Our immune system encounters a plethora of antigens including viruses and bacteria. We negate these challenges in part by generating a diverse set of antibodies. The diversity comes through stitching together various segments of genes through a process called V-D-J (segment) recombination. But VDJ recombination fails to generate sufficient antibody diversity to negate nature’s antigenic challenges. Somatic hypermutation (SHM) and class switch recombination (CSR) add additional diversity. Both SHM and CSR depend upon the DNA mutagenesis activity of Activation Induced Deaminase (AID). Unfortunately, during the process of SHM, AID can accidentally generate mutations that ultimately lead to deleterious chromosomal translocations, deletions, and mutagenesis, which may culminate in malignancies. Thus, loss of AID function leads to immunodeficiency syndromes when insufficient diversity is generated but aberrant AID activity leads to cancer. In our laboratory, we investigate various regulatory mechanisms that promote AID activity during CSR and SHM but prevent it from causing oncogenic chromosomal translocations. In the process of addressing critical question regarding antibody diversification mechanisms, we investigate the fields of DNA recombination, non-coding RNA processing, DNA damage repair and epigenetic.
Description of Current Research
Our research elucidates the function of non-coding RNA (ncRNA) transcription and processing in the control of DNA recombination and mutagenesis in somatic cells. Our laboratory has developed a pipeline in which transiently expressed regulatory ncRNAs can be identified and functionally characterized. We use conditional models for the ncRNA degradation complex RNA exosome, high throughput RNA-sequencing, and bioinformatics tools to identify novel long intergenic ncRNAs, enhancer RNAs, and antisense RNAs. Using this pipeline, we have localized transcription of B-lymphocyte genome antisense RNAs to where chromosomal translocations occur.Mechanisms that promote transcription termination of these antisense RNAs, when perturbed, generate ssDNA structures ideally suited for creating chromosomal translocations either due to DNA mutator activity of the oncogene Activation Induced cytidine Deaminase (AID) or due to other mechanisms such as transcription/replication complex collisions. Biological functionality of the transcription of these lncRNAs are attributed to maintenance of genomic organization and structure. For example, lncRNA-CSR, a ncRNA expressed at an enhancer sequence distal to the immunoglobulin heavy chain locus (IgH), physically interacts with a super-enhancer sequence to promote its activity in IgH locus recombination.Taken together, our research has advanced the mechanistic understanding of non-coding RNA transcription occurring inside topological domains of enhancer/super-enhancer/promoter interaction networks. In addition, our laboratory has identified a direct role for RNA exosome and RNA polymerase II ubiquitination in AID-dependent mutagenesis of the IgH locus during DNA recombination events, promoting IgH class switch recombination in B cells. Conclusions from our research provides a critical understanding of immune system disregulation and oncogenesis. Below are some examples of focus point projects being pursued in the laboratory.
Activation induced deaminase (AID) mediated mutations on Both strands of DNA substrate
Activation Induced cytidine Deaminase (AID) initiates Immunoglobulin (Ig) heavy chain (IgH) class switch recombination (CSR) and Ig variable region somatic hyper mutation (SHM) in B lymphocytes by deaminating cytidines on template and non-template strands of transcribed DNA substrates. However, the mechanism of AID access to the template DNA strand, particularly when hybridized to a nascent RNA transcript, has been an enigma. We now implicate the RNA exosome, a cellular RNA processing/degradation complex, in targeting AID to both DNA strands.
In B lineage cells activated for CSR, the RNA exosome associates with AID, accumulates on IgH switch regions in an AID dependent fashion, and is required for optimal CSR. Moreover, both the cellular RNA exosome complex and a recombinant RNA exosome core complex impart robust AID and transcription dependent DNA deamination of both strands of transcribed SHM substrates in vitro. Our findings reveal a role for non-coding RNA surveillance machinery in generating antibody diversity.
RNA exosome substrate antisense transcription, AID and B Cell genomic mutagenesis mechanisms
The immunoglobulin diversification processes of somatic hyper mutation and class switch recombination critically rely on transcription coupled targeting of AID to Ig loci in activated B lymphocytes. AID catalyzes deamination of cytidine deoxynucleotides on exposed single stranded DNA. In addition to driving immunoglobulin diversity, promiscuous targeting of AID mutagenic activity poses a deleterious threat to genomic stability. Recent genome wide studies have uncovered pervasive AID activity throughout the B cell genome.
It is increasingly apparent that AID activity is frequently targeted to genomic loci undergoing early transcription termination where RNA exosome promotes the resolution of stalled transcription complexes via co transcriptional RNA degradation mechanisms. In our laboratory, we study consequences of eukaryotic transcription that lead to RNA exosome recruitment, and ultimately AID mediated mutagenesis of the B cell genome.
Long non-coding RNA and enhancer RNA transcription that control distal DNA regulatory element interactions
We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem (ES) cells by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of novel long noncoding RNAs (lncRNAs) and enhancer RNAs (eRNAs). Unexpectedly, eRNA expressing regions accumulate Rloop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNAexpressing element (lncRNACSR) engaged in long range DNA interactions and regulating IgH 3’ regulatory region superenhancer function.
CRISPR-Cas9 mediated ablation of lncRNACSR transcription decreases its chromosomal looping mediated association with the IgH 3’regulatory region superenhancerand leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers, by resolving deleterious transcription coupled secondary DNA structures, while also regulating long range superenhancer chromosomal interactions important for cellular function.
RNA polymerase II stalling regulation
Activation Induced Deaminase (AID) function is essential for initiation of class switch recombination (CSR) and somatic hyper mutation (SHM) in B lymphocytes. AID deaminates cytidine residues on substrate sequences in the immunoglobulin(Ig) locus via a transcription dependent mechanism. This activity is stimulated by the eleven subunit cellular noncoding RNA 3’- 5’ exonucleolytic processing complex, RNA exosome. The mechanism by which the RNA exosome recognizes the free 3’end of immunoglobulin locus specific RNA substrates and stimulates AID DNA deamination activity on its in vivo substrate sequences is an important question.
Here we report that the cellular HECT E3-ligase, Nedd4, supports AID function during CSR by controlling RNA polymerase II associated AID/RNA exosome complex function in B cells. We observe that Nedd4 ubiquitinates RNA pol II for degradation, thus providing a mechanism for generation of Ig locus specific 3’end free germline transcripts. In this study we link noncoding RNA processing following RNA polymerase I pausing with regulation of the mutator AID protein.