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Investigating transcriptional regulation of viral and cellular genes by EBV EBNA 2

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posted on 2023-06-08, 11:03 authored by Richard Palermo
Epstein-Barr virus (EBV) is linked to the development of several human malignancies. Epstein-Barr Nuclear Antigen 2 (EBNA 2) is required for the immortalisation and continued proliferation of EBV-infected B-cells. EBNA 2 is a transcriptional regulator of both viral and cellular genes. The viral C promoter (Cp), regulated by EBNA 2, drives transcription of an ~120 kb pre-mRNA that is differentially spliced to generate messages encoding all the other EBNAs required for immortalisation. To study the regulation of Cp-transcript elongation, we used a pair of EBV-infected cell-lines to compare the transcriptional complexes associated with the Cp transcriptional unit and two shorter EBNA 2-regulated viral genes, LMP1 and LMP2A. Interestingly, we found an accumulation of RNA Polymerase II (Pol II) in association with the pausing factors DSIF and NELF at Cp, which were absent at the LMP gene locus. Further experiments revealed that C promoter sequences have a much higher propensity to occlude nucleosome formation, promoting TBP recruitment and Pol II accumulation. We also found highlevel recruitment of the Pol II C-terminal domain (CTD) kinase, pTEFb at Cp, increased Pol II Serine 2 CTD phosphorylation and retention at promoter-distal regions. Furthermore pTEFb recruitment at Cp was facilitated by association with the bromodomain protein Brd4 and Pol II pausing. By sustaining a nucleosome-free region and recruiting high levels of pTEFb, Cp elongation appears highly adapted to ensure production of the long EBNA-encoding transcript required to establish and maintain B-cell immortalisation. In additional studies we examined the association of methylated forms of EBNA 2 with viral and cellular genes. EBNA 2 is modified by asymmetric (aDMA) or symmetric (sDMA) arginine di-methylation in the arginine-glycine repeat region. We found that aDMA-modified EBNA 2 preferentially bound promoters to regulate gene expression, implicating this modification as a key regulator of EBNA 2 activity.


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