The generation of high-affinity antibodies of different isotypes forms the molecular basis of all long-term protective immunity against pathogens and vaccines. High affinity and isotype switching is acquired by the mutagenic antibody maturation pathways that are activated in B cells upon antigen encounter. At the molecular level, antibody maturation involves transcription-dependent mutagenesis of the antibody-encoding immunoglobulin genes. Mutagenesis is governed by dynamic changes in long-range chromatin interactions, extending up to 300 kb, between immunoglobulin genes and various regulatory elements. In our group, we study the molecular mechanisms of antibody maturation using a combination of genetics, molecular biology, biochemistry and genomics.
The specific aim of this project is to understand how the interplay of chromatin architecture and transcription regulates mutagenesis and isotype switching in human B cells. The project involves genetic manipulation of B cell lines, including gene knockouts and knock-ins, to alter chromatin architecture and transcriptional patterns at immunoglobulin genes, and determine how these changes affect mutagenesis and isotype switching.
Techniques and skills
(1) CRISPR-based editing, flow cytometry and quantitative PCR, to generate cell lines. (2) Tri-C, a chromatin conformation capture technique which measures multiway interactions between immunoglobulin genes, enhancers and architectural elements. (3) Precision run-on sequencing (PRO-seq), which measures the levels of nascent transcription genome-wide. (4) Chromatin immunoprecipitation with sequencing (ChIP-seq) to determine the occupancy transcription cofactors and architectural proteins. (5) Mutational analyses using deep sequencing methods. All these methods are established in the lab. Additionally, the student will learn to generate hypotheses, design experiments, troubleshoot, analyse data, and develop presentation skills.
Objectives for each year:
Year 1: (1) Generation and characterization of cell lines. (2) Begin PRO-seq and Tri-C.
Year 2: Complete PRO-seq, Tri-C and ChIP-seq. Analyse data in collaboration with bioinformaticians.
Year 3: Finalize the study, write the manuscript and submit for publication.
Molecular mechanisms of antibody maturation in human B cells.
Costea, J., Schoeberl, U. E., Malzl, D., Von der Linde, M., Fitz, J., Makharova, M., Goloborodko, A., Pavri, R. A de novo transcription-dependent TAD boundary underpins critical multiway interactions during antibody class switch recombination. Molecular Cell (2023). DOI: 10.1016/j.molcel.2023.01.014 Peycheva, M., Neumann, T., Malzl, D., Nazarova, M., Schoeberl, U. and Pavri, R. DNA replication timing directly regulates the frequency of oncogenic chromosomal translocations. Science 377, 6612 (2022). DOI: 10.1126/science.abj5502 Fitz, J., Neumann, T., Steininger, M., Wiedemann, E-M., Cantoran Garcia, A., Athanasiadis, A., Schoeberl, U. and Pavri, R. Spt5-mediated enhancer transcription directly couples enhancer activation with physical promoter interaction. Nature Genetics 52, 505–515 (2020). DOI: 10.1038/s41588-020-0605-6
Sharif H, Acharya S, Dhondalay GKR, Varricchi, Shoshanna Krasner-Macleod SK, Laisuan W, Switzer A, Lenormand M, Kashe E, Parkin RV, Yi Y, Koc M, Fedina O, Vilà-Nadal G, Marone G, Eifan A, Scadding GW, Fear DJ, Nadeau KC, Durham SR, Shamji MH. Altered chromatin landscape in circulating T follicular helper and regulatory cells following grass pollen subcutaneous and sublingual immunotherapy. (2021). J Allergy Clin Immunol, 147(2):663-676 (2021). DOI: 10.1016/j.jaci.2020.10.035 Ramadani F, Bowen H, Gould HJ, Fear DJ. Transcriptional Analysis of the Human IgE-Expressing Plasma Cell Differentiation Pathway. Front Immunol, 10, p.402 (2019). DOI: 10.3389/fimmu.2019.00402 Recaldin T, Hobson PS, Mann, EH, Ramadani F, Cousins DJ, Lavender P. and Fear DJ. miR-29b directly targets activation-induced cytidine deaminase in human B cells and can limit its inappropriate expression in naïve B cells. Mol Immunol. 101:419-428 (2018). DOI: 10.1016/j.molimm.2018.07.028