UCLA

The human adenovirus early region 1A (E1A) has been extensively studied as a model transcriptional regulator to uncover the molecular mechanisms that regulate viral and cellular gene expression. While it was initially thought that early viral gene activation by E1A was primarily due to association of CR3 with Mediator, it was recently demonstrated by Pelka et al. that an interaction between HATs p300/CBP and a region of E1A including CR3 also stimulates transcription (1). To further analyze this mechanism, we assayed the association of YFP labeled p300 with truncated E1A proteins fused to mCherry labeled LacI bound to a LacO array in nuclei. Either of two highly acidic regions flanking CR3 were identified as being required for E1A association with p300 in vivo. Infection of primary human airway epithelial cells with Ad recombinants expressing alanine substitutions in both acidic regions resulted in greatly decreased expression of E2 and E4 RNAs. ChIP-seq for H3K18/27ac and Pol2 pre-initiation complex (PIC) components revealed decreased levels of H3 acetylation at the E2early, E3, and E4 promoters and defective TBP and Pol2 assembly at the E2early promoter after infection with the Ad mutant. GRO-seq in infected cells demonstrated decreased release of promoter-proximal paused Pol2 at E4 by p300 binding mutant E1A. Our results demonstrate that transcription of E2early is regulated during Pol2 PIC assembly mediated by H3 acetylation and E4 is regulated during the transition to productive transcriptional elongation, both of which are dependent on the presence of these acidic regions of E1A.