Adenoviruses have taught us much about transcriptional regulation and cell cycle control because the cells they commonly infect are end-differentiated non-cycling cells. In order for these cells to be adequate hosts for viral replication, the virus must force the cells into the cell cycle. Adenoviruses express the small e1a protein immediately upon infection, which is responsible for initiating cell replication. Small e1a interacts with both the transcriptional co- activators p300/CBP and the transcriptional repressor RB-family proteins in order to induce epigenetic reprogramming that results in activation of cell cycle genes and inactivation of genes detrimental to adenoviral replication.
This work investigated how the specific interactions between e1a and p300/CBP or e1a and RB-family proteins affect the distribution of the H3K18ac marker throughout the genome and the subsequent changes in expression.
Cell cycle arrested cells were infected with adenovirus e1a mutants that either cannot interact with p300/CBP, or cannot interact with RB-family proteins. The genome wide distribution of the histone marker H3K18ac following infection with wild-type e1a or the e1a mutants, compared to mock infected cells, was determined by chromatin immunoprecipitation with anti-H3K18ac antibody followed by massive parallel sequencing (ChIP-seq). Correlations between H3K18ac and expression levels were established through whole transcriptome sequencing (RNA-seq).
We have found that a simple model can only begin to describe the complex interactions between e1a and it's cellular partners. The e1a interaction with p300/CBP appears to be more important for global hypoacetylation than the interaction with RB-family proteins, but surprisingly, the e1a and RB interaction is required for hyperacetylation and activation of cell cycle promoters.
The second half of this work focuses on the construction and validation of an adenovirus "helper virus" for targeting the Helper Dependent Adenovirus/Epstein Barr virus (HDAd/EBV) hybrid system targeted to Hematopoietic stem cells by a chimeric Ad5/35 fiber. The challenges associated with developing an adenovirus replication permissive cell line (HEK293) with adequate levels of FLPe expression to limit helper virus contamination in the HDAd/EBV vector stock are also discussed.