Epstein-Barr Virus (EBV) causes lymphomas and lymphoproliferative diseases in HIV infected and immune suppressed people. These tumor cells frequently express Latency III EBV Nuclear Antigens (EBNAs) and Latent Membrane Proteins (LMP). In vitro, EBV converts Resting B Lymphocytes (RBLs) to continuously proliferating Lymphoblasts Cell Lines (LCLs) by expressing the same viral proteins. EBV conversion of RBLs to LCLs is therefore a relevant model that can be genetically manipulated to investigate EBV's role in growth transformation. LCL growth depends on EBV transcription factors (TFs) EBNA2, EBNALP, EBNA3A, EBNA3C and LMP1 activated NF-?B. Recently, we found that all the essential EBNAs and LMP1 activated NF-kB subunits converge to a small number of enhancers sites. Of these enhancers, 187 had markedly higher and broader histone H3K27ac signals, characteristic of super-enhancers, and were designated EBV super-enhancers (ESE). Super-enhancers (SE) govern cell transcription, development, phenotype, and oncogenesis. We found ESE- associated genes included the MYC and BCL2 oncogenes. ESEs were enriched for B cell TF motifs and had high STAT5A and NFAT co-occupancy. ESE associated genes were more highly expressed than other LCL genes. Disrupting ESEs by the bromodomain inhibitor JQ1 or conditionally inactivating an EBV oncoprotein or NF-kB decreased MYC or BCL2 expression and arrested LCL growth. To further characterize the ESEs, we will examine their (1) DNA elements, (2) protein compositions, and (3) associated enhancer RNAs (eRNAs). We will use reporter assays and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to identify the DNA elements essential for ESE activity. We will use BioTAP-XL crosslinking, affinity purification followed by mass spectrometry to characterize the ESE proteomic components. We will also use Global Run On followed by deep sequencing (GRO-seq) to identify enhancer RNAs affected by EBV super-enhancers and useshort hairpin RNAs (shRNAs), short interferring RNAs (siRNAs) or Locked Nucleic Acid anti-sense oligonucleotides (LNAs) knock down to determine their significance. The experiments here in use integrative approaches to elucidate the molecular mechanism by which ESEs activate key oncogenic divers. These studies will identify opportunities for therapeutic intervention.
Epstein-Barr Virus (EBV) associated malignancies remain to be a significant threat to human health. These studies will elucidate the molecular basis through which EBV super-enhancers activate oncogenic drivers to immortalize normal B lymphocytes into lymphoblasts and identify potential targets for intervention.
|Liang, Jun; Zhou, Hufeng; Gerdt, Catherine et al. (2016) Epstein-Barr virus super-enhancer eRNAs are essential for MYC oncogene expression and lymphoblast proliferation. Proc Natl Acad Sci U S A 113:14121-14126|