The impact of transcriptional mutagenesis on cellular homeostasis

Abstract

DNA is exposed to chemical and physical agents that represent a continuous threat to its integrity by formation of DNA lesions. Despite the existence and efficiency of DNA repair systems in cells, some lesions may not be removed, and interfere not only with the fidelity of DNA replication but also with DNA transcription. In fact, RNA polymerase can bypass non-bulky lesions, such as 8-oxoguanine (8-oxoG) and O6-methylguanine (O6-meG) and, due to base misincorporation, lead to the production of mutated RNA through a process referred to as transcriptional mutagenesis (TM). Although the concept of TM is well known, the biological consequences are still a recent discovery. While the few existing studies of TM have begun to shed light on the process, the role these seemingly transient errors might play in disease processes, especially in tumorigenesis, is currently unknown.

The overall aim of this thesis was to explore to which extent lesion-induced TM influences protein function and how this may affect cellular homeostasis in vivo. Using plasmids containing single site-specific DNA lesions placed within probe genes with established links between specific mutations and subsequent phenotypes, we confirmed that 8-oxoG and O6-meG induced TM in mammalian cells. Further, we explored the effects of TM on splicing fidelity in vivo (Paper I) and found that TM in regulatory sequences of splicing signals resulted in activation of alternative splicing sites, thus leading to the production of disease associated splice forms and/or disrupting physiological ratios between alternatively spliced isoforms. In addition, we examined effects of TM on p53 and its tumor suppressor function in human cells (Paper II). We found that expression of mutant R248W p53 due to TM was sufficient to reduce p53’s transactivation capacity of several target genes, which are required for its tumor suppressive function. Moreover, we showed that TM of p53 reduced its tumor suppressor function by impairing both proper cell cycle control and induction of apoptosis resulting in stimulated proliferation and survival. A genome-wide gene expression analysis further revealed that TM of p53 at codon 248 deregulated both the transactivation and downregulation of numerous target genes, which are crucial for its tumor suppressor function (Paper III). These deregulated genes were involved in regulation of several cellular processes, such as cell-cycle arrest, apoptosis, and DNA damage response.

To conclude, the work presented here shed light on biological effects of TM in vivo and provides evidence for possible mechanisms by which TM might contribute to human disease development. We showed for the first time that lesion-induced TM could activate alternative splicing sites in vivo, thus reducing splicing fidelity and resulting in aberrant splicing. In addition, the work presented in this thesis, together with the results from other studies, strongly suggest that TM could be a contributing mechanism in the multistep process of tumorigenesis by inactivating a tumor suppressor or activating an oncogene thus stimulating proliferation and survival of an already initiated pre-neoplastic cell.