Investigating the role of chromatin-associated RNA processing in Early B-cell development

Columbia University Health Sciences, New York NY

Investigators

Abstract

Project Summary B-cells are unique among mammalian cells because of their ability to undergo programmed genomic rearrangements. The first of these rearrangements is called VDJ recombination and is primarily responsible for generating the vast diversity of antibodies seen in nature. This is critical for the development of the adaptive immune system, but generating double-stranded DNA breaks is an enormous risk to the cell, therefore VDJ recombination must be tightly regulated. My lab has previously shown that VDJ recombination depends on the RNA exosome’s 3’ RNA processing activity. After losing the RNA exosome Pro-B-cells accumulate non-coding RNAs throughout the IgH locus. My project seeks to build upon these findings by finding the exact mechanism through which the RNA exosome and RNA helicases act during VDJ recombination. I will use novel mouse models to investigate RNA helicases that have been shown to act on chromatin-associated RNAs that I hypothesize play a role in VDJ recombination. I will confirm these results in a RAG-inducible in-vitro system to confirm that off-target genomic instability is not causing the defect in VDJ recombination and investigate the transcriptional status of RNA processing deficient Pro-B-cells. After these experiments, I will perform experiments to identify the status of the chromatin-associated RNA at the Ig loci and how they affect the frequency and structure of double-strand breaks at recombination signal sequences. Finally, I will attempt to rescue the defect by targeting a dCas9-RNAseH construct to sites with increased chromatin-associated RNAs in helicase or exosome-deficient backgrounds, showing that the helicases and the exosome play an active and direct role in VDJ recombination, independent of their role elsewhere in the genome.