The cell cycle is a tightly regulated process and proper control of the cell cycle progression is essential for maintaining genomic integrity and preventing uncontrolled cellular proliferation. At the heart of this regulation is the tumor suppressor protein p53, which protects tissues from developing tumors by inducing cell cycle arrest and enabling DNA repair or apoptosis. A central way for p53 to cause cell cycle arrest is to transcriptionally activate the CDK inhibitor p21WAF1/Cip1. Elevated p21 prevents phosphorylation and subsequent inactivation of the pocket proteins RB, p107, and p130. In their hypophosphorylated state, the pocket proteins form transcriptional repressor complexes by binding to E2F proteins. E-cyclins are crucial cyclins for cell cycle progression and exhibit their highest expression during G1/S phases. Oncogenic cyclin E/CDK2 complex activation is a common feature in human malignancies and leads to DNA replication stress and genome instability. Since the discovery of E-cyclins, the focus of literature was more directed towards the transcriptional regulation of CCNE1, and the transcriptional regulation of CCNE2 remains elusive. This thesis investigates the transcriptional regulation of CCNE2, with emphasis on identifying important DNA elements and transcription factors involved in its cell cycle- and p53-dependent transcriptional regulation. By utilizing luciferase reporter assays, DNA affinity purifications, mRNA, and protein analysis, two E2F elements within the CCNE2 promoter were identified as important transcriptional regulators of CCNE2. These E2F elements bind RB and p130 and play a synergistic role in mediating p53- and cell cycle-dependent transcriptional regulation of CCNE2. In addition, a NRF1 binding site within the CCNE2 promoter was identified as a specific activator of CCNE2’s transcription that binds the NRF1 protein. Notably, the NRF1 protein is shown to be cell cycle-regulated, but not affected by p53 induction. Furthermore, I show that the transcriptional downregulation of CCNE2 is predominantly mediated by RB, following the p53-p21-RB/E2F signaling pathway and requiring the two E2F sites. This thesis uncovers the molecular mechanisms governing CCNE2 transcriptional regulation. The identification of two E2F elements and a NRF1 binding sites as important regulatory elements in the CCNE2 promoter improves our understanding of the p53- and cell cycle-dependent transcriptional regulation of cell cycle genes. Furthermore, this work provided a mechanistic explanation of how p53, through the pathway of p53-p21-RB/E2F, and via the two E2F elements identified, represses the transcription of oncogenes, such as CCNE2, to halt the cell cycle and prevent tumor formation.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:92610 |
| Date | 15 July 2024 |
| Creators | Azzahrani, Khaled |
| Contributors | Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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