approach of using B cell derived iPS cells with conditional Dicer and DGCR8 alleles will recapitulate our ability to investigate the role of non-coding RNAs in the later stages of B cell development and give us an opportunity to study the role of non-coding RNAs in somatic hypermutation and class switch recombination in the context of several uniquely rearranged immunoglobulin loci.
The precise mechanisms that govern the allele, cell type and stage specific nature of chromatin accessibility at immunoglobulin loci in lymphocytes remains elusive. We examine the role of miRNAs and other non-coding RNAs in B cell development and function. In particular, we aim to investigate the role of Dicer-dependent nuclear RNAs in the control of accessibility of immunoglobulin loci a process that presents a novel perspective in mammalian gene regulation.