Background: VDJ recombination, Class switch recombination (CSR) and somatic hypermutation (SHM) are three B lymphocyte specific processes that mediate antibody gene diversification. VDJ recombination requires the DNA double strand generation by the Recombination activation genes (RAG1 an RAG2) where as CSR and SHM requires the single-strand DNA break activity of the Activation Induced Deaminase (AID) enzyme. Both RAG1/2 and AID activities are coupled with noncoding RNA transcription at sites of DNA break/mutation. The properties of the ncRNAs generated at sites of programmed DNA breaks are poorly characterized in B cells. Recently advances in biology has provided compelling evidence that post-transcriptional and co-transcriptional modification of ncRNAs determine a component of RNA epigenomics and have significant role in driving cellular development and function. In this application, supported by preliminary data generated in our laboratory, we are evaluating the role of RNA modification N6-methyladenosine (m6A) and its associated enzymes METTL3 and METTL14 in B cell development, function and genomic integrity. Objective/Hypothesis: In his proposal, we will determine how RNA methylation m6A on transcripts generated in the IgH locus and the rest of the B cell genome controls programmed DNA recombination, antibody gene diversification and prevents chromosomal instability.
Specific Aims :
Aim 1 :
Aim 1 : Are m6A modifying enzymes Mettl3 and Mettl14 important for class switch recombination, somatic hypermutation, and/or for preventing genomic stability in B cells? ;
AIM 2 : What is the mechanism by which m6A modification promotes IgH DNA recombination and/or prevents genomic instability? AIM 3: Do RNA methylation and coupled RNA surveillance pathways have a role in early B cell development and during VDJ recombination? Study Design: Using cell lines and mouse models, we will study mechanism by which RNA m6A methylation plays a role in programmed DNA recombination and protection of B cell genomic integrity. We will evaluate the mechanism of formation of single-strand DNA structures formation and RNA surveillance at sites of AID activity in matured B cells and the mechanism by which inhibitory RNAs are degraded at sites of RAG activity in B cells during VDJ recombination. We will also evaluate the molecular mechanism by which RNA modification promotes programmed DNA rearrangements. We use a combination of mouse genetics, genomics, biochemistry and 3D-STORM microscopy to accomplish the goals of the proposed project. Disease Relevance: B cells are a central component of the adaptive immune response, but also prone to undergo leukemias and lymphomas when antibody gene diversification processes are not well controlled. Proposed studies leads to a better understanding of the mechanism of antibody gene diversification but also educate us how B cell cancer (specially in context of DLBCL and Multiple Myeloma) are prevented.
During B cell development, rapid transcription of B cell lineage specific genes and the immunoglobulin loci coding and noncoding segments are essential for generation of antibodies that are specific for neutralization of many antigens we encounter. The transcriptional and post transcriptional regulation of the noncoding RNAs generated during B cell development is important since these ncRNAs not only play an important role in controlling gene expression, genome organization and DNA rearrangement mechanisms, but when not controlled properly cause genomic instability. In this application, we evaluate the role of N6-methyladenosine (m6A) modification of various ncRNAs expressed in the B cell genome in promoting antibody gene diversification and conserving B cell genomic integrity.