Date of Award

January 2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Barry Milavetz

Abstract

Modifications of histone proteins play an important role in transcription. These modifications regulate how chromatin is compacted by the histone proteins and either made available for transcription or prevents transcription machinery from proceeding. The modifications can be added or removed by histone acetyltransferases and histone methyltransferases for the former, and histone deacetylates and demethylases for the latter. To explore the dynamicity of these modifications we used the simple but effective Simian Virus 40 (SV40) model. SV40 continues to be an important tool in understanding eukaryotic transcription. It experiences similar processes as cellular chromatin, utilizing host proteins to propagate its own growth. This hijacking includes using host histone modifying enzymes to modify its own histone tails. We have shown existing modifications during wild – type infection, but in this study, we use inhibitors for these modifying enzymes to observe the effects of changing the modifications compared to normal conditions. Utilizing ChIP to measure modified histones, we are able to demonstrate the decrease or increase of histone modifications. These measurements are then used along with ChIP-seq data to visualize where the modifications are on the genome. Furthermore, we evaluated total RNA produced to evaluate the effects of the inhibitors on overall RNA levels of small T-antigen and large T-antigen. Finally, we utilize RNA-seq to seek an understanding to the overall effects the inhibitors are having on SV40 genes. Surprisingly, we observed that DMSO as a solvent should be avoided when studying epigenetics as it had dramatic effects on not only the cell after 30 minutes, but the virus' chromatin structure and early transcription. This made it difficult for us to make any conclusions from treatment with DMSO. We did try alternative solvents which gave us minimal differences, however; we did observe and can conclude that impacting one histone modifying enzyme has an impact on other histone modifications and early transcription. We see a reduction in the amount of T - antigen spliced during early transcription compared to the untreated samples and the alternative solvent treated samples, and we observed a difference in histone modification location compared to the untreated samples. We conclude that DMSO is having an effect on SV40 chromatin structure that is likely causing the change in early transcription. The drugs themselves also appear to have an effect when dissolved in the alternative solvent on chromatin structure, however; the impact on early transcription is minimal.

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