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Adenovirus small e1a regulates host cell transcription

  • Author(s): Nava, Miguel
  • Advisor(s): Berk, Arnold J
  • et al.
Abstract

ABSTRACT OF THE DISSERTATION

Adenovirus small e1a regulates host cell transcription

by

Miguel Nava

Microbiology, Immunology and Molecular Genetics

University of California, Los Angeles, 2015

Professor Arnold J. Berk, Chair

The study of adenoviruses has contributed immensely to the understanding of biology. Adenoviruses have been used as a model system to explore the mechanism of cellular transformation. To that end, specific viral encoded genes from the E1A and E1B transcriptional units were shown to be necessary for oncogenic and cellular transformation. Subsequent studies identified E1A and E1B regions encoded proteins that were necessary for transformation and also identified the cellular factors that bound to E1A and E1B encoded proteins. A specific E1A isoform, small e1a, exerts its oncogenic potential by co-opting two key cellular factors- P300 and RB.

The advent and wide use of next generation sequencing has facilitated the dissection of transcriptomic questions. Experiments using this technology have confirmed much of the original work about the transcriptional changes that occur following infection with adenoviruses. In addition, new sequencing technology also generates copious amounts of data that can be scrutinized in various manners and lead to new hypotheses. The majority of the work contained in this dissertation employed the use of next generation sequencing.

Prior to the wide use of next generation sequencing, researchers utilized tiling array platforms to determine factor binding to limited regions of the genome (ie promoter regions). Using ChIP-chip studies our collaborators and we described how e1a expression resulted in a redistribution of cellular factors known as pocket proteins (RB, p107 and p130) and lysine acetylases p300/CBP. Specifically, we observed that p130, RB and p300 accumulated near the promoters of genes whose expression was repressed 24 hours p.i. Although e1a was no longer present at the promoters of those repressed genes 24 hours p.i., prior data generated by others had found evidence of an e1a-p300-RB trimolecular complex.

With next generation sequencing becoming the norm in analyzing the modulation of transcription, we applied this technology to address the changes in cellular transcription that occur after infection with adenoviruses that (of the E1A region) only express small e1a (dl1500), an E1A deletion mutant (dl312), an e1a-P300 binding mutant (e1aP300b-) and an RB binding mutant (e1aRBb-). Using these adenoviruses we conducted RNA-seq before and after infection. Concomitantly, we conducted ChIP-seq for pol2, P300, RB, H3K18ac, H3K27ac, H3K9ac and H3K4me1. We determined that e1a-P300 and e1a-RB interactions are important for the activation and repression of certain clusters of cellular genes. We found that e1a repression of some genes depended on both e1a-P300 and e1a-RB interactions. Moreover, P300 and RB accumulated through the length of these genes following infection with an adenovirus that expresses wt e1a. These results are described in the insertion of Ferrari et al. (2014) as chapter 2 of this dissertation.

In Ferrari et al. (2014) we only reported ChIP-seq for pol2 from mock and dl1500 infected cells. We have extended ChIP-seq for pol2 from cells infected with adenoviruses that express wt small e1a, e1aP300b-, e1aRBb- or dl312. Results from these additional pol2 ChIP-seq experiments will be discussed in chapter 3. Furthermore, the use of next generation sequencing in the context of an adenoviral infection has also permitted us to map the binding of pol2, histone modifications (e.g. H3K18ac) and viral transcripts to the viral genome. In chapter 4 of this dissertation we aligned reads from our ChIP-seq data to the viral genome.

Recently, we transitioned to human foreskin fibroblasts (HFFs) for our studies and as a result have had to repeat some of the studies originally conducted in IMR90 cells. During 3H-thymidine incorporation experiments to determine whether e1a wt and e1a mutants drove G0/G1 HFF cells into S-phase, we observed that infection with e1aRBb- resulted in 3H-thymidine incorporation similar to e1a wt. This was surprising because e1aRBb- failed to induce genes involved in cell cycle progression. We conducted additional analyses and studies to determine why e1aRBb- caused 3H-thymidine incorporation without induction of S-phase genes. This is the subject of chapter 5.

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