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. 2021 Mar 11;10:e63512. doi: 10.7554/eLife.63512

Figure 1. Promoter H3K18/27 acetylation activated by E1A activation domain (E1A-AD)-CBP/p300 interaction stimulates paused polymerase II (Pol2) release at adenovirus promoter E4.

(a, bottom) Map of the major human adenovirus 5 early E2, E3, and E4 mRNAs. Deletions in the E3 regions of dl312 and the E1A-DM vector are shown by cross-hatched horizontal bars. Shared E3 sequence used as the E3 gene body is indicated by the gray bar above E3. Vertical stripes highlighted in yellow indicate promoter-proximal regions. Global Run-On sequencing (GRO-seq) counts from primary human bronchial-tracheal epithelial cells infected with wt+dl312 or DM vectors at 12 hr post-infection were plotted on the Ad5 genome with H3K18ac, H3K27ac, and H3K9ac ChIP-seq data (Hsu et al., 2018). GRO-seq tracks are shown for the two viral DNA strands (+, transcribed to the right; and –, transcribed to the left), with two different y-axis scales to allow visualization of high- and low-amplitude peaks. The double-headed arrows in the GRO-seq plots in the E4 region refer to gene body regions discussed in the text. (b) Average fold change in pause index for E2early, E3, and E4 in cells expressing DM-E1A compared to wt E1A. Pause index is the ratio of reads in the promoter region (transcription start site [TSS] to +200) to reads in the gene body (+200 to TTS). Error bars represent standard deviation of three biological replicates. Paired t-test comparing wt E1A and DM-E1A for E2, E3, and E4. * indicates a significant difference (p-value<0.05) between cells expressing wt E1A and DM-E1A. ‘ns’ indicates no significant difference.

Figure 1—source data 1. qRT-PCR for E2early, E3, and E4 pre-mRNA transcripts in cells treated with DMSO or A-485.

Figure 1.

Figure 1—figure supplement 1. Small and large E1A protein interaction and E1A mutants.

Figure 1—figure supplement 1.

(a) E1A interaction maps for large E1A (289 aa) and small e1a (243 aa). Colored portions and horizontal black bars above them represent regions of E1A that interact with the indicated cellular protein. Dashed lines connect conserved regions of E1A that bind the indicated targets together. Conserved regions (CR) 1–4 are indicated in brackets. (b) Sequences of Ad5 mutants from E1A (aa 121–223), with alanine substitutions in red. (c) Western blot showing wt and DM large E1A protein levels at 12 hr post-infection (p.i). Human bronchial-tracheal epithelial cells were coinfected with the wt E1A-expressing virus at an MOI (multiplicity of infection) of 5 and dl312 at an MOI (multiplicity of infection) of 95 or infected with the DM virus alone at an MOI (multiplicity of infection) of 100. Ku-86 protein levels were used as loading controls.