Abstract
How histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing colocalization at an integrated lacO array of fusions of LacI-mCherry to E1A fragments with YFP-p300. Viruses with mutations in E1A subdomains were constructed and analyzed for kinetics of early viral RNA expression and association of acetylated H3K9, K18, K27, TBP, and RNA polymerase II (Pol II) across the viral genome. The results indicate that this E1A interaction with p300 is required for H3K18 and H3K27 acetylation at the E2early, E3, and E4 promoters and is required for TBP and Pol II association with the E2early promoter. In contrast, H3K18/27 acetylation was not required for TBP and Pol II association with the E3 and E4 promoters but was required for E4 transcription at a step subsequent to Pol II preinitiation complex assembly.IMPORTANCE Despite a wealth of data associating promoter and enhancer region histone N-terminal tail lysine acetylation with transcriptional activity, there are relatively few examples of studies that establish causation between these histone posttranslational modifications and transcription. While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300, as reported previously (P. Pelka, J. N. G. Ablack, J. Torchia, A. S. Turnell, R. J. A. Grand, J. S. Mymryk, Nucleic Acids Res 37:1095-1106, 2009, https://doi.org/10.1093/nar/gkn1057), and that the resulting acetylation of H3K18/27 affects varied steps in transcription at different viral promoters.
Highlights
How histone acetylation promotes transcription is not clearly understood
Pelka et al reported that p300 interacts with large early region 1A (E1A)(aa 139 –204), a region lacking the well-characterized large and small E1A p300/CBP binding site formed from the E1A N-terminal ϳ12 aa plus E1A(aa 54 – 82) in CR1 [7, 25]
And of considerable interest, TBP and polymerase II (Pol II) association with the E4 promoter were only minimally decreased by the double mutant (DM) mutations (Fig. 8), even though E4 RNA expression by the DM was nearly eliminated (Fig. 4c). These results suggest that the DM mutations block E4 transcription at a step subsequent to preinitiation complex (PIC) assembly. Together these results demonstrate that the E2early promoter is regulated in part by p300/CBP-mediated acetylation of H3K18/27, which are required for a high level of Pol II PIC assembly, while H3K9ac appears to be sufficient for PIC assembly and moderate transcription at the E3 promoter
Summary
How histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing colocalization at an integrated lacO array of fusions of LacI-mCherry to E1A fragments with YFP-p300. The 13S and 12S transcripts are generated from two 5= splice sites, separated by 138 bp and a common 3= splice site, and are most abundant early after infection and in transformed cells They encode the 289-amino-acid (aa) large E1A and the 243-aa small e1a proteins, respectively [2]. The association of p300/CBP with small e1a inhibits H3K18/27 acetylation to about 30% the level in uninfected cells [5] This is thought to be the primary mechanism by which adenovirus represses host cell genes that would otherwise interfere with productive viral infection [5, 8]. Mutant E1As with amino acid substitutions in the C4 Zn-finger region (Fig. 1) that are defective for activation [18] did jvi.asm.org 2
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