The regulation of E2F

Burden, Morwenna J. (2000) The regulation of E2F. PhD thesis, University of Glasgow.

Abstract

The cellular transcription factor E2F plays a critical role in co-ordinating the transcription of target genes necessary for cell cycle progression. E2F interacts with important regulators of the cell cycle, such as the Rb tumour suppresser protein, related proteins p107 and p130 and cyclins and cyclin- dependent kinases. The cellular E2F activity is a heterodimer consisting of a DP family member, of which three members have been characterised and an E2F family member, of which six family members have been isolated. Murine DP-3 differs from the other DP proteins due to striking complexity at the RNA level. RNA analysis has shown that extensive processing gives rise to four different DP-3 proteins, alpha, beta, gamma? and sigma. These variants arise via two open reading frame changes, including the insertion of the E region, a 16 residue sequence within the DNA binding domain of DP-3 alpha and sigma, which forms part of a nuclear localisation signal. Additionally, the insertion of single glutamine codon has been noted in the DP-3gamma isoform. Extensive splicing within the 5'UTR of DP-3 results in translation initiation at two different methionine codons. Since DP-1 is the major component of the E2F DNA binding activity in mammalian cells and the role of DP-3 remains unclear, the aim of this study was to investigate the expression of DP-3 at the RNA and protein level, in an attempt to understand the role of DP-3 in the E2F heterodimer. In an effort to understand the post-transcriptional control of DP-3, the murine DP-3 gene was isolated. Analysis of the exon/intron arrangement of the DP-3 gene and comparison of the DP-3 genomic and cDNA sequences provided insight into the post-transcriptional regulation of DP-3, in particular at sites such as the E region. Additionally, comparison with the murine DP-1 gene revealed striking conservation in genomic organisation, suggesting that they are ancestrally related. Analysis of DP-3 RNA via Northern blotting was performed to study the expression pattern of the different DP-3 RNAs. A range of different mouse tissues and tissue culture cell lines were tested for the presence of DP-3 RNA and DP-3 RNA abundance was analysed during cellular processes such as differentiation. An investigation into the effects of the different DP-3 5'UTRs on translational regulation of DP-3 protein expression was performed using chimeric DP-3 5'UTR-luciferase reporter constructs. The translational potential of each 5'UTR was analysed by transient transfection in a range of mammalian cells and was found to be different for each 5'UTR. The translational ability of each 5'UTR was also analysed in vitro. The effect of the tumour suppresser p53 on the translational ability of the DP-3 5'UTRs was also assessed. p53 is known to influence the translation of both its own RNA and that of cdk4. Analysis suggested that p53 might influence the translation of specific DP-3 isoforms. To aid the detection of DP-3 protein, anti-peptide polyclonal antibodies were made. These were used to study DP-3 expression by western blotting in a range of different mammalian cells and by immunostaining. These results imply that the expression of DP-3 is highly regulated at the post-transcriptional level. Although a definite role for DP-3 in E2F mediated processes has yet to be assigned, these results provide insight into the control of DP-3 expression, which may ultimately be linked to the role of DP-3 in cells.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Cellular biology.
• Subjects: Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: La Thangue, Professor Nick
• Date of Award: 2000
• Depositing User: Enlighten Team
• Unique ID: glathesis:2000-71274
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 May 2019 10:49
• Last Modified: 28 Oct 2022 08:31
• Thesis DOI: 10.5525/gla.thesis.71274
• URI: https://theses.gla.ac.uk/id/eprint/71274