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The role of the EBNA2-EBF1 complex in EBV driven B cell transformation
The role of the EBNA2-EBF1 complex in EBV driven B cell transformation
The Epstein Barr virus (EBV) infection of B cells is associated with various malignancies, e.g. Burkitt’s lymphoma, Hodgkin’s lymphoma, lymphoproliferative diseases. Upon infection, EBV induces a complex viral and cellular gene expression program causing the transformation of naïve resting B cells to lymphoblastoid cell lines (LCLs) in vitro. These are continuously proliferating cultures and used to study molecular mechanisms of the transformation process. EBV nuclear antigen (EBNA) 2 is one of the first genes expressed after EBV infection and a key regulator of the transformation process. It preferentially binds to B cell specific enhancers and promotors via cellular adaptor proteins. CBF1 is the main DNA anchor but the knowledge about the contribution of B cell specific transcription factors (TFs) to EBNA2’s activity is limited. The early B cell factor (EBF) 1, a key TF during B cell development, has been identified as an EBNA2 co-factor. By identifying the α1-helix in the EBNA2 N-terminal dimerization (END) domain as a crucial region for the interaction with EBF1, I was able to generate mutant EBV lacking this region (EBVΔα1) in order to study the role of the EBNA2-EBF1 complexes during the transformation of infected B cells. Analysis of cellular processes and RNA expression revealed that EBVΔα1 was impaired in reprogramming cellular gene expression leading to a cell cycle arrest at the early S phase and impaired metabolism. Nevertheless, it was possible to establish long-term LCLΔα1 cultures on CD40 ligand expressing feeder cells. The analysis of primary EBVΔα1 infected B cells and LCLΔα1 revealed reduced expression of MYC and LMP1, which are important EBNA2 target genes and required for optimal cell proliferation and survival. Moreover, reverse genetics identified 3 classes of chromatin binding sites 1) EBF1 independent EBNA2 binding, 2) EBF1 dependent EBNA2 binding, 3) sites at which EBF1 and EBNA2 required the complex formation in order to bind. These data indicated that a functional EBNA2-EBF1 complex is required for optimal B cell transformation upon EBV infection. Subsequently, two CRISPR/Cas9 based genome editing approaches were explored to establish conditional EBF1 LCLs to further delineate the contribution of EBF1 to the EBNA2 induced transformation process of EBV infected B cells.
Epstein-Barr virus, EBNA2, EBF1, protein complex, RNA sequencing
Beer, Sophie
2021
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Beer, Sophie (2021): The role of the EBNA2-EBF1 complex in EBV driven B cell transformation. Dissertation, LMU München: Faculty of Biology
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Abstract

The Epstein Barr virus (EBV) infection of B cells is associated with various malignancies, e.g. Burkitt’s lymphoma, Hodgkin’s lymphoma, lymphoproliferative diseases. Upon infection, EBV induces a complex viral and cellular gene expression program causing the transformation of naïve resting B cells to lymphoblastoid cell lines (LCLs) in vitro. These are continuously proliferating cultures and used to study molecular mechanisms of the transformation process. EBV nuclear antigen (EBNA) 2 is one of the first genes expressed after EBV infection and a key regulator of the transformation process. It preferentially binds to B cell specific enhancers and promotors via cellular adaptor proteins. CBF1 is the main DNA anchor but the knowledge about the contribution of B cell specific transcription factors (TFs) to EBNA2’s activity is limited. The early B cell factor (EBF) 1, a key TF during B cell development, has been identified as an EBNA2 co-factor. By identifying the α1-helix in the EBNA2 N-terminal dimerization (END) domain as a crucial region for the interaction with EBF1, I was able to generate mutant EBV lacking this region (EBVΔα1) in order to study the role of the EBNA2-EBF1 complexes during the transformation of infected B cells. Analysis of cellular processes and RNA expression revealed that EBVΔα1 was impaired in reprogramming cellular gene expression leading to a cell cycle arrest at the early S phase and impaired metabolism. Nevertheless, it was possible to establish long-term LCLΔα1 cultures on CD40 ligand expressing feeder cells. The analysis of primary EBVΔα1 infected B cells and LCLΔα1 revealed reduced expression of MYC and LMP1, which are important EBNA2 target genes and required for optimal cell proliferation and survival. Moreover, reverse genetics identified 3 classes of chromatin binding sites 1) EBF1 independent EBNA2 binding, 2) EBF1 dependent EBNA2 binding, 3) sites at which EBF1 and EBNA2 required the complex formation in order to bind. These data indicated that a functional EBNA2-EBF1 complex is required for optimal B cell transformation upon EBV infection. Subsequently, two CRISPR/Cas9 based genome editing approaches were explored to establish conditional EBF1 LCLs to further delineate the contribution of EBF1 to the EBNA2 induced transformation process of EBV infected B cells.