Transcriptomics of E1A and arglu1 in the cell cycle, polymerase pausing, and DNA damage response
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Abstract
Adenovirus E1A protein transform human cells in by disrupting the binding of the retinoblastoma protein (pRb or Rb) to the transcription factor E2F, driving S phase. Although there have been previous studies on the differences between E1A 13S and 12S, the two largest and earliest expressed isoforms, their global effect on human gene expression is less understood. Previously my lab made a mutant of E1A 13S (YCD) and 12S (12S YCD) that deletes residues 2-11 and mutates Y47 and C124, resulting in a loss of binding to pRb and E2F, deficient S phase activation, and reduced viral growth. However, unlike E1A 12S YCD, the 13S YCD mutant was able to drive S phase in a BrdU assay, while 12S had a reduced amount of S phase initiation compared to WT and 13S. To generate hypothesis for how 13S YCD could do this, I analyzed RNA-sequencing of primary human lung fibroblast cells (IMR-90s) infected with wild type adenovirus and three adenovirus mutants, the first expressing only the E1A 13S isoform, the second expressing predominantly E1A 12S, and the last with three mutations in 13S that prevent it from binding to the retinoblastoma protein. I subsequently performed PCA, DEA, and GSEA.
Arglu1 is a human protein that increases the cellular growth rate, the amount of paused RNA polymerase II, and is localized to sites of DNA damage. I further characterized the phenotype of Arglu1 overexpression by showing that Arglu1 overexpressing cells (HTA) increase resistance to the DNA damage inducing genotoxic drug Actinomycin D and have reduced duration of G1 and S-phase but slightly extended G2/M phase. To analyze how Arglu1 increased the cell cycle rate and resistance to DNA damage, I made deletion mutants of Arglu1, stable cell lines overexpressing these mutants, and performed proteomic and ChIP-seq analysis of HTA cells. I found genes involved in DNA damage response, the cell cycle and its phase transitions, and transcription that generate new hypotheses for how Arglu1 affects these pathways.