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Rushad Pavri

In our lab, we study the phenomenon of antibody gene diversification in B lymphocytes – the process by which the immune system generates a vast repertoire of high-affinity antibodies essential to combat pathogenic challenges. We focus on the transcriptional and co-transcriptional mechanisms underlying these events using a combination of RNAi screening, genetic models, and genomic analysis.

Molecular mechanisms of antibody diversification

Research activities

Figure 1: Antibody diversification at the Ig heavy chain locus. B cell activation triggers AID expression, which generates mutations (arrowheads) in specific regions. These lesions are processed through various DNA repair pathways, resulting in SHM and CSR. Mutations at non-Ig genes result in reciprocal translocations between these genes and the Ig locus, leading to genome instability and tumorigenesis.

Antibody diversification occurs at the immunoglobulin (Ig) loci and consists of two processes: somatic hypermutation (SHM) which modulates the antigen-binding properties of the antibody, and class switch recombination (CSR) which generates various antibody isotypes (Figure 1). Antibody maturation is triggered by activation-induced deaminase (AID), which generates mismatches in the Ig DNA sequence by converting cytosines into uracils on single-stranded DNA. AID can also target non-Ig genes, such as the oncogenes, c-myc and Bcl6, albeit to a much lesser extent (Figure 1). These lesions are then processed by various means, involving error-prone repair, double strand breaks (DSBs), and non-homologous end-joining (NHEJ), resulting in SHM and CSR (Figure 1). AID-induced DSBs at non-Ig genes like c-myc may also lead to c-myc/IgH translocations and lymphoid malignancies (Figure 1). In our lab, we are interested in understanding the role of transcription and co-transcriptional factors involved in AID targeting and mutagenesis, and we determine how AID targeting is restricted to specific loci in B cells.

Transcriptional regulation of AID targeting

Figure 2: Model for AID targeting via transcriptional stalling (see text for details). Spt5 recruits AID to the Pol II complex. Stalling at promoter-proximal regions results in AID-mediated mutations in these regions (variable genes and non-Ig genes). Stalling within switch-repetitive regions results in mutations within the regions associated with CSR.

We have shown that the RNA polymerase II (Pol II) stalling and elongation factor, Spt5, is required for CSR and AID targeting. Based on our findings we proposed a model for AID targeting via Spt5 and Pol II stalling (Figure 2), wherein AID is preferentially targeted to regions of Pol II stalling through direct interaction with Spt5. Stalling provides AID with long residence times at, and access to, its ssDNA substrate within the transcription bubble. In this manner, mutations are focused on specific genomic regions which coincide with the known mutation profiles of AID as well as hotspots of AID-dependent translocations (Figure 2).

We are currently developing tools and genetic models to further study the impact of Pol II stalling and Spt5 function in diversifying B cells. Our preliminary findings suggest entirely new modes of gene regulation by Spt5 during B cell activation. We are currently investigating how this influences AID regulation, Ig gene expression, and AID targeting during CSR and SHM.

Cell cycle regulation of antibody diversification

Antibody diversification, both in vivo and in vitro, occurs in highly proliferating B cells. In vitro studies clearly show a correlation between the magnitude of CSR and cell division. The reasons for this are unclear. Cell cycle regulation and/or DNA replication have not been implicated in CSR and SHM so far. Using RNAi screening in B cells, we disclosed the role of cell cycle and DNA replication factors in CSR. Preliminary findings show that these factors associate with AID and Spt5, both in vitro and in vivo, and are localized to AID target genes on a genome-wide basis. We are currently investigating the mechanisms by which these factors regulate CSR, and how CSR is coordinated during the cell cycle in proliferating and hypermutating B cells.

Identification and characterization of novel factors in CSR and SHM

To gain insights into the mechanism of CSR, we performed an RNAi screen for the purpose of identifying new factors involved in this process. We are currently investigating interesting candidates determined from this screen, using mouse genetics, biochemical analyses, and genomics. In addition, we are developing new assays to quantitatively test for SHM in B cells, which will allow us to perform RNAi studies to address the precise role of various putative factors involved in SHM and identify new players in this process.

Our long-term goal is to determine how AID targeting is tightly restricted to specific regions within the transcribed Ig locus, especially during SHM. This enigmatic aspect of the phenomenon eludes a clear explanation, but is critical for the immune response.

Selected Publications

  • Wiedemann, EM., Peycheva, M., Pavri, R. (2016). DNA Replication Origins in Immunoglobulin Switch Regions Regulate Class Switch Recombination in an R-Loop-Dependent Manner. Cell Rep. 17(11):2927-2942
  • Pavri, R., Nussenzweig, MC. (2011). AID targeting in antibody diversity. Adv Immunol. 110:1-26
  • Pavri, R., Gazumyan, A., Jankovic, M., Di Virgilio, M., Klein, I., Ansarah-Sobrinho, C., Resch, W., Yamane, A., Reina San-Martin, B., Barreto, V., Nieland, TJ., Root, DE., Casellas, R., Nussenzweig, MC. (2010). Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5. Cell. 143(1):122-33
  • Pavri, R., Zhu, B., Li, G., Trojer, P., Mandal, S., Shilatifard, A., Reinberg, D. (2006). Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II. Cell. 125(4):703-17
  • Pavri, R., Lewis, B., Kim, TK., Dilworth, FJ., Erdjument-Bromage, H., Tempst, P., de Murcia, G., Evans, R., Chambon, P., Reinberg, D. (2005). PARP-1 determines specificity in a retinoid signaling pathway via direct modulation of mediator. Mol Cell. 18(1):83-96