PhD: University of Illinois, Urbana-Champaign
Development of adaptive immunity, V(D)J recombination.
Development of adaptive immunity, DNA repair and recombination
The power of adaptive immunity stems from the diverse antigen receptor (AgR) gene sequences expressed by the ensemble of B and T cells in the human immune system. The combined repertoires of AgRs contain a vast array of binding specificities resulting in the ability to specifically bind and target any infectious pathogen that invades the body. From a limited amount of genetic material, V(D)J recombination initiates the production of the AgR repertoires during B and T cell development through differential rearrangement of AgR gene segments. V(D)J recombination is initiated by the recombination activating proteins, RAG1 and RAG2, which catalyze site-specific DNA cleavage reactions at the border of selected AgR gene segments. All of the gene segments are flanked by semi-conserved DNA sequences, referred to as recombination signal sequences (RSSs), which target the RAG proteins to appropriate DNA cleavage sites. Programmed generation of DNA double-strand breaks is a risky undertaking, since RAG-mediated off-target events or incorrect DNA repair processes can produce chromosomal aberrations and genomic instability that can result in certain types of leukemias and lymphomas. Nonetheless, successful generation of functional AgR genes through V(D)J recombination is an essential process in the generation of mature B and T cells. Thus, defects in the RAG genes that result in the absence or complete RAG enzymatic inactivity leads to severe combined immunodeficiency disease that is fatal unless successfully treated with hematopoietic stem cell transplantation. Genetic defects producing partially active RAG enzymes can also result in a range of immunodeficiency diseases, and in some cases, autoimmunity. Thus, regulation of the RAG protein complex is essential to guide proper targeting and enzymatic activity in the production of diverse AgR repertoires, while simultaneously preventing excessive DNA cleavage activity at potential off-target RSS-like sites.
In this laboratory, we are seeking to determine the varied mechanisms that regulate V(D)J recombination. As the RSSs are semi-conserved, it is essential that the RAG proteins sufficiently recognize, bind, and cleave at a wide range of variant RSSs such that the majority of AgR gene segments are utilized in the AgR repertoires. However, it is equally imperative that RAG-mediated cleavage at off-target RSS-like sites is prevented. To assess the basis of RAG-RSS interactions, we developed a high-throughput cellular recombination assay to analyze the RSS sequence determinants that promote RAG activity at a level not previously achieved. The greater predictive power of DNA substrate specificity in V(D)J recombination obtained from this method will be useful in determining the extent that RSS allelic variants can lead to altered AgR repertoires that in turn impact adaptive immunity.
In a separate project, we are investigating how the RAG complex is regulated during the DNA damage response (DDR). Continued RAG-mediated cleavage events may be detrimental to DNA repair processes needed for repair of DNA damage produced by exogenous sources or by RAG activity itself. We previously determined that upon exogenous DNA damage, RAG2 is rapidly exported from the nucleus of pre-B cells until DNA repair had been completed, and that pre-B cells expressing export-defective RAG2 showed decreased recovery. Mechanisms that regulate RAG localization and dynamics, as well as identification of RAG interacting partners and DDR-induced post-translational modifications, are a continued focus in the laboratory.
Current Lab Personnel:
- Jennifer Byrum, Ph.D., Research Associate
- William Rodgers, Ph.D., Associate Professor of Research
- Justin Harris, B.S., Graduate Student in Biochemistry and Molecular Biology
- Destiny Simpson, B.S., Graduate Student in Biochemistry and Molecular Biology
- Walker Hoolehan, B.S., Graduate Student in Biochemistry and Molecular Biology
- Hoolehan, W, Harris, JC, Byrum, JN, Simpson, DA, and Rodgers, KK (2022) An updated definition of V(D)J Recombination Signal Sequences revealed by high-throughput recombination assays. Nucleic Acids Research. 50(20):11696-11711; PMCID: PMC9723617
- Byrum JN, Hoolehan WE, Simpson DA, Rodgers W, Rodgers KK (2021) Full length RAG2 expression enhances the DNA damage response in pre-B cells. Immunobiology. 226(3):152089. PMCID: PMC8169632.
- Rodgers W, Byrum JN, Simpson DA, Hoolehan W, Rodgers KK (2019) RAG2 localization and dynamics in the pre-B cell nucleus. PLoS One. 14(5):e0216137. PMCID: PMC6510410.
- Rodgers, KK (2017) Riches in RAGs: Revealing the V(D)J recombinase through high resolution structural studies. Trends Biochem. Sci. 42, 72-84. PMCID: PMC5182142
- Rodgers W, Byrum JN, Sapkota H, Rahman NS, Cail RC, Zhao S, Schatz DG, Rodgers KK (2015) Spatio-temporal regulation of RAG2 following genotoxic stress. DNA Repair. 27:19-27. PMCID: PMC4336829.
- Byrum JN, Zhao S, Rahman NS, Gwyn LM, Rodgers W, Rodgers KK (2015) An interdomain boundary in RAG1 facilitates cooperative binding to RAG2 in formation of the V(D)J recombinase complex. Protein Sci. 24(5):861-73. PMCID: PMC4420534.
Complete List of Published Work in MyBibliography: