Uttiya Basu, PhD - US grants

DESCRIPTION (Provided by the applicant) Abstract: This proposal is directed towards the understanding of various mechanisms by which non-coding RNAs (ncRNA) govern antibody diversification during adaptive immune response. Antibodies are polypeptide complexes produced from B-lymphocytes that are present in the bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign antigens, such as bacteria and viruses. Newly generated B cells migrate from bone-marrow to secondary lymphoid organs where they encounter antigens, and are stimulated to further undergo two Immunoglobulin (Ig) gene alterations known as class switch recombination (CSR) and somatic hypermutation (SHM). CSR is a B cell-specific DNA rearrangement reaction that replaces an Ig heavy chain constant region gene (CH) from C[unreadable] with other downstream CH exons so that secondary isotypes (IgG, IgA etc) with different effector functions are generated. SHM, on the other hand, introduces point mutations into V genes at a very high rate, ultimately leading to increased antibody affinity. Though two distinct processes, CSR and SHM absolutely require transcription through the relevant Ig loci and activity of a single- strand DNA deaminase, Activation Induced cytidine Deaminase (AID). Approach: WE have previously observed that component of the ncRNA-processing pathway, the RNA exosome complex, regulate CSR in ex vivo assays. Here, we use a combination of modern computational biology, proteomics, high-throughput genomics and mouse genetics to identify and functionally characterize ncRNA generated in the Ig locus during affinity maturation. In addition, we will study the function of RNA exosome complex in CSR and SHM in vivo by generating various mouse model systems that harbor loss of function alleles of subunits of the RNA exosome complex. Implication: If successful, this study will be the first to demonstrate the role of ncRNA biogenesis pathway in the genetic and epigenetic control of the Ig locus. Understanding the mechanism of CSR and SHM is of paramount importance. Human patients with mutations in CSR/SHM pathway components suffer with severe immune-deficiencies, whereas aberrant chromosomal alterations in the Ig locus lead to various B and T cel malignancies. Understanding the regulation of the Ig locus via ncRNA will allow generation of directed clinical therapies for treatment of patients suffering various lymphocyte based diseases. Public Health Relevance: In this proposal we identify the mechanism by which non-coding RNA biogenesis pathways regulate antibody affinity maturation via class switch recombination and somatic hypermutation, two processes required for bio-defense via adaptive immune response. Antibodies are polypeptide complexes produced from B-lymphocytes that are present in the bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign antigens, such as bacteria and viruses.

DESCRIPTION (provided by applicant): This proposal is directed towards the understanding of the mechanisms that govern antibody diversification during an adaptive immune response. Antibodies are polypeptide complexes produced from B-lymphocytes that are present in the bodily fluids of vertebrates, and are used by the immune system to identify and neutralize various foreign antigens. Newly generated B cells migrate from bone- marrow to secondary lymphoid organs where they encounter antigens, and are stimulated to further undergo two Immunoglobulin (Ig) gene alterations known as class switch recombination (CSR) and somatic hypermutation (SHM). CSR is a B cell-specific DNA rearrangement reaction that replaces an Ig heavy chain constant region gene (CH) from C¿ with other downstream CH exons so that secondary isotypes (IgG, IgA etc) with different effector functions are generated. SHM, on the other hand, introduces point mutations into V genes at a very high rate, ultimately leading to increased antibody affinity. Though two distinct processes, CSR and SHM absolutely require transcription through the relevant Ig loci and activity of a single-strand DNA deaminase, Activation Induced cytidine Deaminase (AID). AID introduces point mutations in the at specific Ig locus DNA sequences (switch (S) sequences or variable regions (V) genes) that are then converted to DNA lesions (double-strand breaks or mutations) to initiate CSR and SHM. The mechanism by which AID introduces these mutations in the Ig locus in a regulated fashion is an active field of investigation. Our recent studies indicate that AID utilizes the cellular non-codin RNA degradation/processing complex, RNA exosome, to mutate both strands of substrate DNA sequences. Using a combination of modern proteomic approaches, high- throughput genomics and mouse genetics we continue to study the mechanism of function of RNA exosome/AID complex function during CSR and SHM. Understanding the mechanism of AID function is of paramount importance. Human patients with inactivating mutations in the AID gene suffer from Hyper-IgM syndrome (HIGM2), whereas aberrant expression of AID may lead to various B and T cell malignancies. Understanding of AID function in B-lymphocytes will allow treatment of these patients with directed clinical therapies. PUBLIC HEALTH RELEVANCE: This proposal investigates the mechanism of regulation of the proto-oncogene Activation Induced cytidine Deaminase (AID) by the non-coding RNA degradation complex, RNA exosome. AID is essential for initiating the class switch recombination (CSR) and somatic hypermutation (SHM), two processes required for generation of antibodies that participate in bio-defense via adaptive immune response.

PROJECT SUMMARY Background: Class switch recombination and somatic hypermutation are two B lymphocyte specific processes that mediate antibody diversification. Activation Induced Cytidine Deaminase (AID) is essential for initiating both of these processes by deaminating cytidine residues in immunoglobulin (Ig) loci DNA. Despite its specific and indispensible function in the Ig loci, AID has been demonstrated to also deaminate non-Ig locus genes, catalyzing various oncogenic translocations that manifest in tumorogenesis. Recent work has indicated that AID's DNA deamination activity requires its association with transcriptionally stalled RNA polymerase II and the RNA exosome complex. RNA exosome is a cellular non-coding RNA processing and/or degradation macromolecular complex. It is postulated that RNA Exosome's activity is mediated by specific cofactors and sequence characteristics of the target RNA. How RNA exosome's RNA processing activity facilitates AID mediated DNA deamination is a question we seek to address in this application. Objective/Hypothesis: In his proposal, we will determine how RNA exosome activity on transcripts generated in the IgH locus and the rest of the B cell genome generates single-stranded DNA structures following stalling of RNA polII complex and depletion of nucleosomes. Single-strand DNA structures are suitable AID substrates. Specific aims: Aim 1: Are the DIS3 and Exosc10 RNase subunits of RNA exosome complex important for AID activity?; AIM 2: How does RNA exosome substrate antisense RNA (xTSS-RNA and asRNA) promote AID targeting in the B cell genome? AIM 3: To evaluate the role of RNA exosome cofactor Mtr4 (and Senataxin) in the mutagenesis of both strands of DNA in the IgH locus and other regions of the B cell genome. Study Design: Using mouse models that are deficient in RNA exosome RNA degradation activity, we will identify regions in germinal center derived B cell genome that express RNA exosome substrate non-coding RNAs and are also mutated by AID. We will evaluate the mechanism of formation of single-strand DNA structures following localized chromatin remodeling at these identified AID target DNA sequences. We have also generated a mouse model in which the RNA exosome subunit, Exosc3, has been tandem-tagged for high affinity RNA exosome complex purification. Using B cells from these mice, we have purifed RNA exosome complex co-factors and identify them by LC-MS/MS. We will evaluate the role of RNA exosome co-factor(s) in stimulating AID/RNA exosome complex function on both strands of transcribed DNA substrate. Disease Relevance: AID initiates various malignancies in B cells due to its aberrant DNA mutagenesis activity. Proposed studies leads to a better understanding of the mechanisms initiating AID dependent oncogenesis in B lymphocytes (specially in context of DLBCL and Multiple Myeloma), as well as, have direct implications in understanding B lymphocyte based immunodeficiency syndromes like Hyper-IgM syndrome 2.

PROJECT SUMMARY/ ABSTRACT It is becoming increasingly evident that the majority of the mammalian genome has the potential to express non-coding RNAs (ncRNAs). However, the functionality and mechanism(s) of regulation of these ncRNAs are just beginning to be explored. One challenge that biologists encounter is the detection of these ncRNAs, which often tend to be transcriptionally tightly controlled and rapidly degraded. We have recently identified a long noncoding (lnc) RNA expressing locus, known as lncRNA-CSR, that regulates DNA rearrangments in the Immunoglobulin heavy chain (IgH) locus of B cells. Using mouse model systems that lack lncRNA processing/degradation activity along with a combination with high throughput genomics, bioinformatics, and various ChIP-seq based experiments, we are able to predict long-range transcription enhancer function of the lncRNA-CSR locus. In this application, we continue to focus our investigation on the functionality of lncRNA-CSR and three other novel lncRNA expressing loci, that we propose to have a role in orchestrating DNA rearrangment events in germinal center resident B cells. B lymphocytes have the unique ability to undergo programmed somatic mutagenesis of their genomes (at immunoglobulin gene loci) to generate the diversity of antibodies required by our immune system to combat the plethora of antigens we might encounter, a process known as antibody diversification. However, as collateral damage emerging from this very unusual and useful ability to undertake beneficial somatic mutagenesis events is the ability of B cells accidently to mutate their genome at a very low frequency at various inappropriate locations. These accidental mutations are the cause of various B cell malignancies, particularly those that evolve from germinal center derived B cells. Interestingly, cancer-causing translocations in B cells occur at regions of divergent transcription?that is, promoters and enhancers?which exist inside topological domains of superenhancer clusters. We postulate that lncRNA-CSR is responsible for tethering long distant regulatory elements (i.e, promoters and enhancers) in the IgH locus superenhancer cluster to facilitate genome organization, transcription control of regulated genes, and, ultimately, to promote antibody diversification mechanisms without inducing cancer causing DNA alterations.

PROJECT SUMMARY 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.